Axonal Segments Research Articles

Refinement of axonal conduction and myelination in the mouse optic nerve indicate an extended period of postnatal developmental plasticity.

Retinal ganglion cells generate a pattern of action potentials to communicate visual information from the retina to cortical areas. Myelin, an insulating sheath, wraps axonal segments to facilitate signal propagation and when deficient, can impair visual function. Optic nerve development and initial myelination has largely been considered completed by the fifth postnatal week. However, the relationship between the extent of myelination and axonal signaling in the maturing optic nerve is not well characterized. Here, we examine the relationship between axon conduction and elements of myelination using extracellular nerve recordings, immunohistochemistry, western blot analysis, scanning electron microscopy, and simulations of nerve responses. Comparing compound action potentials from mice aged 4-12weeks revealed five functional distinct axonal populations, an increase in the number of functional axons, and shifts toward fast-conducting axon populations at 5 and 8weeks postnatal. At these ages, our analysis revealed increased myelin thickness, lower g-ratios and changes in the 14kDa MBP isoform, while the density of axons and nodes of Ranvier remained constant. At 5 postnatal weeks, axon diameter increased, while at 8weeks, increased expression of a mature sodium ion channel subtype, Nav 1.6, was observed at nodes of Ranvier. A simulation model of nerve conduction suggests that ion channel subtype, axon diameter, and myelin thickness are more likely to be key regulators of nerve function than g-ratio. Such refinement of axonal function and myelin rearrangement identified an extended period of maturation in the normal optic nerve that may facilitate the development of visual signaling patterns.

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy.

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

The effect of a gap concept on peripheral nerve recovery in modified epineurial neurorrhaphy: An experimental study in rats.

Several factors such as surgical approach that only consider topographic anatomy; inadequate fascicular alignment, extraepineurial sprouting in the repair zone; contact of axons with the suture area are the disadvantages of epineurial neurorrhaphy. Accordingly, axonal mismatch, neuroma, and unfavorable nerve recovery become inevitable. Neurotropism is the theory clarifying appropriate matching of the nerve fibers independently without needing surgical approach. The studies comparing the primary nerve repair with the nerve defects bridged in different ways demonstrated better outcomes of nerve recovery in the groups with a nerve gap. In this study, we aimed to demonstrate the effects of the gap concept in primary nerve repair bridged by own epineurium. We hypothesized that this technique will provide better results in terms of peripheral nerve recovery and will significantly eliminate the occurrence of a neuroma, which is quite possible in epineurial neurorrhaphy. A total of 35 Wistar female rats weighing 200 ~ 250 g were randomly divided into five groups each with seven rats. Sham controls constituted Group 1, while the rats with epineural neurorrhaphy were included in Group 2. The remaining three groups were the study groups. In Group 3, after the sciatic nerve transection, epineurium of the distal segment was sleeved and preserved. A 2-mm axonal segment was removed from the epineurium free distal ending and no any procedure was applied to the proximal ending of the transected sciatic nerve. Epineuriums of the both sides were approximated and repaired. In Group 4, a 2-mm axonal segment was removed from the proximal ending of the sciatic nerve after preservation of epineurium and no any procedure was applied to the distal part of sciatic nerve. Epineuriums of the both sides were approximated and repaired. In addition, in Group 5, after epineuriums were sleeved in the both distal and proximal stumps, a 1-mm nerve segment was removed from both endings and epineuriums were repaired in the middle bridging a 2-mm axonal gap again. After a 3months follow-up period Sciatic Functional Index (SFI) was measured by walking track analysis; the area under the evoked compound muscle action potential (CMAP) and latency periods were calculated via electromyographic (EMG) analysis; and histopathological evaluation were performed to compare the parameters of edema, fibrosis, inflammation, vascularization, axonal degeneration, axonal density, myelination, disorganization, and neuroma occurrence. Vascular structures and nerve fibers were counted at ×200 magnification: +1, +2, and +3 indicated the presence of 0-15, 16-30, and >30 structures, respectively. For uncountable parameters (edema, disorganization, myelination, fibrosis, and inflammation): +1 indicated mild, +2 indicated moderate, and +3 indicated severe. The differences between the groups with axonal gap repair and epineural neurorrhaphy were not significant regarding to SFI. The areas under CMAP were as follows: 27.9 ± 5.9 (Δ=12.1%) in Group 1; 16.5 ± 5.5 (Δ=6.3%) in Group 2; 14.1 ± 6.2 (Δ=4.8%) in group 3; 13.8 ± 2.3 (Δ=9.2%) in Group 4, and 22.5 ± 18.3 (Δ=2.2%) in Group 5. Group 5 (1 mm gap in the distal +1 mm gap in the proximal segments) had a significantly better result in terms of the area under CMAP with the value of 22.5 ± 18.3m/Mv (p=.031). Axonal density was 0.9 ± 0.6 (Δ=2.2%) in Group 2, 2.4 ± 0.3 (Δ=5.1%) in Group 3, 2.8 ± 0.1 (Δ=7.7%) in Group 4, and 2.8 ± 0.2 (Δ=4.8%) in Group 5. Myelination was 1.1 ± 0.5 (Δ=3.4%) in group 2, 2.2 ± 0.2 (Δ=6.7%) in group 3, 2.4 ± 0.4 (Δ=6.0%) in Group 4, and 2.7 ± 0.3 (Δ=4.6%) in Group 5. Disorganization was 2.3 ± 0.4 (Δ=4.1%) in Group 2, 1.2 ± 0.2 (Δ=7.7%) in Group 3, 1.3 ± 0.2 (Δ=6.5%) in Group 4, and 1 ± 0.3 (Δ=5.9%) in Group 5. And, neuroma occurrence was found 2.2 ± 0.6 (Δ=2.8%) in Group 2 and 0.3 ± 0.2 (Δ=0.1%) in Group 4 while neuroma was not encountered in Group 3 and Group 5. Comparison between the epineurial neurorrhaphy group and the groups with axonal defect revealed the statistically significant results in the factors of axonal density (p=.001), myelination (p=.028), disorganization (p=.016) and neuroma (p=.001). Creating axonal gap bridged by own epineurium showed favorable results comparing with epineurial neurorrhaphy. Resection of a 1 mm axonal segment from the proximal and distal stumps following the epineurial sleeve procedure and performing the epineurium- only repair can facilitate the nerve regeneration. The feasibility of the described technique has been demonstrated in a small rat model and must be further validated in larger animals before clinical testing.

