Acute effects of hyperglycemia on the peripheral nervous system in zebrafish ( Danio rerio) following nitroreductase-mediated ß-cell ablation.

Diabetic peripheral neuropathy (DPN) is estimated to affect 50% of diabetic patients. Although DPN is highly prevalent, molecular mechanisms remain unknown and treatment is limited to pain relief and glycemic control. We provide a novel model of acute DPN in zebrafish ( Danio rerio) larvae. Beginning 5 days postfertilization (dpf), zebrafish expressing nitroreductase in their pancreatic ß-cells were treated with metronidazole (MTZ) for 48 h and checked for ß-cell ablation 7 dpf. In experimental design, this was meant to serve as proof of concept that ß-cell ablation and hyperglycemia are possible at this time point, but we were surprised to find changes in both sensory and motor nerve components. Compared with controls, neurod+ sensory neurons were often observed outside the dorsal root ganglia in MTZ-treated fish. Fewer motor nerves were properly ensheathed by nkx2.2a+ perineurial cells, and tight junctions were disrupted along the motor nerve in MTZ-treated fish compared with controls. Not surprisingly, the motor axons of the MTZ-treated group were defasciculated compared with the control group, myelination was attenuated, and there was a subtle difference in Schwann cell number between the MTZ-treated and control group. All structural changes occurred in the absence of behavioral changes in the larvae at this time point, suggesting that peripheral nerves are influenced by acute hyperglycemia before becoming symptomatic. Moving forward, this novel animal model of DPN will allow us to access the molecular mechanisms associated with the acute changes in the hyperglycemic peripheral nervous system, which may help direct therapeutic approaches.

A unified model of the excitability of mouse sensory and motor axons.

Non-invasive nerve excitability techniques have provided valuable insight into the understanding of neurological disorders. The widespread use of mice in translational research on peripheral nerve disorders and by pharmaceutical companies during drug development requires valid and reliable models that can be compared to humans. This study established a novel experimental protocol that enables comparative assessment of the excitability properties of motor and sensory axons at the same site in mouse caudal nerve, compared the mouse data to data for motor and sensory axons in human median nerve at the wrist, and constructed a mathematical model of the excitability of mouse axons. In a separate study, ischaemia was employed as an experimental manoeuvre to test the translational utility of this preparation. The patterns of mouse sensory and motor excitability were qualitatively similar to human studies under normal and ischaemic conditions. The most conspicuous differences between mouse and human studies were observed in the recovery cycle and the response to hyperpolarization. Modelling showed that an increase in temperature in mouse axons could account for most of the differences in the recovery cycle. The modelling also suggested a larger hyperpolarization-activated conductance in mouse axons. The kinetics of this conductance appeared to be much slower raising the possibility that an additional or different hyperpolarization-activated cyclic-nucleotide gated (HCN) channel isoform underlies the accommodation to hyperpolarization in mouse axons. Given a possible difference in HCN isoforms, caution should be exercised in extrapolating from studies of mouse motor and sensory axons to human nerve disorders.

Excitability and firing behavior of single slow motor axons transmitting natural repetitive firing of human motoneurons.

Excitability of motor axons is critically important for realizing their main function, i.e., transmitting motoneuron firing to muscle fibers. The present study was designed to explore excitability recovery and firing behavior in single slow axons transmitting human motoneuron firing during voluntary muscle contractions. The abductor digiti minimi, flexor carpi ulnaris, and tibialis anterior were investigated during threshold stimulation of corresponding motor nerves. Motor unit (MU) firing index in response to testing volleys evoking M-responses was used as a physiological measure of axonal excitability and its changes throughout a target interspike interval (ISI) were explored. It was shown that axons displayed an early irresponsive period (within the first ~2-5 ms of a target ISI) that was followed by a responsive period (for the next 5-17 ms of the ISI), in which MUs fired axonal doublets, and a later irresponsive period. At the beginning of the responsive period, M-responses showed small latency delays. However, since at that ISI moment, MUs displayed excitability recovery with high firing index, slight latency changes may be considered as a functionally insignificant phenomenon. The duration of axonal doublet ISIs did not depend on motoneuron firing frequencies (range 4.3-14.6 imp/s). The question of whether or not traditionally described axonal recovery excitability cycle is realistic in natural motor control is discussed. In conclusion, the present approach, exploring, for the first time, excitability recovery in single slow axons during motoneuron natural activation, can provide further insight into axonal firing behavior in normal states and diseases.NEW & NOTEWORTHY Excitability of single slow axons was estimated by motor unit firing index in response to motor nerve stimulation, and its changes throughout a target interspike interval were explored during transmitting human motoneuron natural firing. It was found that axons exhibited early irresponsive, responsive, and later irresponsive periods. Findings question whether the traditionally described axonal excitability recovery cycle is realistic in natural motor control.

