Small-diameter Axons Research Articles

Slow depolarizing electrical stimuli reveal differential time courses of nociceptor recovery after prolonged topical capsaicin in human skin

AbstractBackgroundWe examined de‐functionalization and temporal functional recovery of C‐nociceptor evoked pain after topical 8% capsaicin applied for 4 consecutive days.MethodsCapsaicin and placebo patches were applied to human forearm skin (n = 14). Cold, warmth and heat pain thresholds, pain NRS to electrical and thermal (48°C, 5 s) stimuli and axon reflex flare were recorded weekly for 49 days. Mechanical and heat sensitive (‘polymodal’) nociceptors were activated by single electrical half‐period sinusoidal pulses (0.5 s, 1 Hz). Mechanical and heat insensitive (‘silent’) nociceptors were activated by 4 Hz sinusoidal stimuli.ResultsCapsaicin abolished heat pain. Sensation to electrical sinusoidal stimulation was reduced but never abolished during the treatment. Pain to electrical 1 Hz ‘polymodal’ nociceptor stimulation took longer to recover than pain ratings to 4 Hz 2.5 s sinusoidal stimulation activating ‘polymodal’ and ‘silent’ nociceptors (35 vs. 21 days). Heat pain was indifferent to placebo from day 21–49. Axon reflex flare was abolished during capsaicin and only recovered to ~50% even after 49 days.ConclusionsCapsaicin abolishes heat transduction at terminal nociceptive endings, whereas small‐diameter axons sensitive to sinusoidal electrical stimulation can still be activated. 1 Hz depolarizing stimuli evoke burst discharges, as demonstrated before, and recover slower after capsaicin than single pulses induced by 4 Hz. The difference in recovery suggests differential time course of functional regeneration for C‐nociceptor sub‐types after capsaicin. All sensations recovered completely within 7 weeks in healthy subjects. Our findings contrast analgesia lasting for months in spontaneous neuropathic pain patients treated with 8% capsaicin.SignificanceSinusoidal electrical stimulation can still activate small diameter axons desensitized to heat after 4 consecutive days of topical 8% capsaicin application and reveals differential temporal functional regeneration of C‐nociceptor sub‐types. Electrical sinusoidal stimulation may detect such axons that no longer respond to heat stimuli in neuropathic skin.

Distinct subdivisions of subcortical U-fiber regions in the gyrencephalic ferret brain

The human cerebrum contains a large amount of cortico-cortical association fibers. Among them, U-fibers are short-range association fibers located in white matter immediately deep to gray matter. Although U-fibers are thought to be crucial for higher cognitive functions, the organization within U-fiber regions are still unclear. Here we investigated the properties of U-fiber regions in the ferret cerebrum using neurochemical, neuronal tracing, immunohistochemical and electron microscopic techniques. We found that U-fiber regions can be subdivided into two regions, which we named outer and inner U-fiber regions. We further uncovered that outer U-fiber regions have smaller-diameter axons with thinner myelin compared with inner U-fiber regions. These findings may indicate functional complexity within U-fiber regions in the cerebrum.

Cdon ablation in motor neurons causes age-related motor neuron degeneration and impaired sciatic nerve repair.

The functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging-related muscle wasting. Motor neuron diseases or aging-related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule-downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin-mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown. Hb9-Cre recombinase-driven motor neuron-specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1G93A ) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed. Mice lacking Cdon in motor neurons exhibited middle age onset lethality and aging-related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63±0.35 vs. 3.93±0.22s for f/f, P<0.01) and physical activity. Consistently, neuromuscular junctions of mnKO muscles were incompletely occupied (49.79±5.74 vs. 79.39±3.77% fully occupied neuromuscular junctions for f/f, P<0.0001), suggesting an impaired reinnervation. The transmission electron microscopy analysis revealed that mnKO sciatic nerves had smaller axon diameter (0.88±0.13 vs. 1.43±0.48μm for f/f, P<0.0001) and myelination defects. RNA sequencing of mnKO lumbar spinal cords showed alteration in genes related to neurogenesis, inflammation and cell death. Among the altered genes, ErbB4 and FgfR expressions were significantly altered in mnKO as well as in Cdon-depleted NSC34 motor neuron cells. Consistently, Cdon-depleted NSC34 cells exhibited elevated levels of cleaved Caspase3 and γH2AX proteins, as well as Bax transcription. Cdon-depleted NSC34 cells also exhibited impaired activation of Akt in response to neuregulin-1 (NRG1) treatment. Our current data demonstrate the functional importance of Cdon in motor neuron function and nerve repair. Cdon ablation causes alterations in neurotrophin signalling that leads to motor neuron degeneration.

