Search

Satellite glial cells (SGCs) are homeostatic cells enveloping the somata of peripheral sensory and autonomic neurons. A wide variety of neuronal stressors trigger activation of SGCs, contributing to, for example, neuropathic pain through modulation of neuronal activity. However, compared to neurons and other glial cells of the nervous system, SGCs have received modest scientific attention and very little is known about SGC biology, possibly due to the experimental challenges associated with studying them in vivo and in vitro. Utilizing a recently developed method to obtain SGC RNA from dorsal root ganglia (DRG), we took a systematic approach to characterize the SGC transcriptional fingerprint by using next-generation sequencing and, for the first time, obtain an overview of the SGC injury response. Our RNA sequencing data are easily accessible in supporting information in Excel format. They reveal that SGCs are enriched in genes related to the immune system and cell-to-cell communication. Analysis of SGC transcriptional changes in a nerve injury-paradigm reveal a differential response at 3 days versus 14 days postinjury, suggesting dynamic modulation of SGC function over time. Significant downregulation of several genes linked to cholesterol synthesis was observed at both time points. In contrast, regulation of gene clusters linked to the immune system (MHC protein complex and leukocyte migration) was mainly observed after 14 days. Finally, we demonstrate that, after nerve injury, macrophages are in closer physical proximity to both small and large DRG neurons, and that previously reported injury-induced proliferation of SGCs may, in fact, be proliferating macrophages.

Nav1.7 is a promising drug target for the treatment of pain because individuals with Nav1.7 loss-of-function mutations are insensitive to pain and do not have other serious neurological deficits. However, current peripherally restricted Nav1.7 inhibitors have not performed well in clinical pain trials, which may reflect a lack of understanding of the function of Nav1.7 in the transmission of nociceptive information. Although numerous studies have reported that Nav1.7 has a moderate role in peripheral transduction, the precise contribution of Nav1.7 to axonal propagation in nociceptors is not clearly defined, particularly for afferents innervating deep structures.In this study, we examined the contribution of Nav1.7 to axonal propagation in nociceptors utilising sodium channel blockers in in vivo electrophysiological and calcium imaging recordings from L4 in the mouse. Using the sodium channel blocker TTX (1-10μM) to inhibit Nav1.7 and other TTX-S sodium channels along the sciatic nerve, we first showed that around 2/3rds of nociceptive neurons innervating the skin, but a lower proportion innervating the muscle (45%), are blocked by TTX. In contrast, nearly all large-sized A-fibre cutaneous afferents (95-100%) were blocked by axonal TTX. Characterisation of TTX resistant cutaneous nociceptors revealed that many were polymodal (57%) and capsaicin sensitive (57%).Next, we examined the role of Nav1.7 in axonal propagation in nociceptive neurons by applying the selective channel blocker PF-05198007 (300nM-1μM) to the sciatic nerve between stimulating and recording sites. 100-300nM PF-05198007 blocked propagation in 63% of C-fibre sensory neurons, whereas similar concentrations did not affect propagation in rapidly conducting A-fibre neurons. We conclude that Nav1.7 has an essential contribution to axonal propagation in only around 2/3rds of nociceptive C-fibre neurons, and a lower proportion (≤45%) of nociceptive neurons innervating muscle.

Supplemental Digital Content is Available in the Text. Fibroblast-like synoviocytes (FLS) interact with articular sensory nerve endings. Coculture of tumor necrosis factor (TNF-α)-stimulated FLS and knee-innervating sensory neurons reveals FLS-mediated neuronal sensitization., Pain is a principal contributor to the global burden of arthritis with peripheral sensitization being a major cause of arthritis-related pain. Within the knee joint, distal endings of dorsal root ganglion neurons (knee neurons) interact with fibroblast-like synoviocytes (FLS) and the inflammatory mediators they secrete, which are thought to promote peripheral sensitization. Correspondingly, RNA sequencing has demonstrated detectable levels of proinflammatory genes in FLS derived from arthritis patients. This study confirms that stimulation with tumor necrosis factor (TNF-α) results in expression of proinflammatory genes in mouse and human FLS (derived from osteoarthritis and rheumatoid arthritis patients), as well as increased secretion of cytokines from mouse TNF-α-stimulated FLS (TNF-FLS). Electrophysiological recordings from retrograde labelled knee neurons cocultured with TNF-FLS, or supernatant derived from TNF-FLS, revealed a depolarized resting membrane potential, increased spontaneous action potential firing, and enhanced TRPV1 function, all consistent with a role for FLS in mediating the sensitization of pain-sensing nerves in arthritis. Therefore, data from this study demonstrate the ability of FLS activated by TNF-α to promote neuronal sensitization, results that highlight the importance of both nonneuronal and neuronal cells to the development of pain in arthritis.