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors.

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation.

Effects of temperature on simulated electrotonic potentials and their current kinetics of human motor axons at 20°C-42°C.

To expand our studies on accommodation in human motor nerve axons, the effects of temperature on polarizing nodal and internodal electrotonic potentials and their current kinetics are investigated. The computations use our temperature dependent multi-layered model of the myelinated human motor nerve fiber and the temperature is increased from 20°C to 42°C. The results show that for temperatures from 28°C to 37°C, the polarizing electrotonic potentials almost coincide, as the kinetics of their ionic currents is changed a little. The normal (at 37°C) resting membrane potential is further depolarized or hyperpolarized during hypothermia (≤ 25°C) or hyperthermia (≥ 40°C), respectively and its change is determined by the flow of ionic currents through the internodal axolemma during the polarizing current stimuli. The polarizing electrotonic potentials are more altered during hypothermia and are most altered during hyperthermia. During hyperthermia, the depolarizing nodal and internodal electrotonic potentials are determined by the nodal slow (I Ks ) and internodal fast (I Kf ) and slow (I Ks ) potassium currents. The hyperpolarizing internodal electrotonic potentials are determined by the activation of internodal channels, which are different during hyperthermia at 40°C and 42°C. These potentials are determined by the internodal I Ks current at 40°C and by the internodal inward rectifier (I IR ) and leakage (I Lk ) currents at 42°C. The difference in accommodation to hyperpolarizing currents during focal and uniform hyperthermia at 42°C is discussed. The present results are essential for the interpretation of mechanisms of threshold electrotonus measurements in subjects with symptoms of cooling, warming and fever, which can result from alterations in body temperature.

Theoretical predications of the effects of temperature on simulated adaptive processes in human motor nerve axons at 20°C-42°C.

The effects of temperature on conducting and accommodative processes in the myelinated human motor nerve fiber were previously studied by us in the range of 20°C-42°C. To complete the cycle of our studies on adaptive processes in the fiber, the temperature effects on strength-duration time constant, rheobasic current and recovery cycle are investigated. The computations use our temperature dependent multi-layered model of the fiber and the temperature is increased from 20°C to 42°C. The results show that these excitability parameters are more sensitive to the hypothermia (≤ 25°C) and are most sensitive to the hyperthermia (≥ 40°C), especially at 42°C, than at temperatures in the range of 28°C-37°C. With the increase of temperature from 20°C to 42°C, the strength-duration time constant decreases ~ 8.8 times, while it decreases ~ 2.7% per °C in the range of 28°C-37°C. Conversely, the rheobasic current increases ~ 4.4 times from 20°C to 42°C, while it increases ~ 2.3% per °C in the range of 28°C-37°C. The behavior of relative refractory period and axonal superexcitability in a 100 ms recovery cycle is complex with the increase of temperature. The axonal superexcitability decreases with the increase of temperature during hypothermia. However, it increases rapidly with the increase of temperature during hyperthermia, especially at 42°C and a block of each applied third testing stimulus is obtained. The superexcitability period is followed by a late subexcitability period when the temperatures are in the physiological range of 32°C-37°C. The present results are essential for the interpretation of mechanisms of excitability parameter changes obtained here and measured in healthy subjects with symptoms of cooling, warming and fever, which can result from alterations in body temperature. Our present and previous results confirm that 42°C is the highest critical temperature for healthy subjects.