Neuronal mTOR Outposts: Implications for Translation, Signaling, and Plasticity.

The kinase mTOR is a signaling hub for pathways that regulate cellular growth. In neurons, the subcellular localization of mTOR takes on increased significance. Here, we review findings on the localization of mTOR in axons and offer a perspective on how these may impact our understanding of nervous system development, function, and disease. We propose a model where mTOR accumulates in local foci we term mTOR outposts, which can be found in processes distant from a neuron’s cell body. In this model, pathways that funnel through mTOR are gated by local outposts to spatially select and amplify local signaling. The presence or absence of mTOR outposts in a segment of axon or dendrite may determine whether regional mTOR-dependent signals, such as nutrient and growth factor signaling, register toward neuron-wide responses. In this perspective, we present the emerging evidence for mTOR outposts in neurons, their putative roles as spatial gatekeepers of signaling inputs, and the implications of the mTOR outpost model for neuronal protein synthesis, signal transduction, and synaptic plasticity.

Loss of \u03b24-spectrin impairs Nav channel clustering at the heminode and temporal fidelity of presynaptic spikes in developing auditory brain

Beta-4 (β4)-spectrin, encoded by the gene Sptbn4, is a cytoskeleton protein found at nodes and the axon initial segments (AIS). Sptbn4 mutations are associated with myopathy, neuropathy, and auditory deficits in humans. Related to auditory dysfunction, however, the expression and roles of β4-spectrin at axon segments along the myelinated axon in the developing auditory brain are not well explored. We found during postnatal development, β4-spectrin is critical for voltage-gated sodium channel (Nav) clustering at the heminode along the nerve terminal, but not for the formation of nodal and AIS structures in the auditory brainstem. Presynaptic terminal recordings in Sptbn4geo mice, β4-spectrin null mice, showed an elevated threshold of action potential and increased failures during action potential train at high-frequency. Sptbn4geo mice exhibited a slower central conduction and showed no startle responses, but had normal cochlear function. Taken together, the lack of β4-spectrin impairs Nav clustering at the heminode along the nerve terminal and the temporal fidelity and reliability of presynaptic spikes, leading to central auditory processing deficits during postnatal development.

