Afferent Axons Research Articles

Resurgent neuropathic discharge: an obstacle to the therapeutic use of neuroma resection?

Ectopic discharge ("ectopia") in damaged afferent axons is a major contributor to chronic neuropathic pain. Clinical opinion discourages surgical resection of nerves proximal to the original injury site for fear of resurgence of ectopia and exacerbated pain. We tested this concept in a well-established animal neuroma model. Teased-fiber recordings were made of ectopic spontaneous discharge originating in the experimental nerve-end neuroma and associated dorsal root ganglia in rats that underwent either a single transection (with ligation) of the sciatic nerve or 2 consecutive transections separated by 7, 14, 21, or 30 days. Ectopia emerged in afferent A and C fibers after a single cut with kinetics anticipated from previous studies. When resection was performed during the early period of intense A-fiber activity, a brief period of resurgence was observed. However, resection of neuromas of more than 14 days was followed by low levels of activity with no indication of resurgence. This remained the case in trials out to 60 days after the first cut. Similarly, we saw no indication of resurgent ectopia originating in axotomized dorsal root ganglion neuronal somata and no behavioral reflection of resurgence. In summary, we failed to validate the concern that proximal resection of a problematic nerve would lead to intense resurgent ectopic discharge and pain. As the well-entrenched concept of resurgence is based more on case reports and anecdotes than on solid evidence, it may be justified to relax the stricture against resecting neuromas as a therapeutic strategy, at least within the framework of controlled clinical trials.

Synaptophysin is a selective marker for axons in human cutaneous end organ complexes

BackgroundSmall clear synaptic-like vesicles fill axon terminals of mechanoreceptors. Their functional significance is controversial and probably includes release of neurotransmitters from afferent axon terminals. Synaptophysin, a major protein of the synaptic vesicle membrane, is present in presynaptic endings of the central and peripheral nervous systems. It is also expressed in mechanosensory neurons which extend into skin forming sensory corpuscles. Nevertheless, synaptophysin occurrence in these structures has never been investigated. MethodsHere we used immunohistochemistry to detect synaptophysin in adult human dorsal root ganglia, cutaneous Meissner and Pacinian corpuscles and Merkel cell-neurite complexes from foetal to elderly period. Moreover, we analyzed whether synaptophysin co-localizes with the mechano-gated protein PIEZO2. ResultsSynaptophysin immunoreactivity was observed in primary sensory neurons (36 ± 6%) covering the entire soma size ranges. Axons of Meissner’s and Pacinian corpuscles were positive for synaptophysin from 36 and 12 weeks of estimated gestational age respectively, to 72 years old. Synaptophysin was also detected in Merkel cells (from 14 weeks of estimated gestational age to old age). Additionally in adult skin, synaptophysin and PIEZO2 co-localized in the axon of Meissner and Pacinian corpuscles, Merkel cells as well as in some axons of Merkel cell-neurite complexes. ConclusionPresent results demonstrate that a subpopulation of primary sensory neurons and their axon terminals forming cutaneous sensory corpuscles contain synaptophysin, a typical presynaptic vesicle protein. Although the functional relevance of these findings is unknown it might be related to neurotransmission mechanisms linked to mechanotransduction.

Topographical Organization and Morphology of Substance‐P Axons in the Flat‐Mounts of the Mouse Whole Stomach

More than 50 million Americans suffer from chronic pain. However, the anatomical and physiological mechanisms of peripheral nociceptive processes have not been well elucidated which has seriously impeded the progress of designing novel bioengineering manipulations/treatments for chronic pain. In this study, we performed a comprehensive topographical mapping of pain‐related neural circuitry in the flat‐mount of whole mouse stomach (male, n=8, 3‐5 months). We used Substance P (SP) as a marker for nociceptive axons and applied a combination of state‐of‐the‐art techniques, including flat‐mount tissue processing and immunohistochemistry of the whole stomach, confocal microscopy, Zeiss Imager microscopy to determine the distribution and morphology of SP‐IR axons and terminals in the whole stomach. We found that 1) SP‐IR axons formed extensive terminal networks in both the ventral and dorsal stomachs. 2) SP‐IR axons were much denser in the antrum and corpus regions than in the fundus and cardia. 3) SP‐IR varicose axons ran in parallel with the circular and longitudinal muscle layers. 4) SP‐IR axons innervated blood vessels. 5) SP‐IR varicose axons formed terminals wrapping around individual myenteric neurons. 6) There were no confirmed SP‐IR myenteric neurons. 7) SP‐IR afferent innervation of the stomach showed a similar pattern of SP‐IR axons and terminals between the ventral and dorsal stomachs. In control mice (n=8), we injected tracer DiI into the ventral and dorsal stomach muscular layers or Fluorogold injection (i.p) and found that the main extrinsic source of SP‐IR afferent axons in the stomach was from the T7‐T11 DRG and to a lesser extent the VNG, but not from the celiac ganglia and dorsal motor nucleus of vagus. Our data provide for the first time a comprehensive topographical map for SP‐IR axons and terminals in the whole stomach with single cell/axon/synapse resolution. This work will contribute to a 3D digital representation of a brain‐stomach nociceptive atlas in a stomach scaffold, thus providing a novel anatomical foundation for functional mapping of nociceptive afferent axons and their pathological remodeling in the stomach. The first two authors contributed equally to this work.

