Afferent Axons Research Articles

Peripheral cranio-spinal nerve communication for trapezius muscle control using axonal profiling through immunostaining

Accessory nerve (CNXI) has been known to be the primary conduit for motor control of the trapezius, while the supplementary cervical nerves (C3 and C4) are responsible for processing sensory information from muscle. However, the lack of substantial direct evidence has led to these conclusions being regarded as mere speculation. This study used immunostaining (using antibodies against neurofilament 200 for all axons, choline acetyltransferase for cholinergic axons, tyrosine hydroxylase for sympathetic axons, and alpha 3 sodium potassium ATPase for proprioceptive afferent axons) of human samples to verify the functional contributions of nerves. Study highlights the pivotal role of C3 and C4 in regulating precise movements of trapezius, contributing to motor control, proprioceptive feedback, and sympathetic modulation. CNXI is composed primarily of somatic efferent fibers, with significant numbers of sympathetic or sensory fibers. Furthermore, C3-4 have both cholinergic and non-cholinergic axons, suggesting their involvement in proprioceptive feedback and somatic efferent functions. Although less common, mechanosensors such as nociceptive sensor and sympathetic fibers are also supplied by these cervical nerves. The study demonstrated that these nerves contain motor fibers and significant proprioceptive and sympathetic axons, challenging the long-held notion that CNXI are motor and upper spinal nerves are sensory.

Current perspectives and concerns in corneal neurotization.

This study aimed to comprehensively explore the intricacies of corneal neurotization (CN) and the nuanced factors that set it apart from routine clinical practice, exerting a substantial influence on its success. A symbiotic relationship is evident between corneal innervation and ocular surface health. The loss of corneal innervation results in a potentially challenging corneal condition known as neurotrophic keratopathy (NK). The majority of treatments are primarily focused on preventing epithelial breakdown rather than addressing the underlying pathogenesis. Consequently, to address the impaired corneal sensation (underlying etiology), a novel surgical approach has emerged, namely CN, which involves transferring healthy sensory nerve axons to the affected cornea. This review offers valuable insights into the existing body of supporting evidence for CN, meticulously examining clinical studies, case reports, and experimental findings. The aim is to enhance our understanding of the effectiveness and potential outcomes associated with this innovative surgical technique. The exploration of innovative therapeutic avenues holds promise for revolutionizing the management of NK, offering a potentially permanent solution to a condition once deemed incurable and severely debilitating.

Emergent epileptiform activity in spinal sensory circuits drives ectopic bursting in afferent axons and sensory dysfunction after cord injury.

Spinal cord injury leads to hyperexcitability and dysfunction in spinal sensory processing. As hyperexcitable circuits can become epileptiform, we explored whether such activity emerges in a thoracic spinal cord injury (SCI) contusion model of neuropathic pain. Recordings from spinal sensory axons in multiple below-lesion segmental dorsal roots demonstrated that SCI facilitated the emergence of spontaneous ectopic burst spiking in afferent axons, which were correlated across multiple adjacent dorsal roots. Burst frequency correlated with behavioral mechanosensitivity. The same bursting events were recruited by afferent stimulation, and timing interactions with ongoing spontaneous bursts revealed that recruitment was limited by a prolonged post-burst refractory period. Ectopic bursting in afferent axons was driven by GABAA receptor activation, presumably by conversion of subthreshold GABAergic interneuronal presynaptic axoaxonic inhibitory actions to suprathreshold spiking. Collectively, the emergence of stereotyped bursting circuitry with hypersynchrony, sensory input activation, post-burst refractory period, and reorganization of connectivity represent defining features of an epileptiform network. Indeed, these same features were reproduced in naive animals with the convulsant 4-aminopyridine (fampridine). We conclude that spinal cord injury promotes the emergence of epileptiform activity in spinal sensory networks that promote profound corruption of sensory signaling. This includes hyperexcitability and bursting by ectopic spiking in afferent axons that propagate bidirectionally by reentrant central and peripheral projections as well as sensory circuit hypoexcitability during the burst refractory period. More broadly, the work links circuit hyperexcitability to epileptiform circuit emergence, further strengthening it as a conceptual basis to understand features of sensory dysfunction and neuropathic pain.

The distribution and evolutionary dynamics of dopaminergic neurons in molluscs.

