Axonal Segments Research Articles

Microglial process convergence onto injured axonal swellings, a human postmortem brain tissue study.

Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons following brain injury in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal segments, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals that sustained a TBI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation.

Oligodendrocytes: Myelination, Plasticity, and Axonal Support.

The myelination of axons has evolved to enable fast and efficient transduction of electrical signals in the vertebrate nervous system. Acting as an electric insulator, the myelin sheath is a multilamellar membrane structure around axonal segments generated by the spiral wrapping and subsequent compaction of oligodendroglial plasma membranes. These oligodendrocytes are metabolically active and remain functionally connected to the subjacent axon via cytoplasmic-rich myelinic channels for movement of metabolites and macromolecules to and from the internodal periaxonal space under the myelin sheath. Increasing evidence indicates that oligodendrocyte numbers, specifically in the forebrain, and myelin as a dynamic cellular compartment can both respond to physiological demands, collectively referred to as adaptive myelination. This review summarizes our current understanding of how myelin is generated, how its function is dynamically regulated, and how oligodendrocytes support the long-term integrity of myelinated axons.

Morphological correlates of pyramidal cell axonal myelination in mouse and human neocortex.

The axons of neocortical pyramidal neurons are frequently myelinated. Heterogeneity in the topography of axonal myelination in the cerebral cortex has been attributed to a combination of electrophysiological activity, axonal morphology, and neuronal-glial interactions. Previously, we showed that axonal segment length and caliber are critical local determinants of fast-spiking interneuron myelination. However, the factors that determine the myelination of individual axonal segments along neocortical pyramidal neurons remain largely unexplored. Here, we used structured illumination microscopy to examine the extent to which axonal morphology is predictive of the topography of myelination along neocortical pyramidal neurons. We identified critical thresholds for axonal caliber and interbranch distance that are necessary, but not sufficient, for myelination of pyramidal cell axons in mouse primary somatosensory cortex (S1). Specifically, we found that pyramidal neuron axonal segments with a caliber < 0.24μm or interbranch distance < 18.10μm are rarely myelinated. Moreover, we further confirmed that these findings in mice are similar for human neocortical pyramidal cell myelination (caliber < 0.25μm, interbranch distance < 19.00μm), suggesting that this mechanism is evolutionarily conserved. Taken together, our findings suggest that axonal morphology is a critical correlate of the topography and cell-type specificity of neocortical myelination.

Filtering property of myelinated internode can change neural information representability and might trigger a compensatory action during demyelination

In this paper, for the first time, we showed that an Internode Segment (INS) of a myelinated axon acts as a lowpass filter, and its filter characteristics depend on the number of myelin turns. Consequently, we showed how the representability of a neural signal could be altered with myelin loss in pathological conditions involving demyelinating diseases. Contrary to the traditionally held viewpoint that myelin geometry of an INS is optimised for maximising Conduction Velocity (CV) of Action Potential (AP), our theory provides an alternative viewpoint that myelin geometry of an INS is optimised for maximizing representability of the stimuli a fibre is meant to carry. Subsequently, we show that this new viewpoint could explain hitherto unexplained experimentally observed phenomena such as, shortening of INS length during demyelination and remyelination, and non-uniform distribution of INS in the central nervous system fibres and associated changes in diameter of nodes of ranvier along an axon. Finally, our theory indicates that a compensatory action could take place during demyelination up to a certain number of loss of myelin turns to preserve the neural signal representability by simultaneous linear scaling of the length of an INS and the inner radius of the fibre.

Visualizing Wallerian degeneration in the corticospinal tract after sensorimotor cortex ischemia in mice.

Stroke can cause Wallerian degeneration in regions outside of the brain, particularly in the corticospinal tract. To investigate the fate of major glial cells and axons within affected areas of the corticospinal tract following stroke, we induced photochemical infarction of the sensorimotor cortex leading to Wallerian degeneration along the full extent of the corticospinal tract. We first used a routine, sensitive marker of axonal injury, amyloid precursor protein, to examine Wallerian degeneration of the corticospinal tract. An antibody to amyloid precursor protein mapped exclusively to proximal axonal segments within the ischemic cortex, with no positive signal in distal parts of the corticospinal tract, at all time points. To improve visualization of Wallerian degeneration, we next utilized an orthograde virus that expresses green fluorescent protein to label the corticospinal tract and then quantitatively evaluated green fluorescent protein-expressing axons. Using this approach, we found that axonal degeneration began on day 3 post-stroke and was almost complete by 7 days after stroke. In addition, microglia mobilized and activated early, from day 7 after stroke, but did not maintain a phagocytic state over time. Meanwhile, astrocytes showed relatively delayed mobilization and a moderate response to Wallerian degeneration. Moreover, no anterograde degeneration of spinal anterior horn cells was observed in response to Wallerian degeneration of the corticospinal tract. In conclusion, our data provide evidence for dynamic, pathogenic spatiotemporal changes in major cellular components of the corticospinal tract during Wallerian degeneration.