Topographical Organization and Morphology of Calcitonin Gene‐Related Peptide (CGRP) Axons in the Flat‐Mounts of Whole Mouse Stomach

Nociceptive afferents innervate the stomach and send nociceptive signals centrally to the brain and locally to the enteric nervous system. Nociceptive afferents can be detected with a variety of different markers (e.g., CGRP, SP, TRPV1). However, the distribution and morphological structure of nociceptive axons and terminals have not yet been well determined in the flat‐mounts of the whole stomach. In this study, we used calcitonin gene‐related peptide (CGRP) as a marker to label nociceptive afferent axons and terminals in the in the flat mounts of the whole ventral and dorsal stomachs of mouse (n = 6/each side, 3‐5 months). We applied a combination of state‐of‐the‐art techniques, including confocal microscopy, Zeiss Imager microscopy, flat‐mount tissue processing of the whole organ and immunohistochemistry, Neurolucida 360 tracing and integration of the tracing data into a 3D stomach scaffold to determine the distribution and morphology of CGRP‐IR axons and terminals in the whole stomach. We found that 1) CGRP‐IR axons formed extensive terminal networks in both ventral and dorsal stomachs. 2) CGRP‐IR axons dramatically innervated the blood vessels. 3) In longitudinal and circular muscles, CGRP‐IR varicose axons ran in parallel with the direction of the muscles. 4) CGRP‐IR axons formed a complex network between the longitudinal and circular muscle layers. 5) In the myenteric ganglia, CGRP‐IR axons formed varicose terminal contacts with individual myenteric neurons. 6) We did not observe any significant CGRP‐IR myenteric neurons. 7) CGRP‐IR axon innervation of the blood vessels were traced and digitized and integrated into a 3D scaffold. This is the first time that we provided a topographical map of CGRP‐IR innervation of the whole stomach at single cell/axons resolution. The work provides an anatomical foundation for functional studies of CGRP‐IR axons in various regions of the stomach and their remodeling in diseases. The first two authors contributed equally to this work.

Topographical Distribution and Morphology of CGRP‐IR Axons in the Flat‐Mounts of Whole Mouse and Rat Hearts

To understand and treat cardiac pain, it is important to have an understanding of the topographical distribution of nociceptive axons in the heart. While previous studies have detected nociceptive axons in the sectioned heart, their full distribution has not yet been determined. In this study, we used a nociceptive marker calcitonin gene‐related peptide (CGRP) to determine the distribution of nociceptive afferent axons in the whole heart. We first removed the hearts of male Sprague‐Dawley rats (n=6), and C57BL/6 mice (n=6). The hearts were dissected into left and right atria and ventricles and interventricular septum, and prepared as flat‐mounts. These samples were then processed with immunohistochemical labeling using a primary antibody which binds to CGRP and an Alexa Fluro 488 secondary antibody for fluorescence. The flat‐mounts were imaged with a Zeiss M2 Imager (automatic fluorescence microscope) using a 488 nm excitation wavelength. In both rats and mice, we found that CGRP‐IR nerve bundles entered the heart and innervated the left and right atria and ventricles. For the right/left atria, CGRP‐IR axons entered as large bundles near the superior/inferior vena cava, left pre‐caval vein and the pulmonary veins before bifurcating into small branches, and finally formed single varicose axons and terminals which distributed throughout the tissue, including cardiac ganglia, the SA/AV nodes, auricles, and the blood vessels. In the right/left ventricles and interventricular septum, CGRP‐IR axons entered as large bundles through the base before bifurcating into small branches towards the apex Ultimately, single varicose axons and terminals innervated myocardium and blood vessels. Our data shows the distribution of CGRP‐IR axons in the whole heart at single cell/axon resolution. With this information, we will gain insight into the function of these axons and the pathways that they follow, paving the way for the development of better treatments for cardiac pain and disease. The first two authors contributed equally to this work.