Dopamine is one of the most versatile neurotransmitters in invertebrates. It's distribution and plethora of functions is likely coupled to feeding ecology, especially in Euthyneura (the largest clade of molluscs), which presents the broadest spectrum of environmental adaptations. Still, the analyses of dopamine-mediated signaling were dominated by studies of grazers. Here, we characterize the distribution of dopaminergic neurons in representatives of two distinct ecological groups: the sea angel - obligate predatory pelagic mollusc Clione limacina (Pteropoda, Gymnosomata) and its prey - the sea devil Limacina helicina (Pteropoda, Thecosomata) as well as the plankton eater Melibe leonina (Nudipleura, Nudibranchia). By using tyrosine hydroxylase-immunoreactivity (TH-ir) as a reporter, we showed that the dopaminergic system is moderately conservative among euthyneurans. Across all studied species, small numbers of dopaminergic neurons in the central ganglia contrast to significant diversification of TH-ir neurons in the peripheral nervous system, primarily representing sensory-like cells, which predominantly concentrated in the chemotactic areas and projecting afferent axons to the central nervous system. Combined with α-tubulin immunoreactivity, this study illuminates the unprecedented complexity of peripheral neural systems in gastropod molluscs, with lineage-specific diversification of sensory and modulatory functions.

Quantitative Analysis of CGRP-IR Afferent Axons in the Mouse Stomach Using Zeiss Arivis Vision4D for Automated Tracing

Gastric nociceptive afferents detect and respond to noxious stimuli by transmitting signals from the stomach to the brain. Calcitonin gene-related peptide (CGRP) is a marker for nociceptive afferents and is crucial to gastric functions, including gastric acid secretion, gut motility, and inflammation. However, the topographical quantitative analysis of CGRP-immunoreactive (CGRP-IR) axons in the whole stomach is challenging due to the large amount of axon innervation in various targets within the tissue. Manual tracing of axons is time-consuming, labor-intensive, and extremely tedious, while currently existing automatic neuron tracing programs often need help with large file sizes or tracing inaccuracies. In this study, we used Zeiss Arivis Vision4D, a novel and sophisticated software with a user-guided machine learning capability, for automated tracing of CGRP-IR axons in flat-mounts of the whole mouse stomach muscular layers and thick serial sections of the antrum-pylorus region (C57BL/6J, male, 2-3 months old, n=7). We utilized a combination of techniques, including the Zeiss M2 Imager for automated scanning, Zen 3.3 (blue edition) for image processing, and Vision4D for stitching the entire stomach flat-mounts and sections, 3D fiber tracing, and density analysis. Our results demonstrated that: 1) Vision4D detected and traced almost all CGRP-IR axons of different gastric targets (blood vessels, muscles, myenteric neurons) in different regions (fundus, corpus, antrum) of the stomach. 2) The x-y-z tracing allowed the visualization of axons in space, portraying its continuous trajectory across layers in the muscular flat mounts (longitudinal muscle, myenteric plexus, circular muscle) and cross sections (muscles, submucosa, mucosa). 3) Vision4D also enabled quantitative morphological and laminar analysis based on target and layer separations. The axon density heatmap generated from the tracing data showed high CGRP-IR axon innervation in the blood vessels. After the removal of blood vessels, dense innervation was seen in the upper fundus and antrum-pylorus followed by corpus, indicating the abundance of CGRP in these regions. 4) Vision4D is a powerful tool in handling large data sizes (hundreds of gigabytes of optical sections in more than 300 image frames per stomach) and outstanding in its capability to track nerve fibers in a highly accurate and time effcient manner. This has paved the way for data batch processing to be accessible, as well as shown that multi-marker tracing of the same tissue sample is attainable. 5) The tracing data will be integrated into a stomach scaffold for a 3D map of CGRP-IR afferent axons. This study represents, for the first time, the automatically traced CGRP-IR axons using whole stomach preparations and highlights the potential of Vision4D in creating axon tracing models in other organs and species. This study is supported by NIH 1 U01 NS113867-01, NIH R15 1R15HL137143-01A1 and Carl ZEISS Company. This is the full abstract presented at the American Physiology Summit 2024 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.