Neurorrhaphy in Presence of Polyethylene Glycol Enables Immediate Electrophysiological Conduction in Porcine Model of Facial Nerve Injury.

Facial nerve trauma often leads to disfiguring facial muscle paralysis. Despite several promising advancements, facial nerve repair procedures often do not lead to complete functional recovery. Development of novel repair strategies requires testing in relevant preclinical models that replicate key clinical features. Several studies have reported that fusogens, such as polyethylene glycol (PEG), can improve functional recovery by enabling immediate reconnection of injured axons; however, these findings have yet to be demonstrated in a large animal model. We first describe a porcine model of facial nerve injury and repair, including the relevant anatomy, surgical approach, and naive nerve morphometry. Next, we report positive findings from a proof-of-concept experiment testing whether a neurorrhaphy performed in conjunction with a PEG solution maintained electrophysiological nerve conduction at an acute time point in a large animal model. The buccal branch of the facial nerve was transected and then immediately repaired by direct anastomosis and PEG application. Immediate electrical conduction was recorded in the PEG-fused nerves (n = 9/9), whereas no signal was obtained in a control cohort lacking calcium chelating agent in one step (n = 0/3) and in the no PEG control group (n = 0/5). Nerve histology revealed putative-fused axons across the repair site, whereas no positive signal was observed in the controls. Rapid electrophysiological recovery following nerve fusion in a highly translatable porcine model of nerve injury supports previous studies suggesting neurorrhaphy supplemented with PEG may be a promising strategy for severe nerve injury. While acute PEG-mediated axon conduction is promising, additional work is necessary to determine if physical axon fusion occurs and the longer-term fate of distal axon segments as related to functional recovery.

Mechanical Behavior of Axonal Actin, Spectrin, and Their Periodic Structure: A Brief Review

Actin and spectrin are important constituents of axonal cytoskeleton. Periodic actin-spectrin structures are found in dendrites, initial segment of axon, and main axon. Actin-spectrin periodicity has been hypothesized to be manipulating the axon stability and mechanical behavior. Several experimental and computational studies have been performed focusing on the mechanical behavior of actin, spectrin, and actin-spectrin network. However, most of the actin studies focus on typical long F-actin and do not provide quantitative comparison between the mechanical behavior of short and long actin filaments. Also, most of the spectrin studies focus on erythrocytic spectrin and do not shed light on the behavior of structurally different axonal spectrin. Only a few studies have highlighted forced unfolding of axonal spectrin which are relevant to brain injury scenario. A comprehensive, strain rate dependent mechanical study is still absent in the literature. Moreover, the current opinions regarding periodic actin-spectrin network structure in axon are disputed due to conflicting results on actin ring organization—as argued by recent super-resolution microscopy studies. This review summarizes the ongoing limitations in this regard and provides insights on possible approaches to address them. This study will invoke further investigation into relevant high strain rate response of actin, spectrin, and actin-spectrin network—shedding light into brain pathology scenario such as traumatic brain injury (TBI).Graphic Abstract

Information theoretic modeling of paranodal regions in myelinated axons

As a natural form of nanoscale communication, neuro-spike communication inspires the deployment of nanomachines inside the human body for healthcare. To this end, the identification of failure mechanisms in normal and diseased connections of nervous nano-networks is crucial. Thus, in this paper, we investigate the information transmission through a single myelinated axon segment. We introduce a realistic multi-compartmental model for a single myelinated segment by incorporating the axon's paranodal regions to the model. Next, we characterize the myelinated segment communication channel in terms of attenuation over the range of frequencies. Based on this, we derive the rate per channel use and upper bound on the information capacity. The performance evaluations reveal that our approach provides dramatic correction regarding frequency response. We believe that this result could have a significant effect on the characterization of demyelinated axons from the information and communication technology (ICT) perspective.

Stressed axons craving for glial sugar: links to regeneration?

Stressed axons craving for glial sugar: links to regeneration?

From PNS to CNS: characteristics of anti-neurofascin 186 neuropathy in 16 cases.