Decellularized Biohybrid Nerve Promotes Motor Axon Projections.

Developing nerve grafts with intact mesostructures, superior conductivity, minimal immunogenicity, and improved tissue integration is essential for the treatment and restoration of neurological dysfunctions. A key factor is promoting directed axon growth into the grafts. To achieve this, biohybrid nerves are developed using decellularized rat sciatic nerve modified by in situ polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT). Nine biohybrid nerves are compared with varying polymerization conditions and cycles, selecting the best candidate through material characterization. These results show that a 1:1 ratio of FeCl3 oxidant to ethylenedioxythiophene (EDOT) monomer, cycled twice, provides superior conductivity (>0.2 mS cm-1), mechanical alignment, intact mesostructures, and high compatibility with cells and blood. To test the biohybrid nerve's effectiveness in promoting motor axon growth, human Spinal Cord Spheroids (hSCSs) derived from HUES 3 Hb9:GFP cells are used, with motor axons labeled with green fluorescent protein (GFP). Seeding hSCS onto one end of the conduit allows motor axon outgrowth into the biohybrid nerve. The construct effectively promotes directed motor axon growth, which improves significantly after seeding the grafts with Schwann cells. This study presents a promising approach for reconstructing axonal tracts in humans.

Differences in nerve excitability properties across upper limb sensory and motor axons.

OBJECTIVE: The excitability of motor and sensory axons of the main upper limb nerves were compared to characterise the differences between nerves and provide a guide for future studies in human diseases with median neuropathy at the wrist. METHODS: Axonal excitability studies were undertaken on median and ulnar motor (APB and ADM) and sensory axons (D2 and D5) and the superficial radial axons (D1) using a threshold tracking technique. RESULTS: Compared to the median, ulnar motor axons had reduced early depolarising threshold electrotonus (TEd40(10-20 ms) p = 0.02) and superexcitability (p = 0.03). The ulnar sensory axons required a stronger stimulus (p = 0.02) and had a larger rheobase (p = 0.02) than median axons, but were otherwise comparable. The superficial radial axons were "fanned-in" compared to median, and to a lesser degree ulnar axons, with greater resting I/V slope. Mathematical modelling of the radial and median sensory axons suggested that a 15.1% reduction in conductances between nodal and internodal compartments accounted for 82% of this discrepancy. CONCLUSIONS: The excitability parameters of motor and sensory axons are most comparable between median and ulnar nerves. SIGNIFICANCE: The present study demonstrates the feasibility of, and provides normative data for, axonal excitability recordings of the radial and ulnar nerves. We suggest the use of ulnar recordings as an alternative to the median nerve in the setting of compressive neuropathy at the wrist.

Axons in the Chick Embryo Follow Soft Pathways Through Developing Somite Segments.

During patterning of the peripheral nervous system, motor axons grow sequentially out of the neural tube in a segmented fashion to ensure functional integration of the motor roots between the surrounding cartilage and bones of the developing vertebrae. This segmented outgrowth is regulated by the intrinsic properties of each segment (somite) adjacent to the neural tube, and in particular by chemical repulsive guidance cues expressed in the posterior half. Yet, knockout models for such repulsive cues still display initial segmentation of outgrowing motor axons, suggesting the existence of additional, yet unknown regulatory mechanisms of axon growth segmentation. As neuronal growth is not only regulated by chemical but also by mechanical signals, we here characterized the mechanical environment of outgrowing motor axons. Using atomic force microscopy-based indentation measurements on chick embryo somite strips, we identified stiffness gradients in each segment, which precedes motor axon growth. Axon growth was restricted to the anterior, softer tissue, which showed lower cell body densities than the repulsive stiffer posterior parts at later stages. As tissue stiffness is known to regulate axon growth during development, our results suggest that motor axons also respond to periodic stiffness gradients imposed by the intrinsic mechanical properties of somites.