Identification of Spinal Afferent Innervation in the Rat Heart: Atria and Ventricles: Anterograde Tracing

The spinal afferent innervation of the heart regulates cardiac functions by sending sensory information from the dorsal root ganglia (DRG) to the brain. However, the distribution and morphology of spinal afferents in the whole heart are not well characterized. The challenges here are: 1) diffculty in surgically accessing the upper thoracic DRG to label only cardiac spinal afferents. 2) handling flat-mounts of whole atria and ventricles (thickness ~850 μm) without using sections or partial tissues that could disrupt the continuity of axonal structures. To overcome these challenges, we injected anterograde tracer dextran biotin (DB) into the left DRG (C8-T3) of male SD rats (3-5 months) and allowed them to recover for 14 days for tracer transportation. Flat-mounts of the left and right atria and ventricles were prepared, and DAB (3,3′-Diaminobenzidine) stained, followed by imaging, tracing, and digitization using the Neurolucida system. Our findings are: In the atria, the DB-labeled axons entered the atria potentially through the left precaval vein (LPCV), with predominant innervation of the left side of the atrial wall and extended their projection towards the auricles, middle region of the atrium, and pulmonary veins. Most of the spinal afferent axons were present in the myocardial layer. Varicose DB-labeled axons were also observed on cardiac targets at different regions, and distinct morphological structures were found. In the cardiac muscle, DB-labeled axons ran mainly in the direction of muscle fibers. Also, varicose axons ran along the small vasculature on the atria. Spinal afferents formed varicose contacts with individual principal neurons within some intrinsic cardiac ganglia (ICG). In the ventricles, DB-labeled axons were present in the left and right ventricles, which ramified and expressed spherical-like varicosities on the cardiac muscle. In both the atria and ventricles, spinal afferent axons formed simple, branching, and complex terminal structures. Thus, our work, for the first time, successfully labeled, traced, and digitized the spinal cardiac afferents in the flat-mounts of whole atria and ventricles, which will provide a foundation for specific labeling of cardiac spinal afferent fibers. In addition, it will lead to the future study of topographical cardiac spinal afferent innervation, functional studies, and their remodeling in pathological conditions. This study was supported by NIH HEAL/SPARC U01 NS113867-01, NIH R15 R15HL137143-01A1, and UCF dissertation completion Fellowships. This is the full abstract presented at the American Physiology Summit 2024 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.

Identification of vagal afferent nerve endings in the mouse colon and their spatial relationship with enterochromaffin cells

Understanding how the gut communicates with the brain, via sensory nerves, is of significant interest to medical science. Enteroendocrine cells (EEC) that line the mucosa of the gastrointestinal tract release neurochemicals, including the largest quantity of 5-hydroxytryptamine (5-HT). How the release of substances, like 5-HT, from enterochromaffin (EC) cells activates vagal afferent nerve endings is unresolved. We performed anterograde labelling from nodose ganglia in vivo and identified vagal afferent axons and nerve endings in the mucosa of whole-mount full-length preparations of mouse colon. We then determined the spatial relationship between mucosal-projecting vagal afferent nerve endings and EC cells in situ using 3D imaging. The mean distances between vagal afferent nerve endings in the mucosa, or nearest varicosities along vagal afferent axon branches, and the nearest EC cell were 29.6 ± 19.2 μm (n = 107, N = 6) and 25.7 ± 15.2 μm (n = 119, N = 6), respectively. No vagal afferent endings made close contacts with EC cells. The distances between EC cells and vagal afferent endings are many hundreds of times greater than known distances between pre- and post-synaptic membranes (typically 10–20 nm) that underlie synaptic transmission in vertebrates. The absence of any close physical contacts between 5-HT-containing EC cells and vagal afferent nerve endings in the mucosa leads to the inescapable conclusion that the mechanism by which 5-HT release from ECs in the colonic mucosa occurs in a paracrine fashion, to activate vagal afferents.

A7 CANNABINOID 1 AND 2 RECEPTOR AGONISTS AND MU-OPIOID RECEPTOR AGONISTS SYNERGISTICALLY INHIBIT COLONIC NOCICEPTION DURING ACUTE COLITIS