Long-term tactile hypersensitivity after nerve crush injury in mice is characterized by the persistence of intact sensory axons.

Traumatic peripheral nerve injuries are at high risk of neuropathic pain for which novel effective therapies are urgently needed. Preclinical models of neuropathic pain typically involve irreversible ligation and/or nerve transection (neurotmesis). However, translation of findings to the clinic has so far been unsuccessful, raising questions on injury model validity and clinically relevance. Traumatic nerve injuries seen in the clinic commonly result in axonotmesis (ie, crush), yet the neuropathic phenotype of "painful" nerve crush injuries remains poorly understood. We report the neuropathology and sensory symptoms of a focal nerve crush injury using custom-modified hemostats resulting in either complete ("full") or incomplete ("partial") axonotmesis in adult mice. Assays of thermal and mechanically evoked pain-like behavior were paralleled by transmission electron microscopy, immunohistochemistry, and anatomical tracing of the peripheral nerve. In both crush models, motor function was equally affected early after injury; by contrast, partial crush of the nerve resulted in the early return of pinprick sensitivity, followed by a transient thermal and chronic tactile hypersensitivity of the affected hind paw, which was not observed after a full crush injury. The partially crushed nerve was characterized by the sparing of small-diameter myelinated axons and intraepidermal nerve fibers, fewer dorsal root ganglia expressing the injury marker activating transcription factor 3, and lower serum levels of neurofilament light chain. By day 30, axons showed signs of reduced myelin thickness. In summary, the escape of small-diameter axons from Wallerian degeneration is likely a determinant of chronic pain pathophysiology distinct from the general response to complete nerve injury.

Selective Infrared Neural Inhibition Can Be Reproduced by Resistive Heating

ObjectivesSmall-diameter afferent axons carry various sensory signals that are critical for vital physiological conditions but sometimes contribute to pathologies. Infrared (IR) neural inhibition (INI) can induce selective heat block of small-diameter axons, which holds potential for translational applications such as pain management. Previous research suggested that IR–heating-induced acceleration of voltage-gated potassium channel kinetics is the mechanism for INI. Therefore, we hypothesized that other heating methods, such as resistive heating (RH) in a cuff, could reproduce the selective inhibition observed in INI. Materials and MethodsWe conducted ex vivo nerve-heating experiments on pleural-abdominal connective nerves of Aplysia californica using both IR and RH. We fabricated a transparent silicone nerve cuff for simultaneous IR heating, RH, and temperature measurements. Temperature elevations (ΔT) on the nerve surface were recorded for both heating modalities, which were tested over a range of power levels that cover a similar ΔT range. We recorded electrically evoked compound action potentials (CAPs) and segmented them into fast and slow subcomponents on the basis of conduction velocity differences between the large and small-diameter axonal subpopulations. We calculated the normalized inhibition strength and inhibition selectivity index on the basis of the rectified area under the curve of each subpopulation. ResultsINI and RH showed a similar selective inhibition effect on CAP subcomponents for slow-conducting axons, confirmed by the inhibition probability vs ΔT dose-response curve based on approximately 2000 CAP measurements. The inhibition selectivity indexes of the two heating modalities were similar across six nerves. RH only required half the total electrical power required by INI to achieve a similar ΔT. SignificanceWe show that selective INI can be reproduced by other heating modalities such as RH. RH, because of its high energy efficiency and simple design, can be a good candidate for future implantable neural interface designs.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Use of an invertebrate animal model (Aplysia californica) to develop novel neural interfaces for neuromodulation.

