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There is emerging evidence that Brain Derived Neurotrophic Factor (BDNF), and one of its receptors TrkB, play important roles in the pathogenesis of osteoarthritis (OA) pain. Whilst these studies clearly highlight the potential for targeting BDNF/TrkB signaling to treat OA pain, the mechanism for how BDNF/TrkB signaling contributes to OA pain remains unclear. In this study, we used an animal model of mono-iodoacetate (MIA)-induced OA, in combination with electrophysiology, behavioral testing, Western blot analysis, and retrograde tracing and immunohistochemistry, to identify roles for BDNF/TrkB signaling in the pathogenesis of OA pain. We found that: 1) TrkB is expressed in myelinated medium diameter neurons that innervate the knee joint and bone in naïve animals; 2) peripheral application of BDNF increases the sensitivity of Aδ, but not C knee joint and bone afferent neurons, in response to mechanical stimulation, in naïve animals; 3) BDNF expression increases in synovial tissue in early MIA-induced OA, when pathology is confined to the joint, and in the subchondral bone in late MIA-induced OA, when there is additional damage to the surrounding bone; and 4) TrkB inhibition reverses MIA-induced changes in the sensitivity of Aδ but not C knee joint afferent neurons early in MIA-induced OA, and Aδ but not C bone afferent neurons late in MIA-induced OA. Our findings suggest that BDNF/TrkB signaling may have a role to play in the pathogenesis of OA pain, through effects on knee joint afferent neurons early in disease when there is inflammation confined to the joint, and bone afferent neurons late in disease when there is involvement of damage to subchondral bone. Targeted manipulation of BDNF/TrkB signaling may provide therapeutic benefit for the management of OA pain., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: The medial prefrontal cortex (mPFC) forms output pathways through projection neurons, inversely receiving adjacent and long-range inputs from other brain regions. However, how afferent neurons of mPFC are affected by chronic stress needs to be clarified. In this study, the effects of chronic restraint stress (CRS) on the distribution density of mPFC dendrites/dendritic spines and the projections from the cortex and subcortical brain regions to the mPFC were investigated., Methods: In the present study, C57BL/6 J transgenic (Thy1-YFP-H) mice were subjected to CRS to establish an animal model of depression. The infralimbic (IL) of mPFC was selected as the injection site of retrograde AAV using stereotactic technique. The effects of CRS on dendrites/dendritic spines and afferent neurons of the mPFC IL were investigaed by quantitatively assessing the distribution density of green fluorescent (YFP) positive dendrites/dendritic spines and red fluorescent (retrograde AAV recombinant protein) positive neurons, respectively., Results: The results revealed that retrograde tracing virus labeled neurons were widely distributed in ipsilateral and contralateral cingulate cortex (Cg1), second cingulate cortex (Cg2), prelimbic cortex (PrL), infralimbic cortex, medial orbital cortex (MO), and dorsal peduncular cortex (DP). The effects of CRS on the distribution density of mPFC red fluorescence positive neurons exhibited regional differences, ranging from rostral to caudal or from top to bottom. Simultaneously, CRS resulted a decrease in the distribution density of basal, proximal and distal dendrites, as well as an increase in the loss of dendritic spines of the distal dendrites in the IL of mPFC. Furthermore, varying degrees of red retrograde tracing virus fluorescence signals were observed in other cortices, amygdala, hippocampus, septum/basal forebrain, hypothalamus, thalamus, mesencephalon, and brainstem in both ipsilateral and contralateral brain. CRS significantly reduced the distribution density of red fluorescence positive neurons in other cortices, hippocampus, septum/basal forebrain, hypothalamus, and thalamus. Conversely, CRS significantly increased the distribution density of red fluorescence positive neurons in amygdala., Conclusion: Our results suggest a possible mechanism that CRS leads to disturbances in synaptic plasticity by affecting multiple inputs to the mPFC, which is characterized by a decrease in the distribution density of dendrites/dendritic spines in the IL of mPFC and a reduction in input neurons of multiple cortices to the IL of mPFC as well as an increase in input neurons of amygdala to the IL of mPFC, ultimately causing depression-like behaviors., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections 1,2 . The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization 3-6 , but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Psychological stress has been linked to the development and exacerbation of overactive bladder symptoms, as well as afferent sensitisation in other organ systems. Therefore, we aimed to investigate the effects of water avoidance stress on bladder afferent nerve activity in response to bladder filling and pharmaceutical stimulation with carbachol and ATP in mice. Adult female C57BL/6J mice were exposed to either water avoidance stress (WAS) for 1 h/day for 10 days or normal housing conditions. Voiding behaviour was measured before starting and 24-h after final stress exposure and then animals were euthanised to measure afferent nerve activity in association with bladder compliance, spontaneous phasic activity, contractile responses, as well as release of urothelial mediators. WAS caused increased urinary frequency without affecting urine production. The afferent nerve activity at low bladder pressures (4-7 mmHg), relevant to normal physiological filling, was significantly increased after stress. Both low and high threshold nerves demonstrated enhanced activity at physiological bladder pressures. Urothelial ATP and acetylcholine release and bladder compliance were unaffected by stress as was the detrusor response to ATP (1 mM) and carbachol (1 µM). WAS caused enhanced activity of individual afferent nerve fibres in response bladder distension. The enhanced activity was seen in both low and high threshold nerves suggesting that stressed animals may experience enhanced bladder filling sensations at lower bladder volumes as well as increased pain sensations, both potentially contributing to the increased urinary frequency seen after stress., (© 2021. The Author(s).)

The purpose of this study was to determine the contributions of feedforward and feedback processes on grip force regulation and object orientation during functional manipulation tasks. One patient with massive somatosensory loss resulting from large fiber sensory neuropathy and 10 control participants were recruited. Three experiments were conducted: 1 ) perturbation to static holding; 2 ) discrete vertical movement; and 3 ) functional grasp and place. The availability of visual feedback was also manipulated to assess the nature of compensatory mechanisms. Results from experiment 1 indicated that both the deafferented patient and controls used anticipatory grip force adjustments before self-induced perturbation to static holding. The patient exhibited increased grip response time, but the magnitude of grip force adjustments remained correlated with perturbation forces in the self-induced and external perturbation conditions. In experiment 2 , the patient applied peak grip force substantially in advance of maximum load force. Unlike controls, the patient's ability to regulate object orientation was impaired without visual feedback. In experiment 3 , the duration of unloading, transport, and release phases were longer for the patient, with increased deviation of object orientation at phase transitions. These findings show that the deafferented patient uses distinct modes of anticipatory control according to task constraints and that responses to perturbations are mediated by alternative afferent information. The loss of somatosensory feedback thus appears to impair control of object orientation, whereas variation in the temporal organization of functional tasks may reflect strategies to mitigate object instability associated with changes in movement dynamics. NEW & NOTEWORTHY This study evaluates the effects of sensory neuropathy on the scaling and timing of grip force adjustments across different object handling tasks (i.e., holding, vertical movement, grasping, and placement). In particular, these results illustrate how novel anticipatory and online control processes emerge to compensate for the loss of somatosensory feedback. In addition, we provide new evidence on the role of somatosensory feedback for regulating object orientation during functional prehensile movement.

Neuropathic pain is pain caused by damage to the somatosensory nervous system. Both degenerating injured nerves and neighboring sprouting nerves can contribute to neuropathic pain. However, the mesoscale changes in cutaneous nerve fibers over time after the loss of the parent nerve has not been investigated in detail. In this study, we followed the changes in nerve fibers longitudinally in the toe tips of mice that had undergone spared nerve injury (SNI). Nav1.8-tdTomato, Thy1-GFP and MrgD-GFP mice were used to observe the small and large cutaneous nerve fibers. We found that peripheral nerve plexuses degenerated within 3 days of nerve injury, and free nerve endings in the epidermis degenerated within 2 days. The timing of degeneration paralleled the initiation of mechanical hypersensitivity. We also found that some of the Nav1.8-positive nerve plexuses and free nerve endings in the fifth toe survived, and sprouting occurred mostly from 7 to 28 days. The timing of the sprouting of nerve fibers in the fifth toe paralleled the maintenance phase of mechanical hypersensitivity. Our results support the hypotheses that both injured and intact nerve fibers participate in neuropathic pain, and that, specifically, nerve degeneration is related to the initiation of evoked pain and nerve sprouting is related to the maintenance of evoked pain., (© 2021 Wiley Periodicals LLC.)