Abstract Background Abdominal pain is a debilitating symptom in patients with inflammatory bowel disease. Previously we have shown that combining sub-analgesic doses of cannabinoid 1 receptor (CB1R), but not cannabinoid 2 receptor (CB2R), and mu-opioid receptor (MOR) agonists synergistically inhibits colonic nociception in healthy mice. However, it is unknown whether this combination has analgesic efficacy in a pre-clinical model of colitis. Aims To determine the effects of combining sub-analgesic doses of CBR and MOR agonists on colonic nociception during acute colitis. Methods Colitis was induced in male and female C57BL/6 mice with 2.5% dextran sulfate sodium in drinking water. Extracellular afferent nerve recordings were obtained from ex vivo flat sheet preparations of mouse distal colon. Mechanosensitivity of single afferent axons was assessed via probing of the colon with a 1g von Frey hair before and after superfusion of agonists of CB1R or CB2R plus MOR. To examine effects in vivo, visceromotor response (VMR) to colorectal distention (volume range 20-80 µL) was measured via electromyography. Mice were injected intraperitoneally with vehicle, or a combination of CB1R or CB2R agonist plus morphine 30-minutes prior to VMR experiment. Data were analyzed using a one or two-way ANOVA with Bonferroni test. N denotes number of mice; n denotes number of single afferent axons. Results In afferent nerve recordings, in contrast to healthy mice, the CB2R agonist HU-308 (1 µM and 3 µM) inhibited colonic mechanosensitivity in mice with colitis (1 µM: p<0.01, N=5, n=12; 3 µM: p<0.01, N=7, n=10); a lower concentration (300 nM) had no effect (p=0.52, N=6, n=10). The CB1R agonist ACEA (10 µM) reduced mechanosensitivity during acute colitis (p<0.05, n=11, N=6), whereas 100 nM (p>0.99, n=7, N=5) and 1 µM (p=0.25, n=10, N=6) had no effect. A combination of sub-analgesic concentrations of ACEA (100 nM) and DAMGO (MOR agonist, 1 nM) inhibited colonic mechanosensitivity in healthy mice (p<0.01, N=4, n=8) and during acute colitis (p<0.05, N=6, n=8). While a combination of sub-analgesic concentrations of HU-308 (300 nM) and DAMGO (1 nM) had no effect in healthy mice (p=0.70, N=4, n=8), it inhibited colonic mechanosensitivity during acute colitis (p<0.01, N=8, n=15). In VMR experiments, a combination of a sub-analgesic dose of ACEA (0.3 mg/kg) with morphine (0.3 mg/kg) reduced VMR (p<0.01, N=7) during acute colitis. Similarly, a combination of a sub-analgesic dose of HU-308 (1 mg/kg) and morphine (0.3 mg/kg) reduced VMR (p<0.01, N=6) during acute colitis. Conclusions A CB2R agonist inhibits colonic nociception during acute colitis, but not in healthy mice. A sub-analgesic combination of CB1R and MOR agonists can inhibit pain in healthy and inflamed mice, while combining sub-analgesic CB2R and MOR agonists is only inhibitory during acute colitis. Funding Agencies NRC

Mapping of individual sensory nerve axons from digits to spinal cord with the transparent embedding solvent system.

Achieving uniform optical resolution for a large tissue sample is a major challenge for deep imaging. For conventional tissueclearing methods, loss of resolution and quality in deep regions is inevitable due to limited transparency. Here we describe the Transparent Embedding Solvent System (TESOS) method, which combines tissue clearing, transparent embedding, sectioning and block-face imaging. We used TESOS to acquire volumetric images of uniform resolution for an adult mouse whole-body sample. The TESOS method is highly versatile and can be combined with different microscopy systems to achieve uniformly high resolution. With a light sheet microscope, we imaged the whole body of an adult mouse, including skin, at a uniform 0.8 × 0.8 × 3.5 μm3 voxel resolution within 120 h. With a confocal microscope and a 40×/1.3 numerical aperture objective, we achieved a uniform sub-micron resolution in the whole sample to reveal a complete projection of individual nerve axons within the central or peripheral nervous system. Furthermore, TESOS allowed the first mesoscale connectome mapping of individual sensory neuron axons spanning 5 cm from adult mouse digits to the spinal cord at a uniform sub-micron resolution.

On the Blueprint of the Long Primary Afferent Axons and the Dichotomous Axon Trajectory of Clarke’s Nucleus. A Morphological Tracing Study on the Effect of Hypoxia during Development