Neurofascin (NF) is critical for the formation and maintenance of Ranvier nodes. NF186, the neuronal form of NF, localizes in the initial segment of axon and Ranvier node. NF186 antibody has been detected in demyelinating diseases of both central nervous system (CNS) and peripheral nervous system (PNS). To evaluate the clinical features of patients with anti-NF186 IgG neuropathy. Sixteen patients (16/138) with serum-positive anti-NF186 IgG were included and divided into groups of either CNS or PNS-involved according to their clinical manifestations. Anti-NF186 IgG was detected by cell-based assays. In 7 patients who were confirmed to have CNS involvement, the most frequent symptoms were dizziness (57%) and vision impairment (43%); lesions in centrum semiovale, cerebellum, and meninges were shown by magnetic resonance imaging (MRI). In comparison, limb weakness (78%) and numbness (78%) were the most common symptoms in PNS-involved patients; axonal loss and demyelination were confirmed by nerve conduction examinations. Elevated level of cerebrospinal fluid (CSF) protein was found in 12 cases without statistically significant difference between the CNS and PNS groups. Meanwhile, CSF white blood cell counts were found significantly elevated in CNS-involved patients compared with patients of PNS group. Thirteen patients received immunomodulating treatments, and patients with chronic onset and progressive course showed poor response to the therapies. Patients with anti-NF186 IgG neuropathy showed no specific symptoms or signs. It is worth noting that quite a few patients show CNS-impaired signs only, and cranial MRI is essential for the screening of CNS involvement.

Quetiapine promotes oligodendroglial process outgrowth and membrane expansion by orchestrating the effects of Olig1.

Oligodendroglial lineage cells go through a series of morphological changes before myelination. Prior to myelination, cell processes and membrane structures enlarge by approximately 7,000 times, which is required to support axonal wrapping and myelin segment formation. Failure of these processes leads to maldevelopment and impaired myelination. Quetiapine, an atypical antipsychotic drug, was proved to promote oligodendroglial differentiation and (re)myelination, pending detailed effects and regulatory mechanism. In this study, we showed that quetiapine promotes morphological maturation of oligodendroglial lineage cells and myelin segment formation, and a short-term quetiapine treatment is sufficient to induce these changes. To uncover the underlying mechanism, we examined the effect of quetiapine on the Oligodendrocyte transcription factor 1 (Olig1). We found that quetiapine upregulates Olig1 expression level and promotes nuclear Olig1 translocation to the cytosol, where it functions not as a transcription modulator, but in a way that highly correlates with oligodendrocyte morphological transformation. In addition, quetiapine treatment reverses the negative regulatory effect of the Olig1-regulated G protein-coupled receptor 17 (GPR17) on oligodendroglial morphological maturation. Our results demonstrate that quetiapine enhances oligodendroglial differentiation and myelination by promoting cell morphological transformation. This would shed light on the orchestration of oligodendroglia developmental mechanisms, and provides new targets for further therapeutic research.

Adaptive responses to neurodegenerative stress in glaucoma.

Glaucoma causes loss of vision through degeneration of the retinal ganglion cell (RGC) projection to the brain. The disease is characterized by sensitivity to intraocular pressure (IOP) conveyed at the optic nerve head, through which RGC axons pass unmyelinated to form the optic nerve. From this point, a pathogenic triumvirate comprising inflammatory, oxidative, and metabolic stress influence both proximal structures in the retina and distal structures in the optic projection. This review focuses on metabolic stress and how the optic projection may compensate through novel adaptive mechanisms to protect excitatory signaling to the brain. In the retina and proximal nerve head, the unmyelinated RGC axon segment is energy-inefficient, which leads to increased demand for adenosine-5'-triphosphate (ATP) at the risk of vulnerability to Ca2+-related metabolic and oxidative pressure. This vulnerability may underlie the bidirectional nature of progression. However, recent evidence highlights that the optic projection in glaucoma is not passive but rather demonstrates adaptive processes that may push back against neurodegeneration. In the retina, even as synaptic and dendritic pruning ensues, early progression involves enhanced excitability of RGCs. Enhancement involves depolarization of the resting membrane potential and increased response to light, independent of RGC morphological type. This response is axogenic, arising from increased levels and translocation of voltage-gated sodium channels (NaV) in the unmyelinated segment. During this same early period, large-scale networks of gap-junction coupled astrocytes redistribute metabolic resources to the optic projection stressed by elevated IOP to slow loss of axon function. This redistribution may reflect more local remodeling, as astrocyte processes respond to focal metabolic duress by boosting glycogen turnover in response to axonal activity in an effort to promote survival of the healthiest axons. Both enhanced excitability and metabolic redistribution are transient, indicating that the same adaptive mechanisms that apparently serve to slow progression ultimately may be too expensive for the system to sustain over longer periods.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

SummaryNeurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

GRDN-1/Girdin regulates dendrite morphogenesis and cilium position in two specialized sensory neuron types in C. elegans

Primary cilia are located at the dendritic tips of sensory neurons and house the molecular machinery necessary for detection and transduction of sensory stimuli. The mechanisms that coordinate dendrite extension with cilium position during sensory neuron development are not well understood. Here, we show that GRDN-1, the Caenorhabditis elegans ortholog of the highly conserved scaffold and signaling protein Girdin/GIV, regulates both cilium position and dendrite extension in the postembryonic AQR and PQR gas-sensing neurons. Mutations in grdn-1 disrupt dendrite outgrowth and mislocalize cilia to the soma or proximal axonal segments in AQR, and to a lesser extent, in PQR. GRDN-1 is localized to the basal body and regulates localization of HMR-1/Cadherin to the distal AQR dendrite. However, knockdown of HMR-1 and/or loss of SAX-7/LICAM, molecules previously implicated in sensory dendrite development in C. elegans, do not alter AQR dendrite morphology or cilium position. We find that GRDN-1 localization in AQR is regulated by UNC-116/Kinesin-1, and that correspondingly, unc-116 mutants exhibit severe AQR dendrite outgrowth and cilium positioning defects. In contrast, GRDN-1 and cilium localization in PQR is modulated by LIN-44/Wnt signaling. Together, these findings identify upstream regulators of GRDN-1, and describe new cell-specific roles for this multifunctional protein in sensory neuron development.

Mechanism of axonal degeneration: from molecular signaling to the development of therapeutic applications

Neurons communicate with other cells via long processes, i.e., axons and dendrites, functionally and morphologically specialized tree-like structures. Formation and maintenance of such processes play a crucial role in neuronal functions. Axons are particularly important for construction of neuronal network, and, together with synapses at the end of them, play a central role in transmission of information. Axonal degeneration, a phenomenon that once formed axons lose structural integrity, is most typically observed as "Wallerian degeneration", in which injured axonal segment (distal to the site of injury) degenerates. Different forms of axonal degeneration are also observed in a variety of contexts, including pathogenesis and progression of different neurodegenerative disorders, as well as neuronal network formation during development. Thus, understanding of regulatory mechanism of axonal degeneration is important in many aspects, such as for clarification of neuronal morphogenesis mechanism, and for development of neuroprotective therapy against neurological disorders. Here, I discuss recent progress in the research field of axonal degeneration mechanism.

Schwann cell development: From neural crest to myelin sheath.

Vertebrate nervous system function requires glial cells, including myelinating glia that insulate axons and provide trophic support that allows for efficient signal propagation by neurons. In vertebrate peripheral nervous systems, neural crest-derived glial cells known as Schwann cells (SCs) generate myelin by encompassing and iteratively wrapping membrane around single axon segments. SC gliogenesis and neurogenesis are intimately linked and governed by a complex molecular environment that shapes their developmental trajectory. Changes in this external milieu drive developing SCs through a series of distinct morphological and transcriptional stages from the neural crest to a variety of glial derivatives, including the myelinating sublineage. Cues originate from the extracellular matrix, adjacent axons, and the developing SC basal lamina to trigger intracellular signaling cascades and gene expression changes that specify stages and transitions in SC development. Here, we integrate the findings from in vitro neuron-glia co-culture experiments with in vivo studies investigating SC development, particularly in zebrafish and mouse, to highlight critical factors that specify SC fate. Ultimately, we connect classic biochemical and mutant studies with modern genetic and visualization tools that have elucidated the dynamics of SC development. This article is categorized under: Signaling Pathways > Cell Fate Signaling Nervous System Development > Vertebrates: Regional Development.

Purkinje cell neurotransmission patterns cerebellar basket cells into zonal modules defined by distinct pinceau sizes.