Acupuncture modulates the mesolimbic dopamine (DA) system; an area implicated in drug abuse. However, the mechanism by which peripheral sensory afferents, during acupuncture stimulation, modulate this system needs further investigation. The lateral hypothalamus (LH) has been implicated in reward processing and addictive behaviors. To investigate the role of the LH in mediating acupuncture effects, we evaluated the role of LH and spinohypothalamic neurons on cocaine-induced psychomotor activity and NAc DA release. Systemic injection of cocaine increased locomotor activity and 50 kHz ultrasonic vocalizations (USVs), which were attenuated by mechanical stimulation of needles inserted into HT7 but neither ST36 nor LI5. The acupuncture effects were blocked by chemical lesions of the LH or mimicked by activation of LH neurons. Single-unit extracellular recordings showed excitation of LH and spinohypothalamic neurons following acupuncture. Our results suggest that acupuncture recruits the LH to suppress the mesolimbic DA system and psychomotor responses following cocaine injection.

The mechanism of pain chronicity is largely unknown in lumbar radiculopathy (LR). The anatomical location of nerve injury is one of the important factors associated with pain chronicity of LR. Accumulating evidence has shown constriction distal to the dorsal root ganglion (DRG) caused more severe radiculopathy than constriction proximal to the DRG; thereby, the mechanism of pain chronicity in LR could be revealed by comparing the differences in pathological changes of DRGs between nerve constriction distal and proximal to the DRG. Here, we used 2 rat models of LR with nerve constriction distal or proximal to the DRG to probe how the different nerve injury sites could differentially affect pain chronicity and the pathological changes of DRG neuron subpopulations. As expected, rats with nerve constriction distal to the DRG showed more persistent pain behaviors than those with nerve constriction proximal to the DRG in 50% paw withdraw threshold, weight-bearing test, and acetone test. One day after the operation, distal and proximal nerve constriction showed differential pathological changes of DRG. The ratios of activating transcription factor3 (ATF3)-positive DRG neurons were significantly higher in rats with nerve constriction distal to DRG than those with nerve constriction proximal to DRG. In subpopulation analysis, the ratios of ATF3-immunoreactivity (IR) in neurofilament heavy chain (NFH)-positive DRG neurons significantly increased in distal nerve constriction compared to proximal nerve constriction; although, both distal and proximal nerve constriction presented increased ratios of ATF3-IR in calcitonin gene-related peptide (CGRP)-positive DRG neurons. Moreover, the nerve constriction proximal to DRG caused more hypoxia than did that distal to DRG. Together, ATF3 expression in NHF-positive DRG neurons at the acute stage is a potential bio-signature of persistent pain in rat models of LR.

In 1973, Gibbs, Young, and Smith showed that exogenous cholecystokinin (CCK) administration reduces food intake in rats. This initial report has led to thousands of studies investigating the physiological role of CCK in regulating feeding behavior. CCK is released from enteroendocrine I cells present along the gastrointestinal (GI) tract. CCK binding to its receptor CCK1R leads to vagal afferent activation providing post-ingestive feedback to the hindbrain. Vagal afferent neurons' (VAN) sensitivity to CCK is modulated by energy status while CCK signaling regulates gene expression of other feeding related signals and receptors expressed by VAN. In addition to its satiation effects, CCK acts all along the GI tract to optimize digestion and nutrient absorption. Diet-induced obesity (DIO) is characterized by reduced sensitivity to CCK and every part of the CCK system is negatively affected by chronic intake of energy-dense foods. EEC have recently been shown to adapt to diet, CCK1R is affected by dietary fats consumption, and the VAN phenotypic flexibility is lost in DIO. Altered endocannabinoid tone, changes in gut microbiota composition, and chronic inflammation are currently being explored as potential mechanisms for diet driven loss in CCK signaling. This review discusses our current understanding of how CCK controls food intake in conditions of leanness and how control is lost in chronic energy excess and obesity, potentially perpetuating excessive intake., (Copyright © 2020. Published by Elsevier Inc.)

Cancer neurobiology is an emerging discipline that inevitably unfurls new perspectives in oncology. The role that nerves play in cancer progression resonates with the long-reported dependency of tumors on neuro-molecular mechanisms that remain insufficiently elucidated. Whereas interactions between neurotrophic growth factors and receptors have been heavily studied in the nervous system, their expression in cancers and their impact on tumor cell growth and metastasis through their corresponding signaling pathways has been undervalued. Accumulating evidence suggests that trophic factors released by nerves strongly influence tumor development and that this neural contribution appears to not only play a stimulatory role but also function as an essential part of the tumor's microenvironment. This bidirectional communication between proliferating cells and tumor-infiltrating nerves drives axonogenesis and tumor growth and migration. Acquiring a better understanding of the trophic interactions between primary afferent neurons and invading tumors will guide clinically actionable strategies to prevent tumor-associated axonogenesis, disrupting the chemical crosstalk between neurons and tumors and ultimately decreasing tumor growth and spread., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Dysfunction of the gut-brain axis is one of the potential contributors to the pathophysiology of obesity and is therefore a potential target for treatment. Vagal afferents innervating the gut play an important role in controlling energy homeostasis. There is an increasing evidence for the role of vagal afferents in mediating the anorexigenic effects of glucagon-like peptide-1 (GLP-1), an important satiety and incretin hormone. This study aimed to examine the effect of chronic high fat diet on GLP-1 sensitivity in vagal afferents. C57/BL6 mice were fed either a high-fat or low-fat diet for 6-8 weeks. To evaluate gastrointestinal afferent sensitivity and nodose neurons' response to GLP-1, extracellular afferent recordings and patch clamp were performed, respectively. Exendin-4 (Ex-4) was used as an agonist of the GLP-1 receptor. C-Fos Expression was examined as an indication of afferent input to the nucleus tractus solitarius (NTS). Food intake was monitored in real-time before and after Ex-4 treatment to monitor the consequence of the high fat diet on the satiating effect of GLP-1. In high fat fed (HFF) mice, GLP-1 caused lower activation of intestinal afferent nerves, and failed to potentiate mechanosensitive nerve responses compared to low fat fed (LFF). GLP-1 increased excitability in LFF and this effect was reduced in HFF neurons. Consistent with these findings on vagal afferent nerves, GLP-1 receptor stimulation given systemically, had a reduced satiating effect in HFF compared to LFF mice, and neuronal activation in the NTS was also reduced. The present study demonstrated chronic high fat diet impaired vagal afferent responses to GLP-1, resulting in impaired satiety signaling. GLP-1 sensitivity may account for the impairment of satiety signaling in obesity and thus a therapeutic target for obesity treatment., Competing Interests: Declaration of competing interest Authors have no conflicts of interest to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Head and neck tumors can affect afferent baroreceptor neurons and either interrupt or intermittently increase their signaling, causing blood pressure to become erratic. When the afferent fibers of the baroreflex are injured by surgery or radiotherapy or fail to develop as in familial dysautonomia, their sensory information is no longer present to regulate arterial blood pressure, resulting in afferent baroreflex failure. When the baroreflex afferents are abnormally activated, such as by paragangliomas in the neck, presumably by direct compression, they trigger acute hypotension and bradycardia and frequently syncope, by a mechanism similar to the carotid sinus syndrome. We describe our observations in a large series of 23 patients with afferent baroreflex dysfunction and the cardiovascular autonomic features that arise when the sensory baroreceptor neurons are injured or compressed. The management of afferent baroreceptor dysfunction is limited, but pharmacological strategies can mitigate blood pressure swings, improve symptoms, and may reduce hypertensive organ damage. Although rare, the prevalence of afferent baroreflex dysfunction appears to be increasing in middle-aged men due to human papillomavirus related oropharyngeal cancer., Competing Interests: None., (Thieme. All rights reserved.)

Task-level goals such as maintaining standing balance are achieved through coordinated muscle activity. Consistent and individualized groupings of synchronously activated muscles can be estimated from muscle recordings in terms of motor modules or muscle synergies, independent of their temporal activation. The structure of motor modules can change with motor training, neurological disorders, and rehabilitation, but the central and peripheral mechanisms underlying motor module structure remain unclear. To assess the role of peripheral somatosensory input on motor module structure, we evaluated changes in the structure of motor modules for reactive balance recovery following pyridoxine-induced large-fiber peripheral somatosensory neuropathy in previously collected data in four adult cats. Somatosensory fiber loss, quantified by postmortem histology, varied from mild to severe across cats. Reactive balance recovery was assessed using multidirectional translational support-surface perturbations over days to weeks throughout initial impairment and subsequent recovery of balance ability. Motor modules within each cat were quantified by non-negative matrix factorization and compared in structure over time. All cats exhibited changes in the structure of motor modules for reactive balance recovery after somatosensory loss, providing evidence that somatosensory inputs influence motor module structure. The impact of the somatosensory disturbance on the structure of motor modules in well-trained adult cats indicates that somatosensory mechanisms contribute to motor module structure, and therefore may contribute to some of the pathological changes in motor module structure in neurological disorders. These results further suggest that somatosensory nerves could be targeted during rehabilitation to influence pathological motor modules for rehabilitation. NEW & NOTEWORTHY Stable motor modules for reactive balance recovery in well-trained adult cats were disrupted following pyridoxine-induced peripheral somatosensory neuropathy, suggesting somatosensory inputs contribute to motor module structure. Furthermore, the motor module structure continued to change as the animals regained the ability to maintain standing balance, but the modules generally did not recover pre-pyridoxine patterns. These results suggest changes in somatosensory input and subsequent learning may contribute to changes in motor module structure in pathological conditions.