New tools for monitoring and manipulating neural activity have been developed with steadily improving functionality, specificity, and reliability, which are critical both for mapping neural circuits and treating neurological diseases. This review focuses on the use of an invertebrate animal, the marine mollusk Aplysia californica, in the development of novel neurotechniques. We review the basic physiological properties of Aplysia neurons and discuss the specific aspects that make it advantageous for developing novel neural interfaces: First, Aplysia nerves consist only of unmyelinated axons with various diameters, providing a particularly useful model of the unmyelinated C fibers in vertebrates that are known to carry important sensory information, including those that signal pain. Second, Aplysia's neural tissues can last for a long period in an ex vivo experimental setup. This allows comprehensive tests such as the exploration of parameter space on the same nerve to avoid variability between animals and minimize animal use. Third, nerves in large Aplysia can be many centimeters in length, making it possible to easily discriminate axons with different diameters based on their conduction velocities. Aplysia nerves are a particularly good approximation of the unmyelinated C fibers, which are hard to stimulate, record, and differentiate from other nerve fibers in vertebrate animal models using epineural electrodes. Fourth, neurons in Aplysia are large, uniquely identifiable, and electrically compact. For decades, researchers have used Aplysia for the development of many novel neurotechnologies. Examples include high-frequency alternating current (HFAC), focused ultrasound (FUS), optical neural stimulation, recording, and inhibition, microelectrode arrays, diamond electrodes, carbon fiber microelectrodes, microscopic magnetic stimulation and magnetic resonance electrical impedance tomography (MREIT). We also review a specific example that illustrates the power of Aplysia for accelerating technology development: selective infrared neural inhibition of small-diameter unmyelinated axons, which may lead to a translationally useful treatment in the future. Generally, Aplysia is suitable for testing modalities whose mechanism involves basic biophysics that is likely to be similar across species. As a tractable experimental system, Aplysia californica can help the rapid development of novel neuromodulation technologies.

Evidence That ITPR2-Mediated Intracellular Calcium Release in Oligodendrocytes Regulates the Development of Carbonic Anhydrase II + Type I/II Oligodendrocytes and the Sizes of Myelin Fibers.

Myelination of neuronal axons in the central nervous system (CNS) by oligodendrocytes (OLs) enables rapid saltatory conductance and axonal integrity, which are crucial for normal brain functioning. Previous studies suggested that different subtypes of oligodendrocytes in the CNS form different types of myelin determined by the diameter of axons in the unit. However, the molecular mechanisms underlying the developmental association of different types of oligodendrocytes with different fiber sizes remain elusive. In the present study, we present the evidence that the intracellular Ca2+ release channel associated receptor (Itpr2) contributes to this developmental process. During early development, Itpr2 is selectively up-regulated in oligodendrocytes coinciding with the initiation of myelination. Functional analyses in both conventional and conditional Itpr2 mutant mice revealed that Itpr2 deficiency causes a developmental delay of OL differentiation, resulting in an increased percentage of CAII+ type I/II OLs which prefer to myelinate small-diameter axons in the CNS. The increased percentage of small caliber myelinated axons leads to an abnormal compound action potentials (CAP) in the optic nerves. Together, these findings revealed a previously unrecognized role for Itpr2-mediated calcium signaling in regulating the development of different types of oligodendrocytes.

Optical neural stimulation using the thermoplasmonic effect of gold nano-hexagon.

The use of nanoparticle photothermal effect as adjuvants in neuromodulation has recently received much attention, with many open questions about new nanostructures' effect on the action potential. The photothermal properties of hexagonal gold nanoparticles are investigated in this work, including the absorption peak wavelength and light-heat conversion rate, using both experimental and simulation methods. Furthermore, the ability to use these nanostructures in axonal neural stimulation and cardiac stimulation by measuring temperature changes of gold nano-hexagons under 532 nm laser irradiation is studied. In addition, their thermal effect on neural responses is investigated by modeling small-diameter unmyelinated axons and heart pacemaker cells. The results show that the increase in temperature caused by these nano-hexagons can successfully stimulate the small diameter axon and produce an action potential. Experiments have also demonstrated that the heat created by gold nano-hexagons affects toad cardiac rhythm and increases T wave amplitude. An increase in T wave amplitude on toad heart rhythm shows the thermal effect of nano hexagons heat on heart pacemaker cells and intracellular ion flows. This work demonstrates the feasibility of utilizing these nanostructures to create portable and compact medical devices, such as optical pacemakers or cardiac stimulation.