The primary afferent system in the rat’s spinal cord starts to develop in the third last week of gestation. First, the pseudounipolar DRG neurons extend their centripetal long primary axons, targeting rostral supra-segmental nuclei in the spinal cord. Meanwhile, the subsequent innervation of the juxta- and intra-segmental spinal levels enables the three subdivisions to commence integrating a complex network with the body periphery. This process may continue to refine and adapt the system life-long. The experimental data elucidated the steps involved in developing the cytoarchitecture by separating the axons of the long and intermediate subdivisions from the short subdivision. Here, we present a blueprint of the features of the long primary afferent axons developing in sequential waves. The pioneering long afferent axons targeted the dorsal gracile nuclei at spring tide and Clarke’s nuclei at neap tide in ventrally bent trajectories. The paradigm’s myelotomy blocked these pioneering fibers from stepping down the developmental cascade, rendering an unknown phenotype. This reflected a hypothetical transition hub stationed on the assembly line, delineating a critical period. The paradigm also affected the neuropil’s ripening independently from the long primary afferent system. The data disclosed that fetal hyposaturation yielded an in vivo genomic engineering capability. Fetal tissue was susceptible to hyposaturation, showing remarkable versatility early in fetal life. The translational impact may favor research into the elusive etiology of clinical syndromes concerning the afferent system relating to fetal hyposaturation.

The Positive Effect of Renal Denervation Persists even Through Regrowth of Afferent Axons Improving Renal Afferent Nerve Activity During High Sodium Intake in Rats

The Positive Effect of Renal Denervation Persists even Through Regrowth of Afferent Axons Improving Renal Afferent Nerve Activity During High Sodium Intake in Rats

Alterations of the nervous system in patients with vibration disease

Introduction. An important aspect of prevention is the dynamic monitoring of workers exposed to the vibration in the workplace, which not only forms the basis for the diagnosis of occupational intoxication, but also ensures timely resolution of issues of examination of working capacity.
 Materials and methods. Twenty one male patient with an established diagnosis of vibration disease (VD) associated with exposure to local vibration. A dynamic survey was conducted, 3 observation periods were studied.
 Results. Over the 3rd period of the examination, the latency of N10 and N30 increased when compared with the 1st period. In periods 2 and 3, the duration of N13–N18 increased compared to period 1. The rate of pulse conduction (RPC) along the ulnar nerve decreased during period 2 when compared with period 1. Over the period 3, a decrease in RPC along the tibial nerve was revealed when compared with periods 1 and 2. During periods 2 and 3, the amplitude of the ulnar nerve action potential decreased when compared with period 1, along the afferent axons of the ulnar nerve the RPC decreased during period 2 when compared with 1. In period 3, the RPC along the median nerve decreased when compared with period 1.
 Limitations. The limitation of this study is that the dynamics of changes depending on the degree of vibration disease has not been analyzed. This task was not implemented due to the small number of observations in the sample. 
 Conclusion. The progression of vibration disease syndromes and the degree of severity of vibration disease in dynamics were noted. The time of activation of neurons of the somatosensory zone of the cerebral cortex and the time of passage of the pulse from neurons of the cervical thickening to the thalamic structures in dynamics was found to slow down. There were increased disturbances in the conduction of the pulse along the axons at the level of the brachial plexus and from the brachial plexus to the lower parts of the brain stem. Demyelinating changes in the motor component of the peripheral nerves of the upper and lower extremities were aggravated in patients.

Functional knockout of the TRPV1 channel has no effect on the exercise pressor reflex in rats.

The role played by the transient receptor potential vanilloid 1 (TRPV1) channel on the thin fibre afferents evoking the exercise pressor reflex is controversial. To shed light on this controversy, we compared the exercise pressor reflex between newly developed TRPV1+/+ , TRPV1+/- and TRPV1-/- rats. Carotid arterial injection of capsaicin (0.5μg), evoked significant pressor responses in TRPV1+/+ and TRPV1+/- rats, but not in TRPV1-/- rats. In acutely isolated dorsal root ganglion neurons innervating the gastrocnemius muscles, capsaicin evoked inward currents in neurons isolated from TRPV1+/+ and TRPV1+/- rats but not in neurons isolated from TRPV1-/- rats. The reflex was evoked by stimulating the tibial nerve in decerebrated rats whose femoral artery was either freely perfused or occluded. We found no difference between the reflex in the three groups of rats regardless of the patency of the femoral artery. For example, the peak pressor responses to contraction in TRPV1+/+ , TRPV1+/- and TRPV1-/- rats with patent femoral arteries averaged 17.1 ± 7.2, 18.9 ± 12.4 and 18.4 ± 8.6mmHg, respectively. Stimulation of the tibial nerve after paralysis with pancuronium had no effect on arterial pressure, findings which indicated that the pressor responses to contraction were not caused by electrical stimulation of afferent tibial nerve axons. We also found that expression levels of acid-sensing ion channel 1 and endoperoxide 4 receptor in the L4 and 5 dorsal root ganglia were not upregulated in the TRPV1-/- rats. We conclude that TRPV1 is not needed to evoke the exercise pressor reflex in rats whose contracting muscles have either a patent or an occluded arterial blood supply. KEY POINTS: A reflex arising in contracting skeletal muscle contributes to the increases in arterial blood pressure, cardiac output and breathing evoked by exercise. The sensory arm of the reflex comprises both mechanoreceptors and metaboreceptors, of which the latter signals that blood flow to exercising muscle is not meeting its metabolic demand. The nature of the channel on the metaboreceptor sensing a mismatch between supply and demand is controversial; some believe that it is the transient receptor potential vanilloid 1 (TRPV1) channel. Using genetically engineered rats in which the TRPV1 channel is rendered non-functional, we have shown that it is not needed to evoke the metaboreflex.