Background/aims: We examined the effects of proton pump inhibitors (PPIs) on gastric antral ulcers induced by non-steroidal anti-inflammatory drugs in re-fed mice and the role of capsaicin-sensitive afferent nerves (CSANs) in the protective effects of PPIs on the antral mucosa., Methods: Male mice were administered indomethacin after 2 h of re-feeding of diet after a 24-h fast, and gastric lesions were examined 24 h after indomethacin dosing. The effects of PPIs (lansoprazole and omeprazole), histamine H 2 -receptor antagonists (H 2 -RAs, famotidine, ranitidine), capsaicin and misoprostol on the formation of antral ulcers induced by indomethacin were examined. Functional ablation of CSANs was caused by pretreatment of mice with a high dose of capsaicin., Results: Indomethacin produced lesions selectively in the gastric antrum in re-fed conditions. Formation of antral ulcers was not affected by H 2 -RAs, but inhibited by PPIs, capsaicin and misoprostol. The anti-ulcer effect of lansoprazole was 30 times stronger than that of omeprazole. Antral ulcers induced by indomethacin were markedly aggravated in mice with ablated CSANs. The effects of PPIs and capsaicin on ulcer formation were inhibited by ablation of CSANs, pretreatment with a capsaicin receptor antagonist (capsazepine/ruthenium red) and an inhibitor of nitric oxide synthesis (L-NAME). However, the inhibitory effect of misoprostol was not prevented by the ablation of CSANs or drugs., Conclusions: The results suggested that CSANs play an important role in protection of the antral mucosa and that both lansoprazole and omeprazole are capable of preventing NSAID-induced antral ulcers by activating CSANs.

The role of proprioceptive feedback on motor lateralization remains unclear. We asked whether motor lateralization is dependent on proprioceptive feedback by examining a rare case of proprioceptive deafferentation (GL). Motor lateralization is thought to arise from asymmetries in neural organization, particularly at the cortical level. For example, we have previously provided evidence that the left hemisphere mediates optimal motor control that allows execution of smooth and efficient arm trajectories, while the right hemisphere mediates impedance control that can achieve stable and accurate final arm postures. The role of proprioception in both of these processes has previously been demonstrated empirically, bringing into question whether loss of proprioception will disrupt lateralization of motor performance. In this study, we assessed whether the loss of online sensory information produces deficits in integrating specific control contributions from each hemisphere by using a reaching task to examine upper limb kinematics in GL and five age-matched controls. Behavioral findings revealed differential deficits in the control of the left and right hands in GL and performance deficits in each of GL's hands compared with controls. Computational simulations can explain the behavioral results as a disruption in the integration of postural and trajectory control mechanisms when no somatosensory information is available. This rare case of proprioceptive deafferentation provides insights into developing a more accurate understanding of handedness that emphasizes the role of proprioception in both predictive and feedback control mechanisms. NEW & NOTEWORTHY The role of proprioceptive feedback on the lateralization of motor control mechanisms is unclear. We examined upper limb kinematics in a rare case of peripheral deafferentation to determine the role of sensory information in integrating motor control mechanisms from each hemisphere. Our empirical findings and computational simulations showed that the loss of somatosensory information results in an impaired integration of control mechanisms, thus providing support for a complementary dominance hypothesis of handedness.

The transient receptor potential melastatin type 8 (TRPM8) receptor channel is expressed in primary afferent neurons where it is the main transducer of innocuous cold temperatures and also in a variety of tumors, where it is involved in progression and metastasis. Modulation of this channel by intracellular signaling pathways has therefore important clinical implications. We investigated the modulation of recombinant and natively expressed TRPM8 by the Src kinase, which is known to be involved in cancer pathophysiology and inflammation. Human TRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by Src which is expressed either heterologously or endogenously. Src action on TRPM8 potentiates its activity, as treatment with PP2, a selective Src kinase inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold-induced channel activation. RNA interference directed against the Src kinase diminished the extent of PP2-induced functional downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured rat dorsal root ganglion neurons, and this action was antagonized by the protein tyrosine phosphatase inhibitor pervanadate, confirming that TRPM8 activity is sensitive to the cellular balance between tyrosine kinases and phosphatases. This positive modulation of TRPM8 by Src kinase may be relevant for inflammatory pain and cancer signaling., (© 2019 Wiley Periodicals, Inc.)

Spared corticospinal tract (CST) and proprioceptive afferent (PA) axons sprout after injury and contribute to rewiring spinal circuits, affecting motor recovery. Loss of CST connections post-injury results in corticospinal signal loss and associated reduction in spinal activity. We investigated the role of activity loss and injury on CST and PA sprouting. To understand activity-dependence after injury, we compared CST and PA sprouting after motor cortex (MCX) inactivation, produced by chronic MCX muscimol microinfusion, with sprouting after a CST lesion produced by pyramidal tract section (PTx). Activity suppression, which does not produce a lesion, is sufficient to trigger CST axon outgrowth from the active side to cross the midline and to enter the inactivated side of the spinal cord, to the same extent as PTx. Activity loss was insufficient to drive significant CST gray matter axon elongation, an effect of PTx. Activity suppression triggered presynaptic site formation, but less than PTx. Activity loss triggered PA sprouting, as PTx. To understand injury-dependent sprouting further, we blocked microglial activation and associated inflammation after PTX by chronic minocycline administration after PTx. Minocycline inhibited myelin debris phagocytosis contralateral to PTx and abolished CST axon elongation, formation of presynaptic sites, and PA sprouting, but not CST axon outgrowth from the active side to cross the midline. Our findings suggest sprouting after injury has a strong activity dependence and that microglial activation after injury supports axonal elongation and presynaptic site formation. Combining spinal activity support and inflammation control is potentially more effective in promoting functional restoration than either alone., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Neonatal tissue injury disrupts the balance between primary afferent-evoked excitation and inhibition onto adult spinal projection neurons. However, whether this reflects cell-type-specific alterations at synapses onto ascending projection neurons, or rather is indicative of global changes in synaptic signaling across the mature superficial dorsal horn (SDH), remains unknown. Therefore the present study investigated the effects of neonatal surgical injury on primary afferent synaptic input to adult mouse SDH interneurons using in vitro patch-clamp techniques. Hindpaw incision at postnatal day (P)3 significantly diminished total primary afferent-evoked glutamatergic drive to adult Gad67-GFP and non-GFP neurons, and reduced their firing in response to sensory input, in both males and females. Early tissue damage also shaped the relative prevalence of monosynaptic A- versus C-fiber-mediated input to mature GABAergic neurons, with an increased prevalence of Aβ- and Aδ-fiber input observed in neonatally-incised mice compared with naive littermate controls. Paired presynaptic and postsynaptic stimulation at an interval that exclusively produced spike timing-dependent long-term potentiation (t-LTP) in projection neurons predominantly evoked NMDAR-dependent long-term depression in naive Gad67-GFP interneurons. Meanwhile, P3 tissue damage enhanced the likelihood of t-LTP generation at sensory synapses onto the mature GABAergic population, and increased the contribution of Ca 2+ -permeable AMPARs to the overall glutamatergic response. Collectively, the results indicate that neonatal injury suppresses sensory drive to multiple subpopulations of interneurons in the adult SDH, which likely represents one mechanism contributing to reduced feedforward inhibition of ascending projection neurons, and the priming of developing pain pathways, following early life trauma. SIGNIFICANCE STATEMENT Mounting clinical and preclinical evidence suggests that neonatal tissue damage can result in long-term changes in nociceptive processing within the CNS. Although recent work has demonstrated that early life injury weakens the ability of sensory afferents to evoke feedforward inhibition of adult spinal projection neurons, the underlying circuit mechanisms remain poorly understood. Here we demonstrate that neonatal surgical injury leads to persistent deficits in primary afferent drive to both GABAergic and presumed glutamatergic neurons in the mature superficial dorsal horn (SDH), and modifies activity-dependent plasticity at sensory synapses onto the GABAergic population. The functional denervation of spinal interneurons within the mature SDH may contribute to the "priming" of developing pain pathways following early life injury., (Copyright © 2019 the authors.)