Interactions among diameter, myelination, and the Na/K pump affect axonal resilience to high-frequency spiking

Axons reliably conduct action potentials between neurons and/or other targets. Axons have widely variable diameters and can be myelinated or unmyelinated. Although the effect of these factors on propagation speed is well studied, how they constrain axonal resilience to high-frequency spiking is incompletely understood. Maximal firing frequencies range from ∼1 Hz to >300 Hz across neurons, but the process by which Na/K pumps counteract Na+ influx is slow, and the extent to which slow Na+ removal is compatible with high-frequency spiking is unclear. Modeling the process of Na+ removal shows that large-diameter axons are more resilient to high-frequency spikes than are small-diameter axons, because of their slow Na+ accumulation. In myelinated axons, the myelinated compartments between nodes of Ranvier act as a "reservoir" to slow Na+ accumulation and increase the reliability of axonal propagation. We now find that slowing the activation of K+ current can increase the Na+ influx rate, and the effect of minimizing the overlap between Na+ and K+ currents on spike propagation resilience depends on complex interactions among diameter, myelination, and the Na/K pump density. Our results suggest that, in neurons with different channel gating kinetic parameters, different strategies may be required to improve the reliability of axonal propagation.

Tension Sensor Based on Fluorescence Resonance Energy Transfer Reveals Fiber Diameter-Dependent Mechanical Factors During Myelination.

Oligodendrocytes (OLs) form a myelin sheath around neuronal axons to increase conduction velocity of action potential. Although both large and small diameter axons are intermingled in the central nervous system (CNS), the number of myelin wrapping is related to the axon diameter, such that the ratio of the diameter of the axon to that of the entire myelinated-axon unit is optimal for each axon, which is required for exerting higher brain functions. This indicates there are unknown axon diameter-dependent factors that control myelination. We tried to investigate physical factors to clarify the mechanisms underlying axon diameter-dependent myelination. To visualize OL-generating forces during myelination, a tension sensor based on fluorescence resonance energy transfer (FRET) was used. Polystyrene nanofibers with varying diameters similar to neuronal axons were prepared to investigate biophysical factors regulating the OL-axon interactions. We found that higher tension was generated at OL processes contacting larger diameter fibers compared with smaller diameter fibers. Additionally, OLs formed longer focal adhesions (FAs) on larger diameter axons and shorter FAs on smaller diameter axons. These results suggest that OLs respond to the fiber diameter and activate mechanotransduction initiated at FAs, which controls their cytoskeletal organization and myelin formation. This study leads to the novel and interesting idea that physical factors are involved in myelin formation in response to axon diameter.

The role of aquaporin-4 in optic nerve head astrocytes in experimental glaucoma.

To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null). C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression, and transport block were also analyzed. B6 mice had abundant AQP4 expression in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal AQP4in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p = 0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6). Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

Neuroinflammation in the normal-appearing white matter (NAWM) of the multiple sclerosis brain causes abnormalities at the nodes of Ranvier.

Changes to the structure of nodes of Ranvier in the normal-appearing white matter (NAWM) of multiple sclerosis (MS) brains are associated with chronic inflammation. We show that the paranodal domains in MS NAWM are longer on average than control, with Kv1.2 channels dislocated into the paranode. These pathological features are reproduced in a model of chronic meningeal inflammation generated by the injection of lentiviral vectors for the lymphotoxin-α (LTα) and interferon-γ (IFNγ) genes. We show that tumour necrosis factor (TNF), IFNγ, and glutamate can provoke paranodal elongation in cerebellar slice cultures, which could be reversed by an N-methyl-D-aspartate (NMDA) receptor blocker. When these changes were inserted into a computational model to simulate axonal conduction, a rapid decrease in velocity was observed, reaching conduction failure in small diameter axons. We suggest that glial cells activated by pro-inflammatory cytokines can produce high levels of glutamate, which triggers paranodal pathology, contributing to axonal damage and conduction deficits.