Distribution and morphology of calcitonin gene-related peptide (CGRP) innervation in flat mounts of whole rat atria and ventricles

Calcitonin gene-related peptide (CGRP) is widely used as a marker for nociceptive afferent axons. However, the distribution of CGRP-IR axons has not been fully determined in the whole rat heart. Immunohistochemically labeled flat-mounts of the right and left atria and ventricles, and the interventricular septum (IVS) in rats for CGRP were assessed with a Zeiss imager to generate complete montages of the entire atria, ventricles, and septum, and a confocal microscope was used to acquire detailed images of selected regions. We found that 1) CGRP-IR axons extensively innervated all regions of the atrial walls including the sinoatrial node region, auricles, atrioventricular node region, superior/inferior vena cava, left pre-caval vein, and pulmonary veins. 2) CGRP-IR axons formed varicose terminals around individual neurons in some cardiac ganglia but passed through other ganglia without making appositions with cardiac neurons. 3) Varicose CGRP-IR axons innervated the walls of blood vessels. 4) CGRP-IR axons extensively innervated the right/left ventricular walls and IVS. Our data shows the rather ubiquitous distribution of CGRP-IR axons in the whole rat heart at single-cell/axon/varicosity resolution for the first time. This study lays the foundation for future studies to quantify the differences in CGRP-IR axon innervation between sexes, disease models, and species.

Spinal afferent innervation in flat-mounts of the rat stomach: anterograde tracing

The dorsal root ganglia (DRG) project spinal afferent axons to the stomach. However, the distribution and morphology of spinal afferent axons in the stomach have not been well characterized. In this study, we used a combination of state-of-the-art techniques, including anterograde tracer injection into the left DRG T7-T11, avidin–biotin and Cuprolinic Blue labeling, Zeiss M2 Imager, and Neurolucida to characterize spinal afferent axons in flat-mounts of the whole rat stomach muscular wall. We found that spinal afferent axons innervated all regions with a variety of distinct terminal structures innervating different gastric targets: (1) The ganglionic type: some axons formed varicose contacts with individual neurons within myenteric ganglia. (2) The muscle type: most axons ran in parallel with the longitudinal and circular muscles and expressed spherical varicosities. Complex terminal structures were observed within the circular muscle layer. (3) The ganglia-muscle mixed type: some individual varicose axons innervated both myenteric neurons and the circular muscle, exhibiting polymorphic terminal structures. (4) The vascular type: individual varicose axons ran along the blood vessels and occasionally traversed the vessel wall. This work provides a foundation for future topographical anatomical and functional mapping of spinal afferent axon innervation of the stomach under normal and pathophysiological conditions.

Efferent axons in the zebrafish lateral line degenerate following sensory hair cell ablation