Chapter 15 - The phrenic neuromuscular system

The phrenic neuromuscular system consists of the phrenic motor nucleus in the mid-cervical spinal cord, the phrenic nerve, and the diaphragm muscle. This motor system helps sustain breathing throughout life, while also contributing to posture, coughing, swallowing, and speaking. The phrenic nerve contains primarily efferent phrenic axons and afferent axons from diaphragm sensory receptors but is also a conduit for autonomic fibers. On a breath-by-breath basis, rhythmic (inspiratory) depolarization of phrenic motoneurons occurs due to excitatory bulbospinal synaptic pathways. Further, a complex propriospinal network innervates phrenic motoneurons and may serve to coordinate postural, locomotor, and respiratory movements. The phrenic neuromuscular system is impacted in a wide range of neuromuscular diseases and injuries. Contemporary research is focused on understanding how neuromuscular plasticity occurs in the phrenic neuromuscular system and using this information to optimize treatments and rehabilitation strategies to improve breathing and related behaviors.

A single baroreceptor unit consists of multiple sensors

Arterial baroreceptors (BRs) play a vital role in the regulation of the cardiopulmonary system. What is known about how these sensors operate at the subcellular level is limited, however. Until recently, one afferent axon was considered to be connected to a single baroreceptor (one-sensor theory). However, in the lung, a single airway mechanosensory unit is now known to house many sensors (multiple-sensor theory). Here we tested the hypothesis that multiple-sensor theory also operates in BR units, using both morphological and electrophysiological approaches in rabbit aortic arch (in whole mount) labeled with Na+/K+-ATPase, as well as myelin basic protein antibodies, and examined microscopically. Sensory structures presented in compact clusters, similar to bunches of grapes. Sensory terminals, like those in the airways, formed leaf-like or knob-like expansions. That is, a single myelinated axon connected with multiple sensors forming a network. We also recorded single-unit activities from aortic baroreceptors in the depressor nerve in anesthetized rabbits and examined the unit response to a bolus intravenous injection of phenylephrine. Unit activity increased progressively as blood pressure (BP) increased. Five of eleven units abruptly changed their discharge pattern to a lower activity level after BP attained a plateau for a minute or two (when BP was maintained at the high level). These findings clearly show that the high discharge baroreceptor deactivates after over-excitation and unit activity falls to a low discharge sensor. In conclusion, our morphological and physiological data support the hypothesis that multiple-sensory theory can be applied to BR units.

Abstract MP23: Arterial Depressor Responses Induced By Neuromodulation Of Deep Peroneal Nerve In Spontaneously Hypertensive Rats

Hypertension affects nearly half of the US population but only 43% achieved blood pressure control with medication alone. Medical devices for hypertension include implantable lead electrodes that stimulate the carotid baroreceptors with promising results, albeit with significant adverse complications. To address these limitations, we have proposed the use of deep peroneal nerve stimulation (DPNS), which elicited a depressor response in anesthetized, breathing supported, spontaneously hypertensive rats (SHR). In this study, we further define the electrical stimulation parameters that optimize the DPNS depressor response, and demonstrated that increasing the pulse duration from 0.15 ms to 1ms, of 1.0 mA pulses at 2 Hz for 10 sec, significantly reduced the mean arterial pressure (MAP) by 8±4 mmHg (p<0.005; n=4) in this animal model. DPNS also caused an immediate increase in renal nerve activity (RNA; p< 0.004, n=5), which may represent afferent sensory axons from the kidney, although this possibility needs to be further investigated. In a separate cohort of anesthetized SHR animals, breathing spontaneously, we demonstrated that optimal DPNS stimulation reduced the MAP from 121±3 to 108±4; p=0.02; n=10). To confirm if DPNS is able to evoke a depressor response in fully awake SHR animals, we developed a novel miniaturized wireless microchannel electrode (w-μCE) with a L-shaped microchannel, through which the DPN slides and locks into a recording/stimulation chamber, causing no discomfort to the animal during locomotion. Two weeks after implantation of the w-μCE neural stimulation device, animals were movement-retrained to received wireless DPNS for 10 min daily for 2 weeks. Blood pressure was measured by tail-cuff at baseline, 10 days after device implantation, and 1 and 2-hr 15 days after DPNS. After two weeks of DPNS, the acute neuromodulation treatment reduced the initial systolic BP of 154±20 mmHg to 127±7 and 119±2 mmHg at 1 and 2 hr; respectively (p< 0.001, n=15-19 measurements; n=2 animals). These results provide evidence of the effectiveness and reliability of DPN neuromodulation as a possible treatment for drug-resistant hypertension.