As mitochondrial dysfunction is evident in neurodegenerative disorders that are accompanied by pain, we generated inducible mutant mice with disruption of mitochondrial respiratory chain complex IV, by COX10 deletion limited to sensory afferent neurons through the use of an Advillin Cre-reporter. COX10 deletion results in a selective energy-deficiency phenotype with minimal production of reactive oxygen species. Mutant mice showed reduced activity of mitochondrial respiratory chain complex IV in many sensory neurons, increased ADP/ATP ratios in dorsal root ganglia and dorsal spinal cord synaptoneurosomes, as well as impaired mitochondrial membrane potential, in these synaptoneurosome preparations. These changes were accompanied by marked pain hypersensitivity in mechanical and thermal (hot and cold) tests without altered motor function. To address the underlying basis, we measured Ca 2+ fluorescence responses of dorsal spinal cord synaptoneurosomes to activation of the GluK1 (kainate) receptor, which we showed to be widely expressed in small but not large nociceptive afferents, and is minimally expressed elsewhere in the spinal cord. Synaptoneurosomes from mutant mice showed greatly increased responses to GluK1 agonist. To explore whether altered nucleotide levels may play a part in this hypersensitivity, we pharmacologically interrogated potential roles of AMP-kinase and ADP-sensitive purinergic receptors. The ADP-sensitive P2Y 1 receptor was clearly implicated. Its expression in small nociceptive afferents was increased in mutants, whose in vivo pain hypersensitivity, in mechanical, thermal and cold tests, was reversed by a selective P2Y 1 antagonist. Energy depletion and ADP elevation in sensory afferents, due to mitochondrial respiratory chain complex IV deficiency, appear sufficient to induce pain hypersensitivity, by ADP activation of P2Y 1 receptors.

Immunohistochemical characterization of primary afferent fibers (intact or after nerve damage) is traditionally performed in thin sections from dorsal root ganglia (DRGs) or in teased fibers, as light scattering in whole-mounts compromises visualization. These procedures are time-consuming, require specific equipment and advanced experimental skills. Lipid-clearing techniques are increasing in popularity, but they have never been used for the peripheral nervous system. We established a modified, inexpensive clearing method based on lipid-removal protocols to make transparent peripheral nerve tissue (inCLARITY). We compared retrograde-labeling and free-floating immunostaining with cryo-sections. Confocal microscopy on whole-mount transparent DRGs showed neurons marked with retrograde tracers applied to experimental neuromas (Retrobeads, Fluoro-ruby, Fluoro-emerald, DiI, and Fluoro-gold). After immunostaining with calcitonin gene-related peptide (peptidergic) or isolectin IB4 (non-peptidergic), nociceptors were visualized. Immunostaining in transparent whole-mount nerves allows simultaneous evaluation of the axotomized branches containing the neuroma and neighboring intact branches as they can be mounted preserving their anatomical disposition and fiber integrity. The goal of our study was to optimize CLARITY for its application in peripheral nerve tissues. The protocol is compatible with the use of retrograde tracers and improves immunostaining outcomes when compared to classical cryo-sectioning, as lack of lipids maximizes antibody penetration within the tissue.

Background: Benign prostatic hyperplasia (BPH) is one of the major causes of lower urinary tract symptoms (LUTS), including storage LUTS such as urinary frequency and urgency. Recently, a growing number of clinical studies indicate that prostatic inflammation could be an important pathophysiological mechanism inducing storage LUTS in patients with BPH. Here we aimed to investigate whether nonbacterial prostatic inflammation in a rat model induced by intraprostatic formalin injection can lead to long-lasting bladder overactivity and changes in bladder afferent neuron excitability., Methods: Male Sprague-Dawley rats were divided into four groups (n = 12 each): normal control group, 1-week prostatic inflammation group, 4-week inflammation group, and 8-week inflammation group. Prostatic inflammation was induced by formalin (10%; 50 µL per lobe) injection into bilateral ventral lobes of the prostate. Voiding behavior was evaluated in metabolic cages for each group. Ventral lobes of the prostate and the bladder were then removed for hematoxylin and eosin (HE) staining to evaluate inflammation levels. Continuous cystometrograms (CMG) were recorded to measure intercontraction intervals (ICI) and voided volume per micturition. Whole-cell patch clamp recordings were performed on dissociated bladder afferent neurons labeled by fluorogold injected into the bladder wall, to examine the electrophysiological properties., Results: Results of metabolic cage measurements showed that formalin-treated rats exhibited significantly (P < 0.05) increases in micturition episodes/12 hours and decrease in voided volume per micturition at every time point post injection. Continuous CMG illustrated the significant ( P < 0.05) higher number of nonvoiding contractions per void and shorter ICI in formalin-treated rats compared with control rats. HE staining showed significant prostatic inflammation, which declined gradually, in prostate tissues of formalin-induced rats. In patch clamp recordings, capsaicin-sensitive bladder afferent neurons from rats with prostatic inflammation had significantly ( P < 0.05) lower thresholds for spike activation and a "multiple" firing pattern compared with control rats at every time point post injection., Conclusions: Formalin-induced prostatic inflammation can lead to long-lasting bladder overactivity in association with bladder afferent neuron hyperexcitability. This long-lasting model could be a useful tool for the study of inflammation-related aspects of male LUTS pathophysiology., (© 2019 Wiley Periodicals, Inc.)

The tooth has an unusual sensory system that converts external stimuli predominantly into pain, yet its sensory afferents in teeth demonstrate cytochemical properties of non-nociceptive neurons. This review summarizes the recent knowledge underlying this paradoxical nociception, with a focus on the ion channels involved in tooth pain. The expression of temperature-sensitive ion channels has been extensively investigated because thermal stimulation often evokes tooth pain. However, temperature-sensitive ion channels cannot explain the sudden intense tooth pain evoked by innocuous temperatures or light air puffs, leading to the hydrodynamic theory emphasizing the microfluidic movement within the dentinal tubules for detection by mechanosensitive ion channels. Several mechanosensitive ion channels expressed in dental sensory systems have been suggested as key players in the hydrodynamic theory, and TRPM7, which is abundant in the odontoblasts, and recently discovered PIEZO receptors are promising candidates. Several ligand-gated ion channels and voltage-gated ion channels expressed in dental primary afferent neurons have been discussed in relation to their potential contribution to tooth pain. In addition, in recent years, there has been growing interest in the potential sensory role of odontoblasts; thus, the expression of ion channels in odontoblasts and their potential relation to tooth pain is also reviewed.

Aims: To examine the effects of RQ-00434739, a novel selective TRPM8 antagonist, on deep body temperature (DBT) and normal bladder sensory function and overactivity and its associated facilitation of mechanosensitive primary bladder single-unit afferent activities (SAAs) induced by intravesical l-menthol or prostaglandin E2 (PGE2) instillation in rats., Main Methods: The effect of RQ-00434739 on DBT was evaluated using intravenous administration of RQ-00434739 (1 mg/kg) or its vehicle under urethane anaesthesia. Cystometry (CMG) was performed on conscious and freely moving rats. SAAs were measured from the left L6 dorsal root under urethane anaesthesia, and the fibers were grouped as Aδ- or C-fiber based on their conduction velocity. For both CMG and SAA measurements, after baseline recording with saline instillation, further recording was performed with intravesical l-menthol (6 mM) or PGE2 (60 μM) instillation after pretreatment with intravenous RQ-00434739 (1 mg/kg) or its vehicle., Key Findings: RQ-00434739 did not significantly affect DBT. In CMG measurements, RQ-00434739 administration increased mean voided volume. Both l-menthol and PGE2 instillation decreased mean voided volume following vehicle pretreatment, whereas such effects were not observed following RQ-00434739 pretreatment. In SAA measurements, either l-menthol or PGE2 instillations increased SAAs of C-fibers, but not SAAs of Aδ-fibers, in the presence of vehicle. RQ-00434739 pretreatment significantly inhibited the l-menthol- and PGE2-induced activation of C-fiber SAAs., Significance: The present results demonstrate that blockade of TRPM8 channels can inhibit the pathological activation of mechanosensitive C-fibers and suggest that RQ-00434739 may be a promising therapeutic drug candidate for bladder hypersensitive disorders without affecting DBT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