The Stathmin-2 membrane-targeting domain is required for axon protection and regulated degradation by DLK signaling

Axon integrity is essential for functional connectivity in the nervous system. The degeneration of stressed or damaged axons is a common and sometimes initiating event in neurodegenerative disorders. Stathmin-2 (Stmn2) is an axon maintenance factor that is depleted in Amyotrophic Lateral Sclerosis, and replenishment of Stmn2 can restore neurite outgrowth in diseased neurons. However, mechanisms responsible for Stmn2-mediated axon maintenance in injured neurons are not known. We used primary sensory neurons to interrogate the role of Stmn2 in the degeneration of severed axons. We discover that membrane association of Stmn2 is critical for its axon-protective activity. Structure-function studies revealed that axonal enrichment of Stmn2 is driven by palmitoylation as well as tubulin interaction. Using live-imaging, we discover that another Stathmin, Stmn3, co-migrates with Stmn2-containing vesicles. We also demonstrate that Stmn3 undergoes regulated degradation through DLK-JNK signaling. The Stmn2 membrane targeting domain is both necessary and sufficient for localization to a specific vesicle population and confers sensitivity to DLK-dependent degradation. Our findings reveal a broader role for DLK in tuning the local abundance of palmitoylated Stathmins in axon segments. Moreover, palmitoylation is a critical component of Stathmin-mediated axon protection, and defining the Stmn2-containing vesicle population will provide important clues toward mechanisms of axon maintenance.

Representing connectivity circuits in autonomic physiology: the SPARC map of organ innervation

The Stimulating Peripheral Activity to Relieve Conditions (SPARC) program is a NIH-funded consortium to improve the understanding of how the autonomic nervous system (ANS) interacts with end organs and the central nervous system. A major goal of SPARC is to use this knowledge to develop the next generation of neuromodulator devices as effective disease therapies. To support neuromodulation planning, the routes of ANS neuron populations (as well as those populations conveying visceral sensing) are to be mapped in terms of the anatomical structures where they pass through or terminate. Such a map would also serve as an organizing scaffold for (i) a growing SPARC repository of electrophysiology recordings and molecular assay data, as well as (ii) the computational simulation of the effect of neural stimulation on any relevant point on the CNS or ANS. The resulting SPARC map represents connectivity as a network of conduits, and is known as the SPARC Connectivity Knowledge Base of the Autonomic Nervous System (SCKAN). SCKAN contains explicit knowledge about CNS-ANS-end organ circuitry derived from SPARC data and scientific literature, in a form that supports computational reasoning. All connections are annotated with standard reference SPARC vocabularies allowing us to integrate datasets that are annotated to the same annotation standard. Circuits on the map represent details of ANS connectivity associated with a particular organ, e.g., bladder control, defensive breathing, modulation of peristalsis or cardiac inotropy/chronotropy. These circuits are created through interviews with SPARC investigators, anatomical experts and the scientific literature. They contain detailed representations of neuron populations giving rise to ANS connections, including mappings of the locations of cell bodies, dendrites, axon segments and synaptic endings. Circuits are modeled using ApiNATOMY, a knowledge model and tool suite specifically created to represent biological connectivity. Furthermore, these detailed circuits are supplemented with general knowledge on connectivity between CNS nuclei, ANS ganglia, nerves, and end organs derived from the scientific literature. To facilitate extracting this knowledge from the literature and maintaining relevance over time, we developed a Natural Language Processing (NLP) pipeline, which extracts connectivity relationships between unique anatomical structures within a sentence from the scientific literature. SCKAN is being used to create a queryable visual atlas of ANS circuitry through the SPARC portal. SPARC Funding: NIH Common Fund, NIH Office of the Director, Award OT2 OD030541 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cholinergic and noradrenergic axonal activity contains a behavioral-state signal that is coordinated across the dorsal cortex.