Development of type I/II oligodendrocytes regulated by teneurin-4 in the murine spinal cord

In the spinal cord, the axonal tracts with various caliber sizes are myelinated by oligodendrocytes and function as high-velocity ways for motor and sensory nerve signals. In some neurological disorders, such as multiple sclerosis, demyelination of small caliber axons is observed in the spinal cord. While type I/II oligodendrocytes among the four types are known to myelinate small diameter axons, their characteristics including identification of regulating molecules have not been understood yet. Here, we first found that in the wild-type mouse spinal cord, type I/II oligodendrocytes, positive for carbonic anhydrase II (CAII), were located in the corticospinal tract, fasciculus gracilis, and the inside part of ventral funiculus, in which small diameter axons existed. The type I/II oligodendrocytes started to appear between postnatal day (P) 7 and 11. We further analyzed the type I/II oligodendrocytes in the mutant mice, whose small diameter axons were hypomyelinated due to the deficiency of teneurin-4. In the teneurin-4 deficient mice, type I/II oligodendrocytes were significantly reduced, and the onset of the defect was at P11. Our results suggest that CAII-positive type I/II oligodendrocytes myelinate small caliber axons in the spinal cord and teneurin-4 is the responsible molecule for the generation of type I/II oligodendrocytes.

SPARC: Modelling a Bidirectional Bioelectric Pelvic Nerve Interface

BackgroundMicturition (urination) and continence are controlled by the autonomic nervous system; disruptions to urinary control due to disease or injury significantly impair patients’ quality‐of‐life. The pelvic nerves (in human, the pelvic splanchnic nerves) provide the primary source of autonomic sensory and motor innervation to the bladder and are prime targets for electrical stimulation to treat over‐ or under‐active bladder. The pelvic nerves in both human and rat are fasciculated, but differ from other well‐studied nerves such as the vagus or sciatic nerves in largely lacking a resistive epineurium. The pelvic nerves also comprise a much higher proportion of smaller‐caliber axons than are currently implemented in existing myelinated axon models.ObjectiveTo determine the significance of the macro‐ and micro‐anatomical properties of the pelvic nerve on the stimulation and recording performance of bioelectric neural interfaces.MethodsThe functional composition of the different fascicles of the pelvic nerve were determined using a combination of immunolabeling and tract tracing from the specific regions of the lower urinary tract or large intestine in male rats. Using this data, a model of the pelvic nerves and electrode array was implemented in SIM4LIFE (zmt.swiss) and EIDORS (eidors.org) to simulate electrical stimulation and recording, respectively. Established models for myelinated and unmyelinated axons were adapted to the small‐diameter axons found in the pelvic nerve and implemented in NEURON (neuron.yale.edu), and parameter sensitivity was established using Sobel’ sensitivity analysis for different fascicle arrangements and orientations.ResultsThe pelvic nerve consists of mixed sensory and motor fascicles, with sensory projections to lower urinary or intestinal tracts projecting in distinct fascicles. Sensitivity profiles to extracellular stimulation depend on the size and orientation of the fascicles, as well as their axon type and diameter. Within‐fascicle selectivity is enhanced in the largest fascicles of the pelvic nerve, and overall thresholds are reduced in the smallest fascicles. The stimulus thresholds and conduction velocities of the small myelinated axons depend on the ultrastructural geometry of the nodal and internodal regions, with less dependence on the juxtanode, in an axon‐type‐dependent manner. Extracellular recording sensitivity profiles for clinically realistic electrodes were computed in EIDORS and validated in SIM4LIFE for predicting spontaneous and electrically evoked activity recordings. Extracellular recording sensitivity and stimulus thresholds were both most closely correlated with axon‐electrode distance.ConclusionThe macroscopic and microscopic structure of the pelvic nerve alters its sensitivity to electrical stimulation, as well as the selectivity of extracellular electrical recording. EIDORS can be used to accurately simulate extracellular recording in complex, heterogenous neural geometries.Support or Funding InformationNIH SPARC 3OT2OD023872‐01S4

Conditional depletion of Fus in oligodendrocytes leads to motor hyperactivity and increased myelin deposition associated with Akt and cholesterol activation.