ATP in the suburothelial layer is released from the bladder urothelium by mechanical stimuli. ATP directly activates purinergic receptors that are expressed on primary bladder afferent neurons and induces the micturition reflex. Although ATP is also released to the bladder lumen from the bladder urothelium, the role of ATP in the bladder lumen is unknown. Recently, clinical studies have reported that urinary ATP levels are much higher in patients with an overactive bladder than healthy controls. These results suggest that ATP in the bladder lumen is also involved in the micturition reflex. In this study, we performed intravesical ATP instillation in the mouse bladder. We evaluated urinary function with novel reliable methods using improved cystometry and ultrasonography, which we previously established. We found that intravesical ATP instillation induced urinary frequency because of activation of bladder afferent nerves without inflammatory changes in the bladder or an increase in post-void residual urine. These results suggest that not only ATP in the suburothelial layer, but also ATP in the bladder lumen, are involved in enhancement of the micturition reflex., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Thyroid hypofunction during early development results in anatomical alterations in the cerebellum, cerebrum, hippocampus and other brain structures. The plastic organization of the nucleus basalis of Meynert (nBM) projections to the whiskers-related somatosensory (wS1) cortex in adolescent pups with maternal thyroid hypofunction and sensory deprivation was assessed through retrograde WGA-HRP labeling., Methods: Congenital hypothyroidism induced by adding PTU (25 ppm) to the drinking water from embryonic day 16 to postnatal day (PND) 60. Pregnant rats were divided to intact and congenital hypothyroid groups. In each group, the total whiskers of pups (4 of 8) were trimmed continuously from PND 0 to PND 60., Results: Following separately WGA-HRP injections into wS1, retrogradely labeled neurons were observed in nBM. The number of labeled neurons in nBM were higher in the congenital hypothyroid and whisker deprived groups compared to their controls (P < 0.05)., Conclusion: Based on our results both congenital hypothyroidism and sensory deprivation may disturb normal development of cortical circuits in of nBM afferents to the wS1 cortex., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The under-reporting of pain and atypical manifestations of painful syndromes within the elderly population have been well documented, however, the specific relationship between pain and aging remains ambiguous. Previous studies have reported degenerative changes in primary afferents with aging. In this study, we questioned whether there is any change in the density of primary afferent endings within the epidermis of aged animals. Rats were categorically assessed in 4 age groups, each representing a key developmental stage across their life span: juvenile (2 months), adult (7 months); aged (18 months), and senescent (24-26 months). The plantar hind paw skin was removed, post-fixed, cut, and immunostained for protein gene product 9.5 and type IV collagen. Rats in the adult aged groups had significantly increased epidermal nerve densities and total lengths of immunoreactive nerve fibers, compared with juvenile as well as senescent rats. However, the paw withdrawal thresholds to punctate mechanical stimulation progressively increased with age, and did not exhibit a clear relationship with epidermal innervation. We conclude a nonlinear, inverted-U shaped relationship between rat plantar epidermal nerve density with aging, which does not correlate with mechanically-induced paw withdrawal behaviors., Perspective: This article presents age-related decreased epidermal innervation in rat hind paw skin, which partly explains mechanisms underlying decreased pain sensitivity in aged subjects. The report may help clinicians to understand that any compromise of pain-sensing pathway can lead to under-reporting of pain, inadequate analgesia, and slower recovery from a painful condition., (Copyright © 2018 The American Pain Society. Published by Elsevier Inc. All rights reserved.)

Several studies have examined spinal reflex modulation during leg cycling in healthy and spinal cord injury (SCI) subjects. However, the effect of cutaneous plantar afferent input on spinal excitability during leg cycling after SCI has not been characterised. The aim of the study was to test the feasibility of using controlled leg cycling in combination with plantar cutaneous electrical stimulation (ES) cycling to assess lower limb spinal sensorimotor excitability in subjects with motor complete or incomplete SCI. Spinal sensorimotor excitability was estimated by measuring cutaneomuscular-conditioned soleus H-reflex activity. Reflex excitability was tested before and after a 10-min ES cycling session in 13 non-injured subjects, 6 subjects with motor incomplete SCI (iSCI) who had moderately impaired gait function, 4 subjects with motor iSCI who had severely impaired gait function, and 5 subjects with motor complete SCI (cSCI). No modulation of soleus H-reflex with plantar cutaneous stimuli was observed after either iSCI or cSCI when compared to non-injured subjects. However, after ES cycling, reflex excitability significantly increased in subjects with iSCI and moderately impaired gait function. ES cycling facilitated spinal sensorimotor excitability only in subjects with motor iSCI with residual gait function. Increased spinal excitability induced with a combination of exercise and afferent stimulation could be adopted with diagnostic and prognostic purposes to reveal the activity-based neurorehabilitation profile of individual subjects with motor iSCI., Trial Registration: ISRCTN 26172500 ; retrospectively registered on 15 July 2016 Graphical abstract ᅟ.

Acid-sensing ion channels (ASIC) are voltage-independent cationic channels that open in response to decrease in extracellular pH. Amongst different subtypes, ASIC3 has received much attention in joint inflammatory conditions including rheumatoid arthritis. There have been a number of studies showing that there is an increase in expression of ASIC3 on nerve afferents supplying joints in response to inflammatory stimulus. Accordingly, a number of selective as well as nonselective ASIC3 inhibitors have shown potential in attenuating pain and inflammation in animal models of rheumatoid arthritis. On the other hand, there have been studies showing that ASIC3 may exert protective effects in joint inflammation. ASIC -/- animals, without ASIC3 genes, exhibit more joint inflammation and destruction in comparison to ASIC +/+ animals. The present review discusses the dual nature of ASIC3 in joint inflammation with possible mechanisms.

Itch and pain are refractory symptoms of many ocular conditions. Ocular itch is generated mainly in the conjunctiva and is absent from the cornea. In contrast, most ocular pain arises from the cornea. However, the underlying mechanisms remain unknown. Using genetic axonal tracing approaches, we discover distinct sensory innervation patterns between the conjunctiva and cornea. Further genetic and functional analyses in rodent models show that a subset of conjunctival-selective sensory fibers marked by MrgprA3 expression, rather than corneal sensory fibers, mediates ocular itch. Importantly, the actions of both histamine and nonhistamine pruritogens converge onto this unique subset of conjunctiva sensory fibers and enable them to play a key role in mediating itch associated with allergic conjunctivitis. This is distinct from skin itch, in which discrete populations of sensory neurons cooperate to carry itch. Finally, we provide proof of concept that selective silencing of conjunctiva itch-sensing fibers by pruritogen-mediated entry of sodium channel blocker QX-314 is a feasible therapeutic strategy to treat ocular itch in mice. Itch-sensing fibers also innervate the human conjunctiva and allow pharmacological silencing using QX-314. Our results cast new light on the neural mechanisms of ocular itch and open a new avenue for developing therapeutic strategies.

Neuropathic pain is caused by peripheral nerve injury (PNI). One hallmark symptom is allodynia (pain caused by normally innocuous stimuli), but its mechanistic underpinning remains elusive. Notably, whether selective stimulation of non-nociceptive primary afferent Aβ fibers indeed evokes neuropathic pain-like sensory and emotional behaviors after PNI is unknown, because of the lack of tools to manipulate Aβ fiber function in awake, freely moving animals. In this study, we used a transgenic rat line that enables stimulation of non-nociceptive Aβ fibers by a light-activated channel (channelrhodopsin-2; ChR2). We found that illuminating light to the plantar skin of these rats with PNI elicited pain-like withdrawal behaviors that were resistant to morphine. Light illumination to the skin of PNI rats increased the number of spinal dorsal horn (SDH) Lamina I neurons positive to activity markers (c-Fos and phosphorylated extracellular signal-regulated protein kinase; pERK). Whole-cell recording revealed that optogenetic Aβ fiber stimulation after PNI caused excitation of Lamina I neurons, which were normally silent by this stimulation. Moreover, illuminating the hindpaw of PNI rats resulted in activation of central amygdaloid neurons and produced an aversion to illumination. Thus, these findings provide the first evidence that optogenetic activation of primary afferent Aβ fibers in PNI rats produces excitation of Lamina I neurons and neuropathic pain-like behaviors that were resistant to morphine treatment. This approach may provide a new path for investigating circuits and behaviors of Aβ fiber-mediated neuropathic allodynia with sensory and emotional aspects after PNI and for discovering novel drugs to treat neuropathic pain.

Despite the relatively high prevalence of migraine or headache, the pathophysiological mechanisms triggering headache-associated peripheral hypersensitivities, are unknown. Since nitric oxide (NO) is well known as a causative factor in the pathogenesis of migraine or migraine-associated hypersensitivities, a mouse model has been established using systemic administration of the NO donor, nitroglycerin (NTG). Here we tried to investigate the time course development of facial or hindpaw hypersensitivity after repetitive NTG injection. NTG (10 mg/kg) was administrated to mice every other day for nine days. Two hours post-injection, NTG produced acute mechanical and heat hypersensitivity in the hind paws. By contrast, cold allodynia, but not mechanical hypersensitivity, occurred in the facial region. Moreover, this hindpaws mechanical hypersensitivity and the facial cold allodynia was progressive and long-lasting. We subsequently examined whether the depletion of capsaicin-sensitive primary afferents (CSPAs) with resiniferatoxin (RTX, 0.02 mg/kg) altered these peripheral hypersensitivities in NTG-treated mice. RTX pretreatment did not affect the NTG-induced mechanical allodynia in the hind paws nor the cold allodynia in the facial region, but it did inhibit the development of hind paw heat hyperalgesia. Similarly, NTG injection produced significant hindpaw mechanical allodynia or facial cold allodynia, but not heat hyperalgesia in transient receptor potential type V1 (TRPV1) knockout mice. These findings demonstrate that different peripheral hypersensitivities develop in the face versus hindpaw regions in a mouse model of repetitive NTG-induced migraine, and that these hindpaw mechanical hypersensitivity and facial cold allodynia are not mediated by the activation of CSPAs.