WS3.3. The Values of H-Reflex and Other Late Responses in Disease

It is relatively easy to record the H reflex from soleus, and many authorities still teach that it can be obtained only from this muscle. Inability to record the soleus H reflex has three possible causes: (i) poor technique; (ii) low central excitability; (iii) loss of afferent fibres due to, e.g., peripheral neuropathy or nerve root pathology. If the reason is (ii), all reflexes, in upper and lower limbs, will be difficult to elicit, and the reflex response should then become apparent during a steady voluntary contraction. A voluntary contraction not only potentiates the reflex but it also decreases the stimulus intensity needed for the reflex, ensures that the reflex involves the contracting muscle, and greatly reduces the attenuation of the response that occurs with higher stimulus rates. It reduces the latency of the reflex responses only slightly (<0.5 ms). During a steady voluntary contraction of the test muscle, H reflexes can be recorded for most limb muscles, and provide information about conduction across the most relevant spinal segments (e.g., C5/6, C6, C6/7, C8/T1, L3/4, L4/5 and S1). In routine diagnostic practice, I assess reflex conduction in perhaps 60% of patients because this provides insight into conduction in afferent and efferent axons over the entire peripheral nerve pathway. In diagnostic practice we focus on pathology that causes loss of reflex function. However, if you can record H reflexes at rest from muscles that normally have no H reflex unless they are contracting voluntarily, there is presumptive evidence of hyperreflexia. If there is EMG evidence of denervation in that muscle, this suggests UMN and LMN changes for a single muscle, and this is support for a diagnosis of motor neurone disease. F waves can provide complementary information about motor axons because the H reflex preferentially involves slowly conduction motor axons from small low-threshold motoneurones, but the F wave measures conduction in larger axons that are not discharged reflexly by the intense afferent volley produced by the supramaximal stimulus.

Sensorimotor nerve lesion of upper airway in patients with obstructive sleep apnea

The pathogenesis of obstructive sleep apnea (OSA) remains controversial. The role of anatomic stenosis is indisputable, and neural regulation of the upper airway remains to be elucidated. The upper airway maintains patency through the upper airway reflex. Lesions in any link of the reflex can increase the collapsibility of the upper airway. In this study, we investigated sensorimotor nerve lesions and their possible relationship with OSA. Tissue samples were obtained from the pharyngopalatine arch in 47 patients with OSA and 45 control participants to examine changes in the expression levels of myelin basic protein (MBP) and agrin through immunohistochemistry and western blotting. Downregulation of MBP in the mucosa reflects myelinated degeneration of mucosal sensory nerve axons, whereas upregulation of agrin in the neuromuscular junction reflects synaptic regeneration following denervation. The two neural factors correlate significantly with polysomnographic parameters, such as the apnea hypopnea index and lowest oxygen saturation. Our findings suggest that sensorimotor nerve damage in the upper airway of patients with OSA may be associated closely with the mechanism of OSA.

Piezo2 mechanosensitive ion channel is located to sensory neurons and nonneuronal cells in rat peripheral sensory pathway: implications in pain.

Piezo2 mechanotransduction channel is a crucial mediator of sensory neurons for sensing and transducing touch, vibration, and proprioception. We here characterized Piezo2 expression and cell specificity in rat peripheral sensory pathway using a validated Piezo2 antibody. Immunohistochemistry using this antibody revealed Piezo2 expression in pan primary sensory neurons of dorsal root ganglia in naïve rats, which was actively transported along afferent axons to both central presynaptic terminals innervating the spinal dorsal horn (DH) and peripheral afferent terminals in the skin. Piezo2 immunoreactivity (IR) was also detected in the postsynaptic neurons of the DH and in the motor neurons of the ventral horn, but not in spinal glial fibrillary acidic protein-positive and Iba1-positive glia. Notably, Piezo2-IR was clearly identified in peripheral nonneuronal cells, including perineuronal glia, Schwann cells in the sciatic nerve and surrounding cutaneous afferent endings, as well as in skin epidermal Merkel cells and melanocytes. Immunoblots showed increased Piezo2 in dorsal root ganglia ipsilateral to plantar injection of complete Freund's adjuvant, and immunostaining revealed increased Piezo2-IR intensity in the DH ipsilateral to complete Freund's adjuvant injection. This elevation of DH Piezo2-IR was also evident in various neuropathic pain models and monosodium iodoacetate knee osteoarthritis pain model, compared with controls. We conclude that (1) the pan neuronal profile of Piezo2 expression suggests that Piezo2 may function extend beyond simply touch or proprioception mediated by large-sized low-threshold mechanosensitive primary sensory neurons; (2) Piezo2 may have functional roles involving sensory processing in the spinal cord, Schwann cells, and skin melanocytes; and (3) aberrant Piezo2 expression may contribute pain pathogenesis.