Fluctuations in brain and behavioral state are supported by broadly projecting neuromodulatory systems. In this study, we use mesoscale two-photon calcium imaging to examine spontaneous activity of cholinergic and noradrenergic axons in awake mice in order to determine the interaction between arousal/movement state transitions and neuromodulatory activity across the dorsal cortex at distances separated by up to 4 mm. We confirm that GCaMP6s activity within axonal projections of both basal forebrain cholinergic and locus coeruleus noradrenergic neurons track arousal, indexed as pupil diameter, and changes in behavioral engagement, as reflected by bouts of whisker movement and/or locomotion. The broad coordination in activity between even distant axonal segments indicates that both of these systems can communicate, in part, through a global signal, especially in relation to changes in behavioral state. In addition to this broadly coordinated activity, we also find evidence that a subpopulation of both cholinergic and noradrenergic axons may exhibit heterogeneity in activity that appears to be independent of our measures of behavioral state. By monitoring the activity of cholinergic interneurons in the cortex, we found that a subpopulation of these cells also exhibit state-dependent (arousal/movement) activity. These results demonstrate that cholinergic and noradrenergic systems provide a prominent and broadly synchronized signal related to behavioral state, and therefore may contribute to state-dependent cortical activity and excitability.

The cholinergic basal forebrain provides a parallel channel for state-dependent sensory signaling to auditory cortex.

Cholinergic basal forebrain (CBF) signaling exhibits multiple timescales of activity with classic slow signals related to brain and behavioral states and fast, phasic signals reflecting behavioral events, including movement, reinforcement and sensory-evoked responses. However, it remains unknown whether sensory cholinergic signals target the sensory cortex and how they relate to local functional topography. Here we used simultaneous two-channel, two-photon imaging of CBF axons and auditory cortical neurons to reveal that CBF axons send a robust, nonhabituating and stimulus-specific sensory signal to the auditory cortex. Individual axon segments exhibited heterogeneous but stable tuning to auditory stimuli allowing stimulus identity to be decoded from population activity. However, CBF axons displayed no tonotopy and their frequency tuning was uncoupled from that of nearby cortical neurons. Chemogenetic suppression revealed the auditory thalamus as a major source of auditory information to the CBF. Finally, slow fluctuations in cholinergic activity modulated the fast, sensory-evoked signals in the same axons, suggesting that a multiplexed combination of fast and slow signals is projected from the CBF to the auditory cortex. Taken together, our work demonstrates a noncanonical function of the CBF as a parallel channel for state-dependent sensory signaling to the sensory cortex that provides repeated representations of a broad range of sound stimuli at all points on the tonotopic map.

Traumatic Axonal Injury in the Optic Nerve: The Selective Role of SARM1 in the Evolution of Distal Axonopathy.

Traumatic axonal injury (TAI), thought to be caused by rotational acceleration of the head, is a prevalent neuropathology in traumatic brain injury (TBI). TAI in the optic nerve is a common finding in multiple blunt-force TBI models and hence a great model to study mechanisms and treatments for TAI, especially in view of the compartmentalized anatomy of the visual system. We have previously shown that the somata and the proximal, but not distal, axons of retinal ganglion cells (RGC) respond to DLK/LZK blockade after impact acceleration of the head (IA-TBI). Here, we explored the role of the sterile alpha and TIR-motif containing 1 (SARM1), the key driver of Wallerian degeneration (WD), in the progressive breakdown of distal and proximal segments of the optic nerve following IA-TBI with high-resolution morphological and classical neuropathological approaches. Wild type and Sarm1 knockout (KO) mice received IA-TBI or sham injury and were allowed to survive for 3, 7, 14, and 21 days. Ultrastructural and microscopic analyses revealed that TAI in the optic nerve is characterized by variable involvement of individual axons, ranging from apparent early disconnection of a subpopulation of axons to a range of ongoing axonal and myelin perturbations. Traumatic axonal injury resulted in the degeneration of a population of axons distal and proximal to the injury, along with retrograde death of a subpopulation of RGCs. Quantitative analyses on proximal and distal axons and RGC somata revealed that different neuronal domains exhibit differential vulnerability, with distal axon segments showing more severe degeneration compared with proximal segments and RGC somata. Importantly, we found that Sarm1 KO had a profound effect in the distal optic nerve by suppressing axonal degeneration by up to 50% in the first 2 weeks after IA-TBI, with a continued but lower effect at 3 weeks, while also suppressing microglial activation. Sarm1 KO had no evident effect on the initial traumatic disconnection and did not ameliorate the proximal optic axonopathy or the subsequent attrition of RGCs, indicating that the fate of different axonal segments in the course of TAI may depend on distinct molecular programs within axons.