Fused in sarcoma (FUS) is a predominantly nuclear multifunctional RNA/DNA-binding protein that regulates multiple aspects of gene expression. FUS mutations are associated with familial amyotrophic lateral sclerosis (fALS) and frontotemporal lobe degeneration (FTLD) in humans. At the molecular level, the mutated FUS protein is reduced in the nucleus but accumulates in cytoplasmic granules. Oligodendrocytes (OL) carrying clinically relevant FUS mutations contribute to non-cell autonomous motor neuron disease progression, consistent with an extrinsic mechanism of disease mediated by OL. Knocking out FUS globally or in neurons lead to behavioral abnormalities that are similar to those present in FTLD. In this study, we sought to investigate whether an extrinsic mechanism mediated by loss of FUS function in OL contributes to the behavioral phenotype. We have generated a novel conditional knockout (cKO) in which Fus is selectively depleted in OL (FusOL cKO). The FusOL cKO mice show increased novelty-induced motor activity and enhanced exploratory behavior, which are reminiscent of some manifestations of FTLD. The phenotypes are associated with greater myelin thickness, higher number of myelinated small diameter axons without an increase in the number of mature OL. The expression of the rate-limiting enzyme of cholesterol biosynthesis (HMGCR) is increased in white matter tracts of the FusOL cKO and results in higher cholesterol content. In addition, phosphorylation of Akt, an important regulator of myelination is increased in the FusOL cKO. Collectively, this work has uncovered a novel role of oligodendrocytic Fus in regulating myelin deposition through activation of Akt and cholesterol biosynthesis.

In vivo quantification of excitation and kilohertz frequency block of the rat vagus nerve

Objective. There is growing interest in treating diseases by electrical stimulation and block of peripheral autonomic nerves, but a paucity of studies on the excitation and block of small-diameter autonomic axons. We conducted in vivo quantification of the strength-duration properties, activity-dependent slowing (ADS), and responses to kilohertz frequency (KHF) signals for the rat vagus nerve (VN). Approach. We conducted acute in vivo experiments in urethane-anaesthetized rats. We placed two cuff electrodes on the left cervical VN and one cuff electrode on the anterior subdiaphragmatic VN. The rostral cervical cuff was used to deliver pulses to quantify recruitment and ADS. The caudal cervical cuff was used to deliver KHF signals. The subdiaphragmatic cuff was used to record compound action potentials (CAPs). Main results. We quantified the input–output recruitment and strength-duration curves. Fits to the data using standard strength-duration equations were qualitatively similar, but the resulting chronaxie and rheobase estimates varied substantially. We measured larger thresholds for the slowest fibres (0.5–1 m s−1), especially at shorter pulse widths. Using a novel cross-correlation CAP-based analysis, we measured ADS of ~2.3% after 3 min of 2 Hz stimulation, which is comparable to the ADS reported for sympathetic efferents in somatic nerves, but much smaller than the ADS in cutaneous nociceptors. We found greater ADS with higher stimulation frequency and non-monotonic changes in CV in select cases. We found monotonically increasing block thresholds across frequencies from 10 to 80 kHz for both fast and slow fibres. Further, following 25 s of KHF signal, neural conduction could require tens of seconds to recover. Significance. The quantification of mammalian autonomic nerve responses to conventional and KHF signals provides essential information for the development of peripheral nerve stimulation therapies and for understanding their mechanisms of action.

The μDBS: Multiresolution, Directional Deep Brain Stimulation for Improved Targeting of Small Diameter Fibers.