High Dietary Fat Consumption Impairs Axonal Mitochondrial Function In Vivo.

Peripheral neuropathy (PN) is the most common complication of prediabetes and diabetes. PN causes severe morbidity for Type 2 diabetes (T2D) and prediabetes patients, including limb pain followed by numbness resulting from peripheral nerve damage. PN in T2D and prediabetes is associated with dyslipidemia and elevated circulating lipids; however, the molecular mechanisms underlying PN development in prediabetes and T2D are unknown. Peripheral nerve sensory neurons rely on axonal mitochondria to provide energy for nerve impulse conduction under homeostatic conditions. Models of dyslipidemia in vitro demonstrate mitochondrial dysfunction in sensory neurons exposed to elevated levels of exogenous fatty acids. Herein, we evaluated the effect of dyslipidemia on mitochondrial function and dynamics in sensory axons of the saphenous nerve of a male high-fat diet (HFD)-fed murine model of prediabetes to identify mitochondrial alterations that correlate with PN pathogenesis in vivo We found that the HFD decreased mitochondrial membrane potential (MMP) in axonal mitochondria and reduced the ability of sensory neurons to conduct at physiological frequencies. Unlike mitochondria in control axons, which dissipated their MMP in response to increased impulse frequency (from 1 to 50 Hz), HFD mitochondria dissipated less MMP in response to axonal energy demand, suggesting a lack of reserve capacity. The HFD also decreased sensory axonal Ca2+ levels and increased mitochondrial lengthening and expression of PGC1α, a master regulator of mitochondrial biogenesis. Together, these results suggest that mitochondrial dysfunction underlies an imbalance of axonal energy and Ca2+ levels and impairs impulse conduction within the saphenous nerve in prediabetic PN.SIGNIFICANCE STATEMENT Diabetes and prediabetes are leading causes of peripheral neuropathy (PN) worldwide. PN has no cure, but development in diabetes and prediabetes is associated with dyslipidemia, including elevated levels of saturated fatty acids. Saturated fatty acids impair mitochondrial dynamics and function in cultured neurons, indicating a role for mitochondrial dysfunction in PN progression; however, the effect of elevated circulating fatty acids on the peripheral nervous system in vivo is unknown. In this study, we identify early pathogenic events in sensory nerve axons of mice with high-fat diet-induced PN, including alterations in mitochondrial function, axonal conduction, and intra-axonal calcium, that provide important insight into potential PN mechanisms associated with prediabetes and dyslipidemia in vivo.

Selective Denervation of the Facial Dermato-Muscular Complex in the Rat: Experimental Model and Anatomical Basis.

The facial dermato-muscular system consists of highly specialized muscles tightly adhering to the overlaying skin and thus form a complex morphological conglomerate. This is the anatomical and functional basis for versatile facial expressions, which are essential for human social interaction. The neural innervation of the facial skin and muscles occurs via branches of the trigeminal and facial nerves. These are also the most commonly pathologically affected cranial nerves, often requiring surgical treatment. Hence, experimental models for researching these nerves and their pathologies are highly relevant to study pathophysiology and nerve regeneration. Experimental models for the distinctive investigation of the complex afferent and efferent interplay within facial structures are scarce. In this study, we established a robust surgical model for distinctive exploration of facial structures after complete elimination of afferent or efferent innervation in the rat. Animals were allocated into two groups according to the surgical procedure. In the first group, the facial nerve and in the second all distal cutaneous branches of the trigeminal nerve were transected unilaterally. All animals survived and no higher burden was caused by the procedures. Whisker pad movements were documented with video recordings 4 weeks after surgery and showed successful denervation. Whole-mount immunofluorescent staining of facial muscles was performed to visualize the innervation pattern of the neuromuscular junctions. Comprehensive quantitative analysis revealed large differences in afferent axon counts in the cutaneous branches of the trigeminal nerve. Axon number was the highest in the infraorbital nerve (28,625 ± 2,519), followed by the supraorbital nerve (2,131 ± 413), the mental nerve (3,062 ± 341), and the cutaneous branch of the mylohyoid nerve (343 ± 78). Overall, this surgical model is robust and reliable for distinctive surgical deafferentation or deefferentation of the face. It may be used for investigating cortical plasticity, the neurobiological mechanisms behind various clinically relevant conditions like facial paralysis or trigeminal neuralgia as well as local anesthesia in the face and oral cavity.