The zebrafish lateral line is a frequently used model to study the mechanisms behind peripheral neuronal innervation of sensory organs and the regeneration thereof. The lateral line system consists of neuromasts, a cluster of protruding hair cells, which are innervated by sensory afferent and modulatory efferent neurons. These flow-sensing hair cells are similar to the hair cells in the mammalian ear. Though, while hair cell loss in humans is irreversible, the zebrafish neuromasts are regarded as the fastest regenerating structure in vertebrates, making them an ideal model to study regeneration. However, one component of the lateral line system, the efferent projections, has largely been omitted in regenerative studies. Here, for the first time, we bring insights into the fate of efferent axons during ablation and regeneration of the hair cells in the zebrafish lateral line. Our behavioral analysis showed functional recovery of hair cells and sensory transmission within 48 h and their regeneration were in line with previous studies. Analysis of the inhibitory efferent projections revealed that in approximately half the cases the inhibitory efferent axons degenerated, which was never observed for the sensory afferent axons. Quantification of hair cells following ablation suggests that the presence of mature hair cells in the neuromast may prevent axon degeneration. Within 120 h, degenerated efferent axons regenerated along the axonal tract of the lateral line. Reanalysis of published single cell neuromast data hinted to a role for Bdnf in the survival of efferent axons. However, sequestering Bdnf, blocking the Trk-receptors, and inhibiting the downstream ERK-signaling, did not induce axon degeneration, indicating that efferent survival is not mediated through neurotrophic factors. To further explore the relation between hair cells and efferent projections, we generated atoh1a mutants, where mature hair cells never form. In larvae lacking hair cells, inhibitory efferent projections were still present, following the tract of the sensory afferent without displaying any innervation. Our study reveal the fate of efferent innervation following hair cell ablation and provide insights into the inherent differences in regeneration between neurons in the peripheral and central nervous system.

Organization and morphology of calcitonin gene-related peptide-immunoreactive axons in the whole mouse stomach.

Nociceptive afferent axons innervate the stomach and send signals to the brain and spinal cord. Peripheral nociceptive afferents can be detected with a variety of markers (e.g., substance P [SP] and calcitonin gene-related peptide [CGRP]). We recently examined the topographical organization and morphology of SP-immunoreactive (SP-IR) axons in the whole mouse stomach muscular layer. However, the distribution and morphological structure of CGRP-IR axons remain unclear. We used immunohistochemistry labeling and applied a combination of imaging techniques, including confocal and Zeiss Imager M2 microscopy, Neurolucida 360 tracing, and integration of axon tracing data into a 3D stomach scaffold to characterize CGRP-IR axons and terminals in the whole mouse stomach muscular layers. We found that: (1) CGRP-IR axons formed extensive terminal networks in both ventral and dorsal stomachs. (2) CGRP-IR axons densely innervated the blood vessels. (3) CGRP-IR axons ran in parallel with the longitudinal and circular muscles. Some axons ran at angles through the muscular layers. (4) They also formed varicose terminal contacts with individual myenteric ganglion neurons. (5) CGRP-IR occurred in DiI-labeled gastric-projecting neurons in the dorsal root and vagal nodose ganglia, indicating CGRP-IR axons were visceral afferent axons. (6) CGRP-IR axons did not colocalize with tyrosine hydroxylase or vesicular acetylcholine transporter axons in the stomach, indicating CGRP-IR axons were not visceral efferent axons. (7) CGRP-IR axons were traced and integrated into a 3D stomach scaffold. For the first time, we provided a topographical distribution map of CGRP-IR axon innervation of the whole stomach muscular layers at the cellular/axonal/varicosity scale.

Abstract 057: The Positive Effect Of Renal Denervation Persists Even Through Regrowth Of Afferent Axons Improving Renal Afferent Nerve Activity During High Sodium Intake In Rats

Introduction: Previous work of ours suggests that pathologically decreased sensitivity of renal afferent neurons (reduced number of highly active tonic neurons) due to high salt diet is normalized 1 wk after renal denervation (DNX). Now we tested the hypothesis that normalized sensitivity of renal neurons persists after DNX even through regrowth of afferent axons after 12 wk. This morphological regrowth of afferent nerves 12wk after renal DNX (postDNX) was shown by us previously. Material and methods: 6 male Sprague Dawley (SD) rats were put on high salt diet (HS; 8% NaCl) for 10 days. In another group of 12 rats on high salt diet (HS) left kidneys were denervated (postDNX) 12 weeks prior to examination. Rats on standard diet were used as controls. Harvested dorsal root ganglion neurons (DRG Th11-L2) with renal afferents were investigated in primary neuronal cell culture using current clamp mode to assess action potential generation during current injection and to characterize neurons as tonic highly active and phasic less active neurons. Results: In renal neurons from rats on HS the relation of tonic to phasic neurons shifted towards less active phasic units (62% tonic neurons in controls [n=72 out of 114] vs. 42% [n=37 out of 86] on HS, (p<0.05, z-test)). Denervation of the left kidney in rats on high salt diet (HS-DNX) led to a recovery of afferent renal DRG neurons after 1 wk; (40% tonic neurons [n=32 out of 80] on HS vs. 72% tonic neurons [n=58 out of 81] on HS+DNX)(p<0.05, z-test)). Even 12 weeks after renal denervation this regained electrophysiological property of tonic firing persisted (43% tonic neurons [n=35 out of 82] on HS vs. 69% tonic neurons [n=46 of 67]on HS-postDNX, p<0.05, z-test)). Conclusion: The reduced proportion of highly active tonic neurons increased 1 wk after renal denervation to control levels. Even 12 weeks after renal denervation, this effect of renal denervation persisted, and the sensitivity of renal neurons to electrical stimuli did not change despite high-salt diet. Hence, the positive effects of renal denervation persisted even through the regrowth of renal afferent axons to the kidney.