Directional deep brain stimulation (DBS) leads have recently been approved and used in patients, and growing evidence suggests that directional contacts can increase the therapeutic window by redirecting stimulation to the target region while avoiding side-effect-inducing regions. We outline the design, fabrication, and testing of a novel directional DBS lead, the μDBS, which utilizes microscale contacts to increase the spatial resolution of stimulation steering and improve the selectivity in targeting small diameter fibers. We outline the steps of fabrication of the μDBS, from an integrated circuit design to post-processing and validation testing. We tested the onboard digital circuitry for programming fidelity, characterized impedance for a variety of electrode sizes, and demonstrated functionality in a saline bath. In a computational experiment, we determined that reduced electrode sizes focus the stimulation effect on small, nearby fibers. Smaller electrode sizes allow for a relative decrease in small-diameter axon thresholds compared to thresholds of large-diameter fibers, demonstrating a focusing of the stimulation effect within small, and possibly therapeutic, fibers. This principle of selectivity could be useful in further widening the window of therapy. The μDBS offers a unique, multiresolution design in which any combination of microscale contacts can be used together to function as electrodes of various shapes and sizes. Multiscale electrodes could be useful in selective neural targeting for established neurological targets and in exploring novel treatment targets for new neurological indications.

Pathologic remodeling in human neuromas: insights from clinical specimens.

Neuroma pathology is commonly described as lacking a clear internal structure, but we observed evidence that there are consistent architectural elements. Using human neuroma samples, we sought to identify molecular features that characterize neuroma pathophysiology. Thirty specimens-12 neuromas-in-continuity (NICs), 11 stump neuromas, two brachial plexus avulsions, and five controls-were immunohistochemically analyzed with antibodies against various components of normal nerve substructures. There were no substantial histopathologic differences between stump neuromas and NICs, except that NICs had intact fascicle(s) in the specimen. These intact fascicles showed evidence of injury and fibrosis. On immunohistochemical analysis of the neuromas, laminin demonstrated a consistent double-lumen configuration. The outer lumen stained with GLUT1 antibodies, consistent with perineurium and microfascicle formation. Antibodies to NF200 revealed small clusters of small-diameter axons within the inner lumen, and the anti-S100 antibody showed a relatively regular pattern of non-myelinating Schwann cells. CD68+ cells were only seen in a limited temporal window after injury. T-cells were seen in neuroma specimens, with both a temporal evolution as well as persistence long after injury. Avulsion injury specimens had similar architecture to control nerves. Seven pediatric specimens were not qualitatively different from adult specimens. NICs demonstrated intact but abnormal fascicles that may account for the neurologically impoverished outcomes from untreated NICs. We propose that there is consistent pathophysiologic remodeling after fascicle disruption. Particular features, such as predominance of small caliber axons and persistence of numerous T-cells long after injury, suggest a potential role in chronic pain associated with neuromas.

(233) Conditional Knockout of Prdm12 from Sensory Neurons in Mice Recapitulates Aspects of Human Congenital Insensitivity to Pain

Painful conditions are a huge medical burden with an estimated prevalence of 32% of the population, and an economic cost of $635 billion, or 4% of the GDP. Currently, the best available medical analgesics are opioids, but due to their vast side effect profile and risk of overdose, these are far from ideal drugs. Recently, the study of genetic mutations leading to congenital insensitivity to pain (CIP) has resulted in submission of novel drugs to the FDA for clinical trial. Here, we show our work on one such mutation of the gene Prdm12. We first characterized the expression pattern of Prdm12 within different sensory neurons present in the dorsal root ganglia (DRG) by performing in-situ hybridization paired with immunohistochemistry, and found it to be expressed in both myelinated and unmyelinated small-diameter axons of the thoracic and lumbar DRGs. We then generated a new strain of mice in which Prdm12 has been conditionally knocked out of neurons in DRG and cranial ganglia, and subjected these mice to a variety of behavioral assays alongside littermate controls. We have shown that the Prdm12cKO mice have reduced sensitivity to nociceptive thermal and mechanical stimuli, and correlated this with a decrease in the number of pain-sensitive IB4+ and CGRP+ Aδ and C fibers in the DRG. We believe that further study of this mouse model, along with investigation of the downstream effectors and genomic targets of PRDM12, will provide a new avenue for the pursuit of analgesic drug discovery.