βIII spectrin controls the planarity of Purkinje cell dendrites by modulating perpendicular axon-dendrite interactions.

The mechanism underlying the geometrical patterning of axon and dendrite wiring remains elusive, despite its crucial importance in the formation of functional neural circuits. The cerebellar Purkinje cell (PC) arborizes a typical planar dendrite, which forms an orthogonal network with granule cell (GC) axons. By using electrospun nanofiber substrates, we reproduce the perpendicular contacts between PC dendrites and GC axons in culture. In the model system, PC dendrites show a preference to grow perpendicularly to aligned GC axons, which presumably contribute to the planar dendrite arborization in vivo We show that βIII spectrin, a causal protein for spinocerebellar ataxia type5, is required for the biased growth of dendrites. βIII spectrin deficiency causes actin mislocalization and excessive microtubule invasion in dendritic protrusions, resulting in abnormally oriented branch formation. Furthermore, disease-associated mutations affect the ability of βIII spectrin to control dendrite orientation. These data indicate that βIII spectrin organizes the mouse dendritic cytoskeleton and thereby regulates the oriented growth of dendrites with respect to the afferent axons.

Editorial: We Are Trigeminal and Different

Rafael Benoliel: The trigeminal nerve supplies all sensory innervation to the head and associated structures. This is “our” nerve, and it is inevitably involved in the multitude of painful disorders that we manage on a daily basis. Despite distinct embryogenic origins and processing capabilities, trigeminal neurons and cell bodies should not be significantly different from their spinal counterparts. The anatomical set-up is similar, with afferent neuronal cell bodies, both spinal and trigeminal, located in the dorsal root (DRG) and the trigeminal (TG) ganglia, respectively. The DRG and TG structures are similar, and they are essentially homologs of each other. The clearest anatomical differences are the proximity of the trigeminal system to the CNS and the relatively long trajectory of afferent axons in the spinal system. Additionally, the TG is the only sensory ganglion of the body that resides within the CNS. Clinical and laboratory observations, however, suggest differences between the trigeminal and spinal systems that may underlie dissimilar functional properties and response to injury or disease. There may be subtle differences between these two systems, the basis and consequences of which are still unclear. One interesting difference: The trigeminal system is the only nerve involved in spontaneous denervation during shedding of deciduous teeth. Is that indicative of any differences that may be of consequence? Evidence in animal models suggests that the trigeminal nerve is more resistant than the spinal system in developing neuropathic pain (NP) following insult or disease. Moreover, sprouting of sympathetic nerves around large ganglionic neurons following injury is not observed in the TG, but is present in the DRG, and cervical sympathectomy does not alter trigeminal NP behavior, but does so in the spinal system. Together with a rich vascular supply to trigeminally innervated regions (ie, the trigeminovascular system), these may explain some differences in the clinical phenotypes of trigeminal vs spinal pain syndromes. For example, clinically diabetic neuropathy is a common source of NP in the limbs and trunk involving classical signs of paresthesia and allodynia. In the trigeminal system, these effects are not pronounced, and there is a sparsity of reports of typical painful diabetic neuropathic pain or indeed neuropathy at all. Insults to trigeminal nerve branches such as microinjuries (eg, tooth extraction, root canal treatment) or macroinjuries rarely result in chronic NP and are consistently reported at a lower prevalence than following injury to the spinal system. Complex regional pain syndrome, a disabling painful disorder resulting from mild to severe nerve injury that commonly presents in the extremities and is accompanied by sensory, vascular, and muscular problems, has no true equivalent with all of these diagnostic features in the trigeminal system. Conversely, pain syndromes such as migraine, cluster headache, and trigeminal neuralgia exist only in the trigeminal system. Taken together, these findings suggest that the spinal and trigeminal systems may differ, particularly in the way they respond to injury. How and why remain a mystery. Gene expression data may offer some insights. Studies in naïve animals reveal differences in expressed genes between the DRG and the TG, indicating that different molecular mechanisms are involved in the baseline functionality of the two systems. The animal data seem to align well with human data on the DRG and TG. It has recently been shown that spinal and trigeminal neuropathies caused by trauma are accompanied by differentially regulated genes within the DRG and TG. The genes involved suggest that neuroinflammatory signaling and other related pathways are involved. These studies are limited due to the fact that they are “whole-ganglion” analyses and do not indicate from which type of neuron or nonneuronal cell present in the ganglia the changes originate. Yet, overall, the message is that the trigeminal system is different. As molecular techniques improve, we may be able to use these data to identify protective genes and pathways that may be novel targets for pain intervention and to elucidate some of the fascinating differences in diseases present exclusively in the trigeminal system.^1–3 Rafael Benoliel Editor-in-Chief