Fluorescence Correlation Spectroscopy Reveals Survival Motor Neuron Oligomerization but No Active Transport in Motor Axons of a Zebrafish Model for Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons that is caused by a deficiency in ubiquitously expressed Survival Motor Neuron (SMN) protein. Two mutually exclusive hypotheses have been discussed to explain increased motor neuron vulnerability in SMA. Reduced SMN levels have been proposed to lead to defective snRNP assembly and aberrant splicing of transcripts that are essential for motor neuron maintenance. An alternative hypothesis proposes a motor neuron-specific function for SMN in axonal transport of mRNAs and/or RNPs. To address these possibilities, we used a novel in vivo approach with fluorescence correlation spectroscopy (FCS) in transgenic zebrafish embryos to assess the subcellular dynamics of Smn in motor neuron cell bodies and axons. Using fluorescently tagged Smn we show that it exists as two freely diffusing components, a monomeric, and a complex-bound, likely oligomeric, component. This oligomer hypothesis was supported by the disappearance of the complex-bound form for a truncated Smn variant that is deficient in oligomerization and a change in its dynamics under endogenous Smn deficient conditions. Surprisingly, our FCS measurements did not provide any evidence for an active transport of Smn in axons. Instead, our in vivo observations are consistent with previous findings that SMN acts as a chaperone for the assembly of snRNP and mRNP complexes.

Excitability of motor and sensory axons in multifocal motor neuropathy.

OBJECTIVE: To assess excitability differences between motor and sensory axons of affected nerves in patients with multifocal motor neuropathy (MMN). METHODS: We performed motor and sensory excitability tests in affected median nerves of 20 MMN patients and in 20 age-matched normal subjects. CMAPs were recorded from the thenar and SNAPs from the 3rd digit. Clinical tests included assessment of muscle strength, two-point discrimination and joint position. RESULTS: All MMN patients had weakness of the thenar muscle and normal sensory tests. Motor excitability testing in MMN showed an increased threshold for a 50% CMAP, increased rheobase, decreased stimulus-response slope, fanning-out of threshold electrotonus, decreased resting I/V slope, shortened refractory period, and more pronounced superexcitability. Sensory excitability testing in MMN revealed decreased accommodation half-time and S2-accommodation and less pronounced subexcitability. Mathematical modeling indicated increased Barrett-Barrett conductance for motor fibers and increase in internodal fast potassium conductance for sensory fibers. CONCLUSIONS: Excitability findings in MMN suggest myelin sheath or paranodal seal involvement in motor fibers and, possibly, paranodal detachment in sensory fibers. SIGNIFICANCE: Excitability properties of affected nerves in MMN differ between motor and sensory nerve fibers.

Interaction of Axonal Chondrolectin with Collagen XIXa1 Is Necessary for Precise Neuromuscular Junction Formation.

Chondrolectin (Chodl) is needed for motor axon extension in zebrafish and is dysregulated in mouse models of spinal muscular atrophy (SMA). However, the mechanistic basis of Chodl function is not known. Here, we use Chodl-deficient zebrafish and mouse mutants to show that the absence of Chodl leads to anatomical and functional defects of the neuromuscular synapse. In zebrafish, the growth of an identified motor axon beyond an "en passant" synapse and later axon branching from synaptic points are impaired, leading to functional deficits. Mechanistically, motor-neuron-autonomous Chodl function depends on its intracellular domain and on binding muscle-derived collagen XIXa1 by its extracellular C-type lectin domain. Our data support evolutionarily conserved roles of Chodl in synaptogenesis and provide evidence for a "synapse-first" scenario of motor axon growth in zebrafish.

Segregation of motor and sensory axons regenerating through bicompartmental tubes by combining extracellular matrix components with neurotrophic factors.