Ramón y Cajal proclaimed the neuron doctrine based on circuit features he exemplified using cerebellar basket cell projections. Basket cells form dense inhibitory plexuses that wrap Purkinje cell somata and terminate as pinceaux at the initial segment of axons. Here, we demonstrate that HCN1, Kv1.1, PSD95 and GAD67 unexpectedly mark patterns of basket cell pinceaux that map onto Purkinje cell functional zones. Using cell-specific genetic tracing with an Ascl1CreERT2 mouse conditional allele, we reveal that basket cell zones comprise different sizes of pinceaux. We tested whether Purkinje cells instruct the assembly of inhibitory projections into zones, as they do for excitatory afferents. Genetically silencing Purkinje cell neurotransmission blocks the formation of sharp Purkinje cell zones and disrupts excitatory axon patterning. The distribution of pinceaux into size-specific zones is eliminated without Purkinje cell GABAergic output. Our data uncover the cellular and molecular diversity of a foundational synapse that revolutionized neuroscience.

Polyethylene glycol-fusion repair of sciatic allografts in female rats achieves immunotolerance via attenuated innate and adaptive responses.

Ablation/segmental loss peripheral nerve injuries (PNIs) exhibit poor functional recovery due to slow and inaccurate outgrowth of regenerating axons. Viable peripheral nerve allografts (PNAs) as growth-guide conduits are immunologically rejected and all anucleated donor/host axonal segments undergo Wallerian degeneration. In contrast, we report that ablation-type sciatic PNIs repaired by neurorrhaphy of viable sciatic PNAs and a polyethylene glycol (PEG)-fusion protocol using PEG immediately restored axonal continuity for many axons, reinnervated/maintained their neuromuscular junctions, and prevented much Wallerian degeneration. PEG-fused PNAs permanently restored many sciatic-mediated behaviors within 2-6weeks. PEG-fused PNAs were not rejected even though host/donors were neither immunosuppressed nor tissue-matched in outbred female Sprague Dawley rats. Innate and adaptive immune responses to PEG-fused sciatic PNAs were analyzed using electron microscopy, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction for morphological features, T cell and macrophage infiltration, major histocompatibility complex (MHC) expression, apoptosis, expression of cytokines, chemokines, and cytotoxic effectors. PEG-fused PNAs exhibited attenuated innate and adaptive immune responses by 14-21days postoperatively, as evidenced by (a) many axons and cells remaining viable, (b) significantly reduced infiltration of cytotoxic and total T cells and macrophages, (c) significantly reduced expression of inflammatory cytokines, chemokines, and MHC proteins, (d) consistently low apoptotic response. Morphologically and/or biochemically, PEG-fused sciatic PNAs often resembled sciatic autografts or intact sciatic nerves. In brief, PEG-fused PNAs are an unstudied, perhaps unique, example of immune tolerance of viable allograft tissue in a nonimmune-privileged environment and could greatly improve the clinical outcomes for PNIs relative to current protocols.

Drosophila Voltage-Gated Sodium Channels Are Only Expressed in Active Neurons and Are Localized to Distal Axonal Initial Segment-like Domains.

In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos-Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para Despite being the only NaV channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (NaV) channel in the fly, para, and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally, para is only expressed in 23 ± 1% of third instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the third instar larval brain shows that para expression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ± 1% of third instar larval neurons may be able to actively fire NaV-dependent APs.

Local modulation of Neurofilament transport at Nodes of Ranvier

Neurofilaments (NFs) are the most abundant cytoskeletal filaments undergoing ‘slow axonal transport’ in axons, and the population of NFs determines the axonal morphology. Both in vitro and ex-vivo experimental evidences show that the caliber of node is much thinner and the number of NFs in the node is much lower than the internode. Based on the Continuity equation, lower population of NFs indicates faster transport velocity. We propose that the local acceleration of NFs transport at node may result from the higher on-track rate or higher transition rate from pausing to running. We construct a segment of axon including both node and internode, and inject NFs by a fixed flux into it continuously. By upregulating transition rate of either or locally at the Node of Ranvier in the ‘6-state’model, we successfully accelerate NFs velocity and reproduce constriction of nodes. Our work demonstrates that local modulation of NF kinetics can change NFs distribution and shape the morphology of Node of Ranvier.