Efficient simulations of stretch growth axon based on improved HH model

ABSTRACT Objective According to recent studies, synaptic connections by axons formed can resist stronger mechanical stresses. A large number of nerve tissues with regular growth can be achieved quickly by appropriately managing the pulling pace and force. However, currently there are few studies on the physiological characteristics of stretching growth axons. Until now, there is no mature technology for determining whether the axons can normally transmit neural signals following continuous mechanical stimulation. In this paper, an improved HH model was proposed to investigate the effect of mechanical stimuli on stretched axons and synapses. Methods First, we add the link between membrane capacitance and diameter to the standard HH model and the fundamental concept of capacitance. Then, unmyelinated stretch growth axons with different lengths were simulated in this model. Results After mechanical pulling stimulation, the improved model successfully replicated the generation and propagation of action potentials in several different axon segments. This increase in length accompanying an increase in diameter due to stretch growth. When the stretching growth axon was stimulated again by traction, the membrane capacitance rapidly increased from the constant value and the inward current was large enough to depolarize the membrane and produce an action potential. After stretching, nerve fibers could still receive signals sent by other neuron cells and complete the signal transmission. Discussion This proposed research can help to optimize stretching axon culture conditions, avoid mechanical force damage to nerve tissue, and achieve a more effective nerve tissue culture process.

Oligodendrocytes Prune Axons Containing α-Synuclein Aggregates In Vivo: Lewy Neurites as Precursors of Glial Cytoplasmic Inclusions in Multiple System Atrophy?

α-Synucleinopathies are spreading neurodegenerative disorders characterized by the intracellular accumulation of insoluble aggregates populated by α-Synuclein (α-Syn) fibrils. In Parkinson's disease (PD) and dementia with Lewy bodies, intraneuronal α-Syn aggregates are referred to as Lewy bodies in the somata and as Lewy neurites in the neuronal processes. In multiple system atrophy (MSA) α-Syn aggregates are also found within mature oligodendrocytes (OLs) where they form Glial Cytoplasmic Inclusions (GCIs). However, the origin of GCIs remains enigmatic: (i) mature OLs do not express α-Syn, precluding the seeding and the buildup of inclusions and (ii) the artificial overexpression of α-Syn in OLs of transgenic mice results in a burden of soluble phosphorylated α-Syn but fails to form α-Syn fibrils. In contrast, mass spectrometry of α-Syn fibrillar aggregates from MSA patients points to the neuronal origin of the proteins intimately associated with the fibrils within the GCIs. This suggests that GCIs are preassembled in neurons and only secondarily incorporated into OLs. Interestingly, we recently isolated a synthetic human α-Syn fibril strain (1B fibrils) capable of seeding a type of neuronal inclusion observed early and specifically during MSA. Our goal was thus to investigate whether the neuronal α-Syn pathology seeded by 1B fibrils could eventually be transmitted to OLs to form GCIs in vivo. After confirming that mature OLs did not express α-Syn to detectable levels in the adult mouse brain, a series of mice received unilateral intra-striatal injections of 1B fibrils. The resulting α-Syn pathology was visualized using phospho-S129 α-Syn immunoreactivity (pSyn). We found that even though 1B fibrils were injected unilaterally, many pSyn-positive neuronal somas were present in layer V of the contralateral perirhinal cortex after 6 weeks. This suggested a fast retrograde spread of the pathology along the axons of crossing cortico-striatal neurons. We thus scrutinized the posterior limb of the anterior commissure, i.e., the myelinated interhemispheric tract containing the axons of these neurons: we indeed observed numerous pSyn-positive linear Lewy Neurites oriented parallel to the commissural axis, corresponding to axonal segments filled with aggregated α-Syn, with no obvious signs of OL α-Syn pathology at this stage. After 6 months however, the commissural Lewy neurites were no longer parallel but fragmented, curled up, sometimes squeezed in-between two consecutive OLs in interfascicular strands, or even engulfed inside OL perikarya, thus forming GCIs. We conclude that the 1B fibril strain can rapidly induce an α-Syn pathology typical of MSA in mice, in which the appearance of GCIs results from the pruning of diseased axonal segments containing aggregated α-Syn.