Chemotherapeutic drugs such as paclitaxel cause painful peripheral neuropathy in many cancer patients and survivors. Although NMDA receptors (NMDARs) at primary afferent terminals are known to be critically involved in chemotherapy-induced chronic pain, the upstream signaling mechanism that leads to presynaptic NMDAR activation is unclear. Group I metabotropic glutamate receptors (mGluRs) play a role in synaptic plasticity and NMDAR regulation. Here we report that the Group I mGluR agonist ( S )-3,5-dihydroxyphenylglycine (DHPG) significantly increased the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of monosynaptic EPSCs evoked from the dorsal root. DHPG also reduced the paired-pulse ratio of evoked EPSCs in spinal dorsal horn neurons. These effects were blocked by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), but not by an mGluR1 antagonist. MPEP normalized the frequency of miniature EPSCs and the amplitude of evoked EPSCs in paclitaxel-treated rats but had no effect in vehicle-treated rats. Furthermore, mGluR5 protein levels in the dorsal root ganglion and spinal cord synaptosomes were significantly higher in paclitaxel- than in vehicle-treated rats. Inhibiting protein kinase C (PKC) or blocking NMDARs abolished DHPG-induced increases in the miniature EPSC frequency of spinal dorsal horn neurons in vehicle- and paclitaxel-treated rats. Moreover, intrathecal administration of MPEP reversed pain hypersensitivity caused by paclitaxel treatment. Our findings suggest that paclitaxel-induced painful neuropathy is associated with increased presynaptic mGluR5 activity at the spinal cord level, which serves as upstream signaling for PKC-mediated tonic activation of NMDARs. mGluR5 is therefore a promising target for reducing chemotherapy-induced neuropathic pain., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Amyotrophic Lateral Sclerosis (ALS) is a fast progressive neurodegenerative disease characterized by muscle denervation, weakening and atrophy, which eventually culminates into death, mainly due to respiratory failure. The traditional view of ALS as a disorder affecting selectively motor neurons throughout the central nervous system has been progressively dispelled by innumerous lines of evidence indicating that other cells but motor neurons may be affected as well. Remarkably, this disorder is not limited to the motor system but rather configures as a systemic disease yielding a plethora of clinical signs. Among this broad clinical spectrum, sensory neuropathy occurring parallel to motor dysfunction is a quite frequent feature within ALS patients, which has spurred the interest of many investigators during the years. In line with this, morphological studies have confirmed that sensory neurons and axons' degeneration may occur in both ALS- experimental models and -patients. Noteworthy, this may have a nonetheless negligible role in ALS -related motor decline, as highlighted by recent studies showing that, degeneration of type I/II proprioceptive fibers is a primary source of alpha-motor neurons' death. These latter in fact, differently from gamma motor neurons, are a direct monosynaptic target of proprioceptive fibers. The present findings contribute to define a novel scenario of sensorimotor ALS pathophysiology where the gamma loop's fine connectivity may play a key role. In support to this view, in the present manuscript we provide a reappraisal on the role of single gamma loop's components in ALS.

Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) suffer from chronic pain that severely affects quality of life. Although the underlying pathophysiology is not well understood, inhibition of bladder sensory afferents temporarily relieves pain. Here, we explored the possibility that optogenetic inhibition of nociceptive sensory afferents could be used to modulate bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the sensory neuron-specific sodium channel (sns) gene to selectively silence these neurons. Optically silencing nociceptive sensory afferents significantly blunted the evoked visceromotor response to bladder distension and led to small but significant changes in bladder function. To study of the role of nociceptive sensory afferents in freely behaving mice, we developed a fully implantable, flexible, wirelessly powered optoelectronic system for the long-term manipulation of bladder afferent expressed opsins. We found that optogenetic inhibition of nociceptive sensory afferents reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naïve mice. These results suggest that selective optogenetic silencing of nociceptive bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain.

Chemotherapeutic drugs, including bortezomib, often cause painful peripheral neuropathy, which is a severe dose-limiting adverse effect experienced by many cancer patients. The glutamate N-methyl-d-aspartate receptors (NMDARs) at the spinal cord level are critically involved in the synaptic plasticity associated with neuropathic pain. In this study, we determined whether treatment with bortezomib, a proteasome inhibitor, affects the NMDAR activity of spinal dorsal horn neurons. Systemic treatment with bortezomib in rats did not significantly affect postsynaptic NMDAR currents elicited by puff application of NMDA directly to dorsal horn neurons. Bortezomib treatment markedly increased the baseline frequency of miniature excitatory postsynaptic currents (EPSCs), which was completely normalized by the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). AP5 also reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation in bortezomib-treated, but not vehicle-treated, rats. Furthermore, inhibition of protein kinase C (PKC) with chelerythrine fully reversed the increased frequency of miniature EPSCs and the amplitude of evoked EPSCs in bortezomib-treated rats. Intrathecal injection of AP5 and chelerythrine both profoundly attenuated mechanical allodynia and hyperalgesia induced by systemic treatment with bortezomib. In addition, treatment with bortezomib induced striking membrane translocation of PKC-βII, PKC-δ, and PKC-ε in the dorsal root ganglion. Our findings indicate that bortezomib treatment potentiates nociceptive input from primary afferent nerves via PKC-mediated tonic activation of presynaptic NMDARs. Targeting presynaptic NMDARs and PKC at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Intestinal parasites alter gastrointestinal (GI) functions like the cholinergic function. Aspiculuris tetraptera is a pinworm frequently observed in laboratory facilities, which infests the mice cecum and proximal colon. However, little is known about the impact of this infection on the GI sensitivity. Here, we investigated possible changes in spontaneous mesenteric nerve activity and on the mechanosensitivity function of worm-free regions of naturally infected mice with A. tetraptera. Infection increased the basal firing of mesenteric afferent nerves in jejunum. Our findings indicate that nicotinic but not muscarinic receptors, similarly affect spontaneous nerve firing in control and infected animals; these axons are mainly vagal. No difference between groups was observed on spontaneous activity after nicotinic receptor inhibition. However, and contrary to the control group, during infection, the muscarinic signaling was shown to be elevated during mechanosensory experiments. In conclusion, we showed for the first time that alterations induced by infection of the basal afferent activity were independent of the cholinergic function but changes in mechanosensitivity were mediated by muscarinic, but not nicotinic, receptors and specifically by high threshold nerve fibers (activated above 20mmHg), known to play a role in nociception. These plastic changes within the muscarinic signaling would function as a compensatory mechanism to maintain a full mechanosensory response and the excitability of nociceptors during infection. These changes indicate that pinworm colonic infection can target other tissues away from the colon., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Early postnatal damage to the mediodorsal thalamus (MD) produces deficits in cognition and behavior believed to be associated with early prefrontal cortical maldevelopment. We assessed the role of MD afferents during development on the morphological and functional maturation of the prefrontal cortex (PFC) and the basolateral amygdala (BLA). Sprague-Dawley rat pups (n = 56) received a bilateral electrolytic lesion of the MD or a MD Sham lesion on postnatal day 4. 7 weeks later, all rats were tested in anxiety-related and cognitive paradigms using the elevated plus maze and novel object recognition tests. Following behavioral testing (P70), rats were killed and the baseline expression of C-Fos protein and the number of GABAergic neurons were evaluated in the PFC and the BLA. The dendritic morphology and spine density in the PFC using Golgi-Cox staining was also evaluated. Adult rats with early postnatal bilateral MD damage exhibited disrupted recognition memory and increased anxiety-like behaviors. The lesion also caused a significant diminution of C-Fos immunolabeling and an increase of the number of GABAergic neurons in the PFC. In the BLA, the number of GABAergic neurons was significantly reduced, associated with an increase in C-Fos immunolabeling. Furthermore, in the PFC the lesion induced a significant reduction in dendritic branching and spine density. Our data are consistent with the hypothesis that the MD plays a role in the development of the PFC and, therefore, may be a good animal model to investigate cognitive symptoms associated with schizophrenia.