Peripherin is a biomarker of axonal damage in peripheral nervous system disease.

Valid, responsive blood biomarkers specific to peripheral nerve damage would improve management of peripheral nervous system (PNS) diseases. Neurofilament light chain (NfL) is sensitive for detecting axonal pathology but is not specific to PNS damage, as it is expressed throughout the PNS and CNS. Peripherin, another intermediate filament protein, is almost exclusively expressed in peripheral nerve axons. We postulated that peripherin would be a promising blood biomarker of PNS axonal damage. We demonstrated that peripherin is distributed in sciatic nerve, and to a lesser extent spinal cord tissue lysates, but not in brain or extra-neural tissues. In the spinal cord, anti-peripherin antibody bound only to the primary cells of the periphery (anterior horn cells, motor axons and primary afferent sensory axons). In vitro models of antibody-mediated axonal and demyelinating nerve injury showed marked elevation of peripherin levels only in axonal damage and only a minimal rise in demyelination. We developed an immunoassay using single molecule array technology for the detection of serum peripherin as a biomarker for PNS axonal damage. We examined longitudinal serum peripherin and NfL concentrations in individuals with Guillain-Barré syndrome (GBS, n = 45, 179 time points), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP, n = 35, 70 time points), multiple sclerosis (n = 30), dementia (as non-inflammatory CNS controls, n = 30) and healthy individuals (n = 24). Peak peripherin levels were higher in GBS than all other groups (median 18.75 pg/ml versus < 6.98 pg/ml, P < 0.0001). Peak NfL was highest in GBS (median 220.8 pg/ml) and lowest in healthy controls (median 5.6 pg/ml), but NfL did not distinguish between CIDP (17.3 pg/ml), multiple sclerosis (21.5 pg/ml) and dementia (29.9 pg/ml). While peak NfL levels were higher with older age (rho = +0.39, P < 0.0001), peak peripherin levels did not vary with age. In GBS, local regression analysis of serial peripherin in the majority of individuals with three or more time points of data (16/25) displayed a rise-and-fall pattern with the highest value within the first week of initial assessment. Similar analysis of serial NfL concentrations showed a later peak at 16 days. Group analysis of serum peripherin and NfL levels in GBS and CIDP patients were not significantly associated with clinical data, but in some individuals with GBS, peripherin levels appeared to better reflect clinical outcome measure improvement. Serum peripherin is a promising new, dynamic and specific biomarker of acute PNS axonal damage.

Synapse-specific diversity of distinct postsynaptic GluN2 subtypes defines transmission strength in spinal lamina I.

The unitary postsynaptic response to presynaptic quantal glutamate release is the fundamental basis of excitatory information transfer between neurons. The view, however, of individual glutamatergic synaptic connections in a population as homogenous, fixed-strength units of neural communication is becoming increasingly scrutinized. Here, we used minimal stimulation of individual glutamatergic afferent axons to evoke single synapse resolution postsynaptic responses from central sensory lamina I neurons in an ex vivo adult rat spinal slice preparation. We detected unitary events exhibiting a NMDA receptor component with distinct kinetic properties across synapses conferred by specific GluN2 subunit composition, indicative of GluN2 subtype-based postsynaptic heterogeneity. GluN2A, 2A and 2B, or 2B and 2D synaptic predominance functioned on distinct lamina I neuron types to narrowly, intermediately, or widely tune, respectively, the duration of evoked unitary depolarization events from resting membrane potential, which enabled individual synapses to grade differentially depolarizing steps during temporally patterned afferent input. Our results lead to a model wherein a core locus of proteomic complexity prevails at this central glutamatergic sensory synapse that involves distinct GluN2 subtype configurations. These findings have major implications for subthreshold integrative capacity and transmission strength in spinal lamina I and other CNS regions.