A specialized reciprocal connectivity suggests a link between the mechanisms by which the superior colliculus and parabigeminal nucleus produce defensive behaviors in rodents

The parabigeminal nucleus (PBG) is the mammalian homologue to the isthmic complex of other vertebrates. Optogenetic stimulation of the PBG induces freezing and escape in mice, a result thought to be caused by a PBG projection to the central nucleus of the amygdala. However, the isthmic complex, including the PBG, has been classically considered satellite nuclei of the Superior Colliculus (SC), which upon stimulation of its medial part also triggers fear and avoidance reactions. As the PBG-SC connectivity is not well characterized, we investigated whether the topology of the PBG projection to the SC could be related to the behavioral consequences of PBG stimulation. To that end, we performed immunohistochemistry, in situ hybridization and neural tracer injections in the SC and PBG in a diurnal rodent, the Octodon degus. We found that all PBG neurons expressed both glutamatergic and cholinergic markers and were distributed in clearly defined anterior (aPBG) and posterior (pPBG) subdivisions. The pPBG is connected reciprocally and topographically to the ipsilateral SC, whereas the aPBG receives afferent axons from the ipsilateral SC and projected exclusively to the contralateral SC. This contralateral projection forms a dense field of terminals that is restricted to the medial SC, in correspondence with the SC representation of the aerial binocular field which, we also found, in O. degus prompted escape reactions upon looming stimulation. Therefore, this specialized topography allows binocular interactions in the SC region controlling responses to aerial predators, suggesting a link between the mechanisms by which the SC and PBG produce defensive behaviors.

Structure-function relation of the developing calyx of Held synapse in vivo.

Key points During development the giant, auditory calyx of Held forms a one‐to‐one connection with a principal neuron of the medial nucleus of the trapezoid body.While anatomical studies described that most of the target cells are temporarily contacted by multiple calyces, multi‐calyceal innervation was only sporadically observed in in vivo recordings, suggesting a structure–function discrepancy.We correlated synaptic strength of inputs, identified in in vivo recordings, with post hoc labelling of the recorded neuron and synaptic terminals containing vesicular glutamate transporters (VGluT).During development only one input increased to the level of the calyx of Held synapse, and its strength correlated with the large VGluT cluster contacting the postsynaptic soma.As neither competing strong inputs nor multiple large VGluT clusters on a single cell were observed, our findings did not indicate a structure–function discrepancy. In adult rodents, a principal neuron in the medial nucleus of the trapezoid (MNTB) is generally contacted by a single, giant axosomatic terminal called the calyx of Held. How this one‐on‐one relation is established is still unknown, but anatomical evidence suggests that during development principal neurons are innervated by multiple calyces, which may indicate calyceal competition. However, in vivo electrophysiological recordings from principal neurons indicated that only a single strong synaptic connection forms per cell. To test whether a mismatch exists between synaptic strength and terminal size, we compared the strength of synaptic inputs with the morphology of the synaptic terminals. In vivo whole‐cell recordings of the MNTB neurons from newborn Wistar rats of either sex were made while stimulating their afferent axons, allowing us to identify multiple inputs. The strength of the strongest input increased to calyceal levels in a few days across cells, while the strength of the second strongest input was stable. The recorded cells were subsequently immunolabelled for vesicular glutamate transporters (VGluT) to reveal axosomatic terminals with structured‐illumination microscopy. Synaptic strength of the strongest input was correlated with the contact area of the largest VGluT cluster at the soma (r = 0.8), and no indication of a mismatch between structure and strength was observed. Together, our data agree with a developmental scheme in which one input strengthens and becomes the calyx of Held, but not with multi‐calyceal competition.

Mapping of Extrinsic Innervation of the Gastrointestinal Tract in the Mouse Embryo.