Segregation of regenerating motor and sensory axons may be a good strategy to improve selective functionality of regenerative interfaces to provide closed-loop commands. Provided that extracellular matrix components and neurotrophic factors exert guidance effects on different neuronal populations, we assessed in vivo the potential of separating sensory and motor axons regenerating in a bicompartmental Y-type tube, with each branch prefilled with an adequate combination of extracellular matrix and neurotrophic factors. The severed rat sciatic nerve was repaired using a bicompartmental tube filled with a collagen matrix enriched with fibronectin (FN) and brain-derived neurotrophic factor (BDNF) encapsulated in poly-lactic co-glycolic acid microspheres (FN + MP.BDNF) in one compartment to preferentially attract motor axons and collagen enriched with laminin (LM) and nerve growth factor (NGF) and neurotrophin-3 (NT-3) in microspheres (LM + MP.NGF/NT-3) in the other compartment for promoting sensory axons regeneration. Control animals were implanted with the same Y-tube with a collagen matrix with microspheres (MP) containing PBS (Col + MP.PBS). By using retrotracer labelling, we found that LM + MP.NGF/NT-3 did not attract higher number of regenerated sensory axons compared with controls, and no differences were observed in sensory functional recovery. However, FN + MP.BDNF guided a higher number of regenerating motor axons compared with controls, improving also motor recovery. A small proportion of sensory axons with large soma size, likely proprioceptive neurons, was also attracted to the FN + MP.BDNF compartment. These results demonstrate that muscular axonal guidance can be modulated in vivo by the addition of fibronectin and BDNF.

Excitability tests using high-density surface-EMG: A novel approach to studying single motor units.

OBJECTIVE: To study excitability of single motor units (MUs) using high-density surface-EMG. METHODS: Motor unit action potentials (MUAPs) were evoked by submaximal stimulation of the median nerve at the wrist and recorded with a 9 × 14 electrode grid on the skin overlying the thenar muscles. For excitability tests of single MUs, the most optimal specific single-channel surface-EMG signal was selected based on the spatiotemporal profile of single MUs. RESULTS: Axonal excitability measures were successfully obtained from 14 single MUs derived from ten healthy subjects. Selecting the optimal single-channel surface-EMG signals minimized interference from other single MUs and improved signal-to-noise ratio. The muscle fiber conduction velocity (MFCV) could also be derived from the unique spatiotemporal profile of single MUs. CONCLUSION: High-density surface-EMG helps to isolate single MUAP responses, making it a suitable technique for assessing excitability in multiple single motor axons per nerve. SIGNIFICANCE: Our method enables the reliable study of ion-channel dysfunction in single motor axons of nerves without any requirement for specific conditions, such as prominent MU loss or enlarged MUAPs due to collateral sprouting.

Focal release of neurotrophic factors by biodegradable microspheres enhance motor and sensory axonal regeneration in vitro and in vivo.

Neurotrophic factors (NTFs) promote nerve regeneration and neuronal survival after peripheral nerve injury. However, drawbacks related with administration and bioactivity during long periods limit their therapeutic application. In this study, PLGA microspheres (MPs) were used to locally release different NTFs and evaluate whether they accelerate axonal regeneration in comparison with free NTFs or controls. ELISA, SEM, UV/visible light microscopy, organotypic cultures of DRG explants and spinal cord slices were used to characterize MP properties and the bioactivity of the released NTFs. Results of organotypic cultures showed that encapsulated NTFs maintain longer bioactivity and enhance neurite regeneration of both sensory and motor neurons compared with free NTFs. For in vivo assays, the rat sciatic nerve was transected and repaired with a silicone tube filled with collagen gel or collagen mixed with PBS encapsulated MPs (control groups) and with free or encapsulated NGF, BDNF, GDNF or FGF-2. After 20 days, a retrotracer was applied to the regenerated nerve to quantify motor and sensory axonal regeneration. NTF encapsulation in MPs improved regeneration of both motor and sensory axons, as evidenced by increased numbers of retrolabeled neurons. Hence, our results show that slow release of NTFs with PLGA MP enhance nerve regeneration.