Opioids are the gold standard for pharmacological treatment of neuropathic pain, but their analgesic effects are unsatisfactory in part due to nerve injury-induced downregulation of opioid receptors in dorsal root ganglia (DRG) neurons. How nerve injury drives such downregulation remains elusive. DNA methyltransferase (DNMT)-triggered DNA methylation represses gene expression. We show here that blocking the nerve injury-induced increase in DRG DNMT3a (a de novo DNMT) rescued the expression of Oprm1 and Oprk1 mRNAs and their respective encoding mu-opioid receptor (MOR) and kappa-opioid receptor (KOR) proteins in the injured DRG. Blocking this increase also prevented the nerve injury-induced increase in DNA methylation in the promoter and 5'-untranslated region of the Oprm1 gene in the injured DRG, restored morphine or loperamide (a peripheral acting MOR preferring agonist) analgesic effects, and attenuated the development of their analgesic tolerance under neuropathic pain conditions. Mimicking this increase reduced the expression of Oprm1 and Oprk1 mRNAs and their coding MOR and KOR in DRG and augmented MOR-gated neurotransmitter release from the primary afferents. Mechanistically, DNMT3a regulation of Oprm1 gene expression required the methyl-CpG-binding protein 1, MBD1, as MBD1 knockout resulted in the decreased binding of DNMT3a to the Oprm1 gene promoter and blocked the DNMT3a-triggered repression of Oprm1 gene expression in DRG neurons. These data suggest that DNMT3a is required for nerve injury-induced and MBD1-mediated epigenetic silencing of the MOR and KOR in the injured DRG. DNMT3a inhibition may serve as a promising adjuvant therapy for opioid use in neuropathic pain management.

The impact of aging on proprioceptive sensory neurons and intrafusal muscle fibers (IMFs) remains largely unexplored despite the central function these cells play in modulating voluntary movements. Here, we show that proprioceptive sensory neurons undergo deleterious morphological changes in middle age (11- to 13-month-old) and old (15- to 21-month-old) mice. In the extensor digitorum longus and soleus muscles of middle age and old mice, there is a significant increase in the number of Ia afferents with large swellings that fail to properly wrap around IMFs compared with young adult (2- to 4-month-old) mice. Fewer II afferents were also found in the same muscles of middle age and old mice. Although these age-related changes in peripheral nerve endings were accompanied by degeneration of proprioceptive sensory neuron cell bodies in dorsal root ganglia (DRG), the morphology and number of IMFs remained unchanged. Our analysis also revealed normal levels of neurotrophin 3 (NT3) but dysregulated expression of the tyrosine kinase receptor C (TrkC) in aged muscles and DRGs, respectively. These results show that proprioceptive sensory neurons degenerate prior to atrophy of IMFs during aging, and in the presence of the NT3/TrkC signaling axis., (© The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic voiding disorder that presents with pain in the urinary bladder and surrounding pelvic region. A growing body of evidence suggests that an increase in the permeability of the urothelium, the epithelial barrier that lines the interior of the bladder, contributes to the symptoms of IC/BPS. To examine the consequence of increased urothelial permeability on pelvic pain and afferent excitability, we overexpressed in the urothelium claudin 2 (Cldn2), a tight junction (TJ)-associated protein whose message is significantly upregulated in biopsies of IC/BPS patients. Consistent with the presence of bladder-derived pain, rats overexpressing Cldn2 showed hypersensitivity to von Frey filaments applied to the pelvic region. Overexpression of Cldn2 increased the expression of c-Fos and promoted the activation of ERK1/2 in spinal cord segments receiving bladder input, which we conceive is the result of noxious stimulation of afferent pathways. To determine whether the mechanical allodynia observed in rats with reduced urothelial barrier function results from altered afferent activity, we examined the firing of acutely isolated bladder sensory neurons. In patch-clamp recordings, about 30% of the bladder sensory neurons from rats transduced with Cldn2, but not controls transduced with GFP, displayed spontaneous activity. Furthermore, bladder sensory neurons with tetrodotoxin-sensitive (TTX-S) action potentials from rats transduced with Cldn2 showed hyperexcitability in response to suprathreshold electrical stimulation. These findings suggest that as a result of a leaky urothelium, the diffusion of urinary solutes through the urothelial barrier sensitizes bladders afferents, promoting voiding at low filling volumes and pain.

Cannabinoid system is a potential target for pain control. Cannabinoid receptor 1 (CB 1 ) activation play a role in the analgesic effect of cannabinoids once it is expressed in primary afferent neurons. This study investigates whether the anti-hyperalgesic effect of CB 1 receptor activation involves P2X 3 receptor in primary afferent neurons. Mechanical hyperalgesia was evaluated by electronic von Frey test. Cannabinoid effect was evaluated using anandamide or ACEA, a non-selective or a selective CB 1 receptor agonists, respectively; AM251, a CB 1 receptor antagonist, and antisense ODN for CB 1 receptor. Calcium imaging assay was performed to evaluated α,β-meATP-responsive cultured DRG neurons pretreated with ACEA. Anandamide or ACEA administered in peripheral tissue reduced the carrageenan-induced mechanical hyperalgesia. The reduction in the carrageenan-induced hyperalgesia induced by ACEA was completely reversed by administration of AM251 as well as by the intrathecal treatment with antisense ODN for CB 1 receptor. Also, ACEA reduced the mechanical hyperalgesia induced by bradykinin and by α,β-meATP, a P2X 3 receptor non-selective agonist, but not by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and chemokine-induced chemoattractant-1 (CINC-1). Finally, CB 1 receptors are co-localized with P2X 3 receptors in DRG small-diameter neurons and the treatment with ACEA reduced the number of α,β-meATP-responsive cultured DRG neurons. Our data suggest that the analgesic effect of CB 1 receptor activation is mediated by a negative modulation of the P2X 3 receptor in the primary afferent neurons., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Axon regeneration in the central nervous system is limited both by inhibitory extracellular cues and by an intrinsically low capacity for axon growth in some CNS populations. Chondroitin sulfate proteoglycans (CSPGs) are well-studied inhibitors of axon growth in the CNS, and degradation of CSPGs by chondroitinase has been shown to improve the extension of injured axons. Alternatively, axon growth can be improved by targeting the neuron-intrinsic growth capacity through forced expression of regeneration-associated transcription factors. For example, a transcriptionally active chimera of Krüppel-like Factor 7 (KLF7) and a VP16 domain improves axon growth when expressed in corticospinal tract neurons. Here we tested the hypothesis that combined expression of chondroitinase and VP16-KLF7 would lead to further improvements in axon growth after spinal injury. Chondroitinase was expressed by viral transduction of cells in the spinal cord, while VP16-KLF7 was virally expressed in sensory neurons of the dorsal root ganglia or corticospinal tract (CST) neurons. After transection of the dorsal columns, both chondroitinase and VP16-KLF7 increased the proximity of severed sensory axons to the injury site. Similarly, after complete crush injuries, VP16-KLF7 expression increased the approach of CST axons to the injury site. In neither paradigm however, did single or combined treatment with chondroitinase or VP16-KLF7 enable regenerative growth distal to the injury. These results substantiate a role for CSPG inhibition and low KLF7 activity in determining the net retraction of axons from sites of spinal injury, while suggesting that additional factors act to limit a full regenerative response., (Copyright © 2016. Published by Elsevier Inc.)

Orofacial pain is associated with peripheral and central sensitization of trigeminal nociceptive neurons. Nerve injury results in release of chemical mediators that contribute to persistent pain conditions. The activation of the transient receptor potential vanilloid 1 (TRPV1), promotes release of calcitonin gene-related peptide (CGRP) and substance P (SP) from trigeminal nerve terminals. CGRP and SP contribute to the development of peripheral hyperalgesia. The expression of SP and CGRP by primary afferent neurons is rapidly increased in response to peripheral inflammation. CGRP receptor activation promotes activation of AMPA receptors, leading to increased firing of neurons which is reflected as central sensitization. In this study we investigated whether inferior alveolar nerve (IAN) injury influences AMPA receptors, CGRP, SP and TRPV1 expression in the trigeminal ganglion (TG). The relative expression of the protein of interest from naive rats was compared to those from injured rats and animals that received low level laser therapy (LLLT). IAN-injury did not change expression of GluA1, GluA2 and CGRP, but increased the expression of TRPV1 and SP. LLLT increases GluA1 and GluA2 expression and decreases TVPV1, SP and CGRP. These results, together with previous behavioral data, suggest that IAN-injury induced changes in the proteins analyzed, which could impact on nociceptive threshold. These data may help to understand the molecular mechanisms of pain sensitization in the TG.