Myelin protein zero - the structural foundation behind peripheral compact myelin.

Schwann cells are the myelinating glia of the peripheral nervous system (PNS). During PNS development, Schwann cell precursors differentiate into mature Schwann cells after the radial sorting of axons and form myelin around selected axonal segments. This multilayered, highly lipid-rich and insulative membrane structure significantly accelerates nerve impulse propagation, which is a pre-requisite for the efficient function of the PNS. During this differentiation process, protein zero (P0), a single-pass transmembrane protein, is expressed in high amounts. P0 is the most abundant PNS myelin protein, at up to 50% of total protein, and its accumulation in membranes eventually forms stable compact myelin. Failure to do so, like in the case of mutated or truncated protein, results in non-compacted myelin and demyelination. This subsequently causes incurable peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), where CMT alone afflicts nearly 3 million people globally. To understand the compaction and maturation of PNS myelin, and the molecular etiology of disease, uncovering the structure-function relationships of P0 are of great importance. We demonstrate the molecular foundation of P0-mediated membrane stacking using a hybrid structural biology approach: on the extracellular side, immunoglobulin (Ig)-like domains form a zipper-like assembly of apposing protein monomers, whereas on the cytoplasmic side, the short, positively charged P0 tail (P0ct) glues membranes together. P0ct undergoes a substantial folding event during membrane binding, and we provide rationale to why membrane stacking is affected in diseases through functional in vitro mutagenesis studies. Additionally, we speculate on how post-translational modifications in P0ct help P0 locate to its target compartments, without inducing premature membrane stacking. Our results shed light on the essential role of P0 in during myelin maturation, and why other proteins fail to replace P0 when its function is compromised.

Mitochondrial transport along RGC axons: Strategies to restore energy homeostasis and visual function

AbstractDeficits in mitochondria anterograde axonal transport is a common feature of neurodegenerative diseases including glaucoma. Whether aberrant expression of components in the transport machinery impinge directly on metabolic stress, neuronal death, and circuit dysfunction, is currently unknown. We used multiphoton microscope live imaging to monitor mitochondrial transport along the axons of retinal ganglion cells (RGC), the neurons that die in glaucoma. We found that high intraocular pressure, the most important risk factor to develop the disease, impairs anterograde movement of mitochondria along RGC axons leading to mitochondria depletion and energy decline. Molecular profiling of motor and adaptor proteins revealed loss of Disrupted in schizophrenia (Disc1) expression in glaucomatous RGC. Adeno‐associated virus (AAV)‐based Disc1 gene delivery rescued anterograde transport and replenished mitochondria in proximal and distal axonal segments. Delivery of a genetically encoded ATP sensor combined with longitudinal live imaging revealed that Disc1 supplementation increased ATP production in stressed neurons. Strikingly, Disc1 gene therapy promoted neuronal survival, reversed abnormal RGC calcium dynamics, and restored light‐evoked retinal responses and visual behaviour. Our study provides proof‐of‐principle that enhancing mitochondrial anterograde transport is an effective strategy to alleviate metabolic stress and glaucomatous neurodegeneration.

Pathology of Initial Axon Segments in Chronic Inflammatory Demyelinating Polyradiculoneuropathy and Related Disorders.

The diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is based on a combination of clinical, electrodiagnostic and laboratory features. The different entities of the disease include chronic immune sensory polyradiculopathy (CISP) and autoimmune nodopathies. It is debatable whether CIDP occurring in the course of other conditions, i.e., monoclonal IgG or IgA gammopathy, should be treated as a separate disease entity from idiopathic CIDP. This study aims to evaluate the molecular differences of the nodes of Ranvier and the initial axon segment (AIS) and juxtaparanode region (JXP) as the potential cause of phenotypic variation of CIDP while also seeking new pathomechanisms since JXP is sequestered behind the paranode and autoantibodies may not access the site easily. The authors initially present the structure of the different parts of the neuron and its functional significance, then discuss the problem of whether damage to the juxtaparanodal region, Schwann cells and axons could cause CIDP or if these damages should be separated as separate disease entities. In particular, AIS's importance for modulating neural excitability and carrying out transport along the axon is highlighted. The disclosure of specific pathomechanisms, including novel target antigens, in the heterogeneous CIDP syndrome is important for diagnosing and treating these patients.