Precise extrinsic afferent (visceral sensory) and efferent (sympathetic and parasympathetic) innervation of the gut is fundamental for gut-brain cross talk. Owing to the limitation of intrinsic markers to distinctively visualize the three classes of extrinsic axons, which intimately associate within the gut mesentery, detailed information on the development of extrinsic gut-innervating axons remains relatively sparse. Here, we mapped extrinsic innervation of the gut and explored the relationships among various types of extrinsic axons during embryonic development in mice. Visualization with characterized intrinsic markers revealed that visceral sensory, sympathetic, and parasympathetic axons arise from different anatomic locations, project in close association via the gut mesentery, and form distinctive innervation patterns within the gut from embryonic day (E)10.5 to E16.5. Genetic ablation of visceral sensory trajectories results in the erratic extension of both sympathetic and parasympathetic axons, implicating that afferent axons provide an axonal scaffold to route efferent axons. Coculture assay further confirmed the attractive effect of sensory axons on sympathetic axons. Taken together, our study provides key information regarding the development of extrinsic gut-innervating axons occurring through heterotypic axonal interactions and provides an anatomic basis to uncover neural circuit assembly in the gut-brain axis (GBA).SIGNIFICANCE STATEMENT Understanding the development of extrinsic innervation of the gut is essential to unravel the bidirectional neural communication between the brain and the gut. Here, with characterized intrinsic markers targeting vagal sensory, spinal sensory, sympathetic, and parasympathetic axons, respectively, we comprehensively traced the spatiotemporal development of extrinsic axons to the gut during embryonic development in mice. Moreover, in line with the somatic nervous system, pretarget sorting via heterotypic axonal interactions is revealed to play critical roles in patterning extrinsic efferent trajectories to the gut. These findings provide basic anatomic information to explore the mechanisms underlying the process of assembling neural circuitry in the gut-brain axis (GBA).

Exogenous Phosphatidylglucoside Improves Cognitive Impairment by Improvement of Neuroinflammation And Neurotrophin Signaling

Alzheimer's disease (AD) is a commonly progressive disorder of dementia. Until now, there are no effective approach to treat the pathological progression of Alzheimer's disease. A novel glucosylated lipids, phosphatidylglucoside (PtdGlc), enriched in the brain, appears to play a important role for brain development. Based on the role of lysoPtdGlc (hydrolytic derivative of PtdGlc)/GPR55-mediated nociceptive afferent axons in the central nervous system, we hypothesized that PtdGlc can aggravated the development of AD. On the contrast, here, we show that long‐term intervention with PtdGlc (0.1% w/w in diet) reduces Aβ and tau pathology, and rescues cognition deficits in APP/PS1 mice. Mechanistically, these improments were linked to the inhibition of APP processing, amelioration of neuroinflammation via the activation of PPARγ, mitigation of oxidative stress, and restoration of the neurotrophin signaling. Our study offers new perspectives for the role of PtdGlc in the Brain, as well as the treatment of AD.

Quantitative Sudies of Spontaneous Electrical Activities in Detrusor Smooth Muscle Cells towards Understanding Urinary Bladder Overactivities

ContextThe urinary incontinence (UI) is defined as the involuntary loss of urine and associated with the enhanced spontaneous contractions of the detrusor smooth muscle (DSM). The spontaneously evoked action potentials (sAPs) in DSM cells initiate and modulate these contractions. The DSM is strongly innervated, connecting approximately 16000 afferent and efferent axons from ganglion neurons. It generates sAPs due to the stochastic nature of purinergic neurotransmitter release from the parasympathetic nerve.ObjectivesThe aim of this current study is to understand the putative relationship between the fluctuating ion channel conductances and stochastically release of ATP in generating sAPs.MethodsThe neurotransmitter current was considered as an independent excitatory conductance in the model where gex(t) and Eex are the one‐variable stochastic process conductance and the reversal potential respectively. In addition, Dex and λ1(t) are known as the diffusion coefficients and Gaussian white noise. The point‐conductance is incorporated into a single DSM cell model based on single cylindrical compartment.ResultsThe elicited AP consists an after depolarization and after hyperpolarization phase. The AP peak amplitude and duration are about 15 mV and 40 ms respectively. The stochastic nature of purinergic neurotransmitter is designed by a point process model. Then, the random injection of point process model is conducted to elicit a series of sAPs and membrane depolarization for 5 seconds. The membrane resting potential is held at − 50 mV with a 3 mV of fluctuation. The stochastically depolarization up to 20 mV activates the T‐type Ca2+ channel first and then the L‐type Ca2+ channel to generate action potential. The Figure 1 A illustrates series of AP generation in the DSM cell due to stochastically depolarizations. The model does not evoke any AP and depolarization, when the conductance of T‐type Ca2+ channel is set to zero (Figure 1 B).ConclusionsFrom the results, it is found that the T‐type Ca2+ channel blocker can be used as a new pharmacological target for the UI. In addition, an extended multidimensional models will a provide windows of insight into the factors that govern excitability and contraction in both normal and unstable bladder.Support or Funding InformationThis work is supported in part by Department of Biotechnology (DBT), India (grant number BT/PR12973/MED/122/47/2016).Effects of T‐type Ca2+ channel blockers on AP generation.Figure 1