Peripheral nerve injury and tissue inflammation result in upregulation of the deubiquitinase USP5, thus causing a dysregulation of T-type calcium channel activity and increased pain sensitivity. Here, we have explored the role of afferent fiber activity in this process. Conditioning stimulation of optogenetically targeted cutaneous TRPV1 expressing nociceptors, but not that of non-nociceptive fibers, resulted in enhanced expression of USP5 in mouse dorsal root ganglia and spinal dorsal horn, along with decreased withdrawal thresholds for thermal and mechanical stimuli that abated after 24 hr. This sensitization was drastically reduced by an interfering peptide that prevented USP5-Cav3.2 association. Sensitization was relieved by pharmacological block of TRPV1 afferents, but not of myelinated neurons. In spinal cord slice recordings, we could optogenetically trigger an activity-dependent potentiation of presynaptic neurotransmission in the spinal dorsal horn that relied on Cav3.2 channel activity. This neuronal-activity-induced USP5 upregulation may underlie a protective, transient sensitization of the pain pathway., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Tension-type headache and other primary headaches may be triggered or aggravated by disorders of pericranial muscles, which is possibly due to convergent or collateral afferent input from meningeal and muscular receptive areas. In rodent models high extracellular concentrations of ATP caused muscle nociception and central sensitization of second order neurons. In a rat model of meningeal nociception we asked if spinal trigeminal activity induced by ATP can be modulated by local anaesthesia of distinct muscles., Methods: Ongoing activity was recorded from spinal trigeminal neurons with afferent input from the cranial dura mater, the temporal muscle and neck muscles. The stable ATP analogue α,β-methylene adenosine 5'-triphosphate (α,β-meATP, 10 mM) was injected into the ipsilateral temporal muscle, 30 min later followed by injection of local anaesthetics (lidocaine, 2 %) into the ipsilateral neck muscles and/or the temporal muscle., Results: Injection of α,β-meATP into the temporal muscle caused progressive increase in ongoing activity of most of the spinal trigeminal neurons within 30 min. Injection of lidocaine into the neck muscles and/or the temporal muscle reduced this activation to previous levels within 10 min., Conclusions: Distinct spinal trigeminal neurons processing meningeal nociceptive information are under the control of convergent afferent input from several pericranial muscles. Blockade of at least one of these inputs can normalize central trigeminal activity. This may explain why therapeutic manipulations of head muscles can be beneficial in primary headaches.

Inflammatory pain is thought to arise from increased transmission from nociceptors and recruitment of 'silent' afferents. To evaluate inflammation-induced changes, mice expressing GCaMP3 in cutaneous sensory neurons were generated and neuronal responses to mechanical stimulation in vivo before and after subcutaneous infusion of an 'inflammatory soup' (IS) were imaged in an unanesthetized preparation. Infusion of IS rapidly altered mechanical responsiveness in the majority of neurons. Surprisingly, more cells lost, rather than gained, sensitivity and 'silent' afferents that were mechanically insensitive and gained mechanosensitivity after IS exposure were rare. However, the number of formerly 'silent' afferents that became mechanosensitive was increased five fold when the skin was heated briefly prior to infusion of IS. These findings suggest that pain arising from inflamed skin reflects a dramatic shift in the balance of sensory input, where gains and losses in neuronal populations results in novel output that is ultimately interpreted by the CNS as pain., Competing Interests: The authors declare that no competing interests exist.

Cocaine- and amphetamine-regulated transcript (CART) is one of the most abundant neuropeptides in vagal afferents, including those involved in regulating feeding. Recent observations indicate that metabolic challenges dramatically alter the neuropeptidergic profile of CART-producing vagal afferents. Here, using confocal microscopy, we reassessed the distribution and regulation of CART(55-102) immunoreactivity in vagal afferents of the male mouse in response to metabolic challenges, including fasting and high-fat-diet feeding. Importantly, the perikarya and axons of vagal C-fibers were labeled using mice expressing channelrodhopsin-2 (ChR2-YFP) in Na v 1.8-Cre-expressing neurons. In these mice, approximately 82% of the nodose ganglion neurons were labeled with ChR2-YFP. Furthermore, ChR2-YFP-labeled axons could easily be identified in the dorsovagal complex. CART(55-102) immunoreactivity was observed in 55% of the ChR2-YFP-labeled neurons in the nodose ganglion and 22% of the ChR2-YFP-labeled varicosities within the area postrema of fed, fasted, and obese mice. The distribution of positive profiles was also identical across the full range of CART staining in fed, fasted, and obese mice. In contrast to previous studies, fasting did not induce melanin-concentrating hormone (MCH) immunoreactivity in vagal afferents. Moreover, prepro-MCH mRNA was undetectable in the nodose ganglion of fasted mice. In summary, this study showed that the perikarya and central terminals of vagal afferents are invariably enriched in CART and devoid of MCH.

Netrin-1 is a neuronal guidance molecule implicated in the development of spinal cord neurons and cortical neurons. In the adult spinal cord, UNC5H (repulsive receptor of netrin-1), but not deleted in colorectal cancer (DCC) (attractive receptor of netrin-1), constitutes a major mode of netrin-1 signal transduction, which may be involved in axon repulsion and inhibits neurite outgrowth. Abnormal sprouting of myelinated afferent fibers in the spinal dorsal horn can cause mechanical allodynia associated with postherpetic neuralgia (PHN, Shingles) and other neuropathic pains. However, whether netrin-1 participates in sprouting of myelinated afferent fibers and mechanical allodynia remains unknown. In an ultropotent TRPV1 agonist resiniferatoxin (RTX)-induced PHN-like model, RTX treatment for 6 weeks increased netrin-1 expression in dorsal horn neurons, including NK-1-positive projection neurons. In human neuroblastoma SH-SY5Y cells, we found that TRPV1 antagonist capsazepine antagonized RTX-induced upregulation of netrin-1. After RTX treatment, UNC5H2 expression was gradually decreased, whereas DCC expression was significantly increased. Silencing netrin-1 in the spinal dorsal horn significantly attenuated RTX-induced mechanical allodynia and sprouting of myelinated fibers into the spinal lamina II. Our results suggest that RTX treatment upregulates netrin-1 expression through activation of TRPV1 receptors and change UNC5H2-rich spinal dorsal horn into a growth-permissive environment by increasing DCC expression, thus enhancing the sprouting of myelinated afferent nerves. Netrin-1 may be targeted for reducing primary afferent sprouting and mechanical allodynia in PHN and other neuropathic pain conditions.

Damage to inner ear afferent terminals is believed to result in many auditory and vestibular dysfunctions. The sequence of afferent injuries and repair, as well as their correlation with vertigo symptoms, remains poorly documented. In particular, information on the changes that take place at the primary vestibular endings during the first hours following a selective insult is lacking. In the present study, we combined histological analysis with behavioral assessments of vestibular function in a rat model of unilateral vestibular excitotoxic insult. Excitotoxicity resulted in an immediate but transient alteration of the balance function that was resolved within a week. Concomitantly, vestibular primary afferents underwent a sequence of structural changes followed by spontaneous repair. Within the first two hours after the insult, a first phase of pronounced vestibular dysfunction coincided with extensive swelling of afferent terminals. In the next 24 h, a second phase of significant but incomplete reduction of the vestibular dysfunction was accompanied by a resorption of swollen terminals and fiber retraction. Eventually, within 1 week, a third phase of complete balance restoration occurred. The slow and progressive withdrawal of the balance dysfunction correlated with full reconstitution of nerve terminals. Competitive re-innervation by afferent and efferent terminals that mimicked developmental synaptogenesis resulted in full re-afferentation of the sensory epithelia. By deciphering the sequence of structural alterations that occur in the vestibule during selective excitotoxic impairment, this study offers new understanding of how a vestibular insult develops in the vestibule and how it governs the heterogeneity of vertigo symptoms., Competing Interests: A.S., A. Broussy, A. Brugeaud and S.G.-N. work for Sensorion, a biopharmaceutical company committed to finding treatments for inner ear disease. C.C. and S.G-N. have shares in the company., (© 2016. Published by The Company of Biologists Ltd.)

Background: Prostatic inflammation is a common histologic finding in men with lower urinary tract symptoms (LUTS). It has been postulated that prostatic inflammation could sensitize afferent neurons innervating the bladder and thereby produce changes in voiding behavior. In support of this, we demonstrate an anatomic basis for pelvic cross-talk involving the prostate and bladder., Methods: Retrograde labeling was performed by an application of a neuro-tracer Fast Blue (FB) to one side of either the anterior prostate (AP), dorsal lateral prostate (DLP)/ventral prostate (VP), bladder, or seminal vesicle (SV)., Results: Examination of dorsal root ganglion (DRG) neuron labeling revealed shared afferent innervation of the prostate and bladder at spinal segments of T13, L1, L2, L6, and S1. Dual labeling was performed by an application of FB and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyaine perchlorate (DiI) to the AP and bladder, respectively. We observed double-labeled DRG neurons at T13, L1, L2, L6, and S1--a finding that proves convergent innervation of prostate and bladder., Conclusions: Our observations demonstrate the potential for neural cross-talk between the prostate and bladder and support a postulated mechanism that prostatic inflammation may induce hyper-sensitization of bladder afferents and produce irritative LUTS., (© 2016 Wiley Periodicals, Inc.)