Normal Cortical Myelination in Galectin-4-Deficient Mice.

Myelin, critical for the correct function of the nervous system, is organized in different patterns that can include long non-myelinated axonal segments. How myelin patterning is regulated remains unexplained. The carbohydrate-binding protein galectin-4 (Gal-4) influences oligodendrocyte differentiation in vitro and is associated with non-myelinable axon segments (NMS) in cultured neurons. In consequence, Gal-4 has been proposed as a myelin patterning regulator, although no in vivo studies have corroborated this hypothesis. We used Gal-4-deficient mice (Lgals4-KO) to study the role of Gal-4 in cortical myelination in vivo. We show that cultured neurons of Lgals4-KO mice form NMS that are regulated as in control neurons. In addition, oligodendrocyte/myelin markers expression measured by biochemical and immunochemical means, and cortical myelin microstructure studied by in-depth image analysis appear unaltered in these animals. Consistently, myelin displays an essentially normal function assessed by in vivo electrophysiology and locomotion analyses. In conclusion, cortical myelin of Lgals4-KO mice does not show any significant defect in composition, organization or function, pointing to a negligible role of Gal-4 in myelination in vivo or, as discussed, to unknown mechanisms that compensate its absence.

Automatic jitter measurement in needle-detected motor unit potential trains

In an active motor unit (MU), the time intervals between the firings of its muscle fibers vary across successive MU activations. This variability is called jitter and is increased in pathological processes that affect the neuromuscular junctions or terminal axonal segments of MUs. Traditionally, jitter has been measured using single fiber electrodes (SFEs) and a difficult and subjective manual technique. SFEs are expensive and reused, implying a potential risk of patient infection; so, they are being gradually substituted by safer, disposable, concentric needle electrodes (CNEs). As CNEs are larger, voltage contributions from individual fibers of a MU are more difficult to detect, making jitter measurement more difficult. This paper presents an automatic method to estimate jitter from trains of motor unit potentials (MUPs), for both SFE and CNE records. For a MUP train, segments of MUPs generated by single muscle fibers (SF MUP segments) are found and jitter is measured between pairs of these segments. Segments whose estimated jitter values are not reliable, according to several SF MUP segment characteristics, are excluded. The method has been tested in several simulation studies that use mathematical models of muscle fiber potentials. The results are very satisfactory in terms of jitter estimation error (less than 10% in most of the cases studied) and mean number of valid jitter estimates obtained per simulated train (greater than 1.0 in many of the cases and less than 0.5 only in the most complicated). A preliminary study with real signals was also performed, using 19 MUP trains from 3 neuropathic patients. Jitter measurements obtained by the automatic method were compared with those extracted from a commercial system (Keypoint) and the edition and supervision of an expert electromyographer. From these measurements 63% were taken from equivalent interval pair sites within the time span of the MUP trains and, as such, were considered as compatible measurements. Differences in jitter of these compatible measurements were very low (mean value of 1.3 μs, mean of absolute differences of 2.97 μs, 25% and 75% percentile intervals of −0.85 and 3.82 μs, respectively). Although new tests with larger number of real recordings are still required, the method seems promising for clinical practice.

Neurofilament Transport Is Bidirectional In Vivo.

Neurofilaments are abundant space-filling cytoskeletal polymers that are transported into and along axons. During postnatal development, these polymers accumulate in myelinated axons causing an expansion of axon caliber, which is necessary for rapid electrical transmission. Studies on cultured nerve cells have shown that axonal neurofilaments move rapidly and intermittently along microtubule tracks in both anterograde and retrograde directions. However, it is unclear whether neurofilament transport is also bidirectional in vivo. Here, we describe a pulse-spread fluorescence photoactivation method to address this in peripheral nerves dissected from hThy1-paGFP-NFM transgenic mice, which express a photoactivatable fluorescent neurofilament protein. Neurofilaments were photoactivated in short segments of myelinated axons in tibial nerves at 2, 4, 8, and 16 weeks of age. The proximal and distal spread of the fluorescence due to the movement of the fluorescent neurofilaments was measured over time. We show that the directional bias and velocity of neurofilament transport can be calculated from these measurements. The directional bias was ∼60% anterograde and 40% retrograde and did not change significantly with age or distance along the nerve. The net velocity decreased with age and distance along the nerve, which is consistent with previous studies using radioisotopic pulse labeling. This decrease in velocity was caused by a decrease in both anterograde and retrograde movement. Thus, neurofilament transport is bidirectional in vivo, with a significant fraction of the filaments moving retrogradely in both juvenile and adult mice.

80-OR: Proinflammatory Macrophage Impairs Retrograde Axonal Transport via Inflammation and Insulin Resistance in Sensory Neurons

Inflammation, insulin resistance (IR) , and deficit of retrograde axonal transport (RAT) are implicated in the pathogenesis of diabetic polyneuropathy (DPN) . Pro-inflammatory macrophage (M1) skewed by diabetic insults causes IR in adipose tissue. The activation of Advanced Glycation End products (AGEs) - Receptor for AGEs (RAGE) signaling is involved in the macrophage polarization into M1, while its contribution to the deficit of RAT in DPN is still unclear. In this study, we investigated the implication of M1-related inflammation to RAT deficit in sensory neurons. We evaluated the activation of macrophage cell line RAW 264.7 (RAW) and neurons isolated from dorsal root ganglia (DRG) of C57BL/6 mice stimulated by 200 μg/ml of AGE. A DRG neurons-RAW co-culture system was established by seeding RAW onto DRG neurons. After 12h co-culturing, we added AGE to the co-culture and stimulated RAW. A DRG neuronal mono-culture was treated with 500 nM of insulin receptor antagonist (BMS-754807) , and 20 ng/ml of TNF-α with/without 50 nM JNK inhibitor (SP600125) . Axonal transport was visualized by Lysotracker and captured by time-lapse microscopy. The movements were quantified on kymographs generated from 100 μm axonal segments. AGE induced the M1 phenotype in RAW with elevated expressions of iNOS and TNF-α mRNA, while no effects on DRG neuron were identified. In the co-culture, the frequency of RAT (RAT%) was significantly reduced when RAW was stimulated by AGE compared to naive RAW (p &amp;lt; 0.001) . In the mono-culture, RAT% was significantly reduced when neurons were treated with BMS-7548 and TNFα (vs. control, both p &amp;lt; 0.001) . TNFα stimulation also increased the phosphorylation of JNK in the axons, a major target of TNFα, which was confirmed by immunofluorescence. The reduction of RAT% by TNFα was rescued by SP600125. In summary, RAT could be impaired by TNF-α and IR, which suggests inflammation induced by M1 in SN can attenuate RAT, which contributes to the DPN pathology. Disclosure S.Osonoi: None. H.Mizukami: None. S.Ogasawara: None. K.Kudoh: None. M.Daimon: None. S.Yagihashi: None. Funding Japan Society for the Promotion of Science (18K16220)

Serotonin transporter binding in major depressive disorder: impact of serotonin system anatomy.

Serotonin transporter (5-HTT) binding deficits are reported in major depressive disorder (MDD). However, most studies have not considered serotonin system anatomy when parcellating brain regions of interest (ROIs). We now investigate 5-HTT binding in MDD in two novel ways: (1) use of a 5-HTT tract-based analysis examining binding along serotonergic axons; and (2) using the Copenhagen University Hospital Neurobiology Research Unit (NRU) 5-HT Atlas, based on brain-wide binding patterns of multiple serotonin receptor types. [11C]DASB 5-HTT PET scans were obtained in 60 unmedicated participants with MDD in a current depressive episode and 31 healthy volunteers (HVs). Binding potential (BPP) was quantified with empirical Bayesian estimation in graphical analysis (EBEGA). Within the [11C]DASB tract, the MDD group showed significantly lower BPP compared with HVs (p = 0.02). This BPP diagnosis difference also significantly varied by tract location (p = 0.02), with the strongest MDD binding deficit most proximal to brainstem raphe nuclei. NRU 5-HT Atlas ROIs showed a BPP diagnosis difference that varied by region (p < 0.001). BPP was lower in MDD in 3/10 regions (p-values < 0.05). Neither [11C]DASB tract or NRU 5-HT Atlas BPP correlated with depression severity, suicidal ideation, suicide attempt history, or antidepressant medication exposure. Future studies are needed to determine the causes of this deficit in 5-HTT binding being more pronounced in proximal axon segments and in only a subset of ROIs for the pathogenesis of MDD. Such regional specificity may have implications for targeting antidepressant treatment, and may extend to other serotonin-related disorders.