Nerve growth factor (NGF) antagonism is on the verge of becoming a powerful analgesic treatment for numerous conditions, including osteoarthritis and lower back pain. This review summarizes the historical research, both fundamental and clinical, that led to our current understanding of NGF biology. We also discuss the surprising number of questions that remain about NGF expression patterns and NGF's various functions and interaction partners in relation to persistent pain and the potential side effects of anti-NGF therapy.

Correct communication between immune cells and peripheral neurons is crucial for the protection of our bodies. Its breakdown is observed in many common, often painful conditions, including arthritis, neuropathies and inflammatory bowel or bladder disease. Here, we have characterised the immune response in a mouse model of neuropathic pain using flow cytometry and cell-type specific RNA sequencing (RNA-seq). We found few striking sex differences, but a very persistent inflammatory response, with increased numbers of monocytes and macrophages up to 3½ months after the initial injury. This raises the question of whether the commonly used categorisation of pain into “inflammatory” and “neuropathic” is one that is mechanistically appropriate. Finally, we collated our data with other published RNA-seq datasets on neurons, macrophages and Schwann cells in naïve and nerve injury states. The result is a practical web-based tool for the transcriptional data-mining of peripheral neuroimmune interactions.http://rna-seq-browser.herokuapp.com/

It is widely accepted that communication between the nervous and immune systems is involved in the development of chronic pain. At each level of the nervous system, immune cells have been reported to accompany and frequently mediate dysfunction of nociceptive circuitry; however the exact mechanisms are not fully understood. One way to speed up progress in this area is to increase interdisciplinary cross-talk. This review sets out to summarize what pain research has already learnt, or indeed might still learn, from examining peripheral and central nociceptive mechanisms using tools and perspectives from other fields like immunology, inflammation biology or the study of stress.

Recent studies have suggested a sexually dimorphic role of spinal glial cells in the maintenance of mechanical hypersensitivity in rodent models of chronic pain. We have used the collagen antibody-induced arthritis (CAIA) mouse model to examine differences between males and females in the context of spinal regulation of arthritis-induced pain. We have focused on the late phase of this model when joint inflammation has resolved but mechanical hypersensitivity persists. While the intensity of substance P, CGRP and galanin immunoreactivity in the spinal cord was not different from controls, the intensity of microglia (Iba-1) and astrocyte (GFAP) markers was elevated in both males and females. Intrathecal administration of the glial inhibitors minocycline and pentoxifylline reversed mechanical thresholds in male, but not female mice. We isolated resident microglia from the lumbar dorsal horns and observed a significantly lower number of microglial cells in females by flow cytometry analysis. However, while genome-wide RNA sequencing results pointed to several transcriptional differences between male and female microglia, no convincing differences were identified between control and CAIA groups.Taken together, these findings suggest that there are subtle sex differences in microglial expression profiles independent of arthritis. Our experiments failed to identify the underlying mRNA correlates of microglial actions in the late phase of the CAIA model. It is likely that transcriptional changes are either subtle and highly localised and therefore difficult to identify with bulk isolation techniques or that other factors, such as changes in protein expression or epigenetic modifications are at play.This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The work cannot be changed in any way or used commercially without permission from the journal.

Supplemental Digital Content is Available in the Text. In vivo GCaMP6s imaging of hind-limb sensory neurons revealed that the number of mechanically responsive sensory neurons triples in bone cancer conditions., Skeletal metastases are frequently accompanied by chronic pain that is mechanoceptive in nature. Mechanistically, cancer-induced bone pain (CIBP) is mediated by peripheral sensory neurons innervating the cancerous site, the cell bodies of which are housed in the dorsal root ganglia (DRG). How these somatosensory neurons encode sensory information in CIBP remains only partly explained. Using a validated rat model, we first confirmed cortical bone destruction in CIBP but not sham-operated rats (day 14 after surgery, designated “late”-stage bone cancer). This occurred with behavioural mechanical hypersensitivity (Kruskal–Wallis H for independent samples; CIBP vs sham-operated, day 14; P < 0.0001). Next, hypothesising that the proportion and phenotype of primary afferents would be altered in the disease state, dorsal root ganglia in vivo imaging of genetically encoded calcium indicators and Markov Cluster Analysis were used to analyse 1748 late-stage CIBP (n = 10) and 757 sham-operated (n = 9), neurons. Distinct clusters of responses to peripheral stimuli were revealed. In CIBP rats, upon knee compression of the leg ipsilateral to the tumour, (1) 3 times as many sensory afferents responded (repeated-measures analysis of variance: P < 0.0001 [vs sham]); (2) there were significantly more small neurons responding (Kruskal–Wallis for independent samples (vs sham): P < 0.0001); and (3) approximately 13% of traced tibial cavity afferents responded (no difference observed between CIBP and sham-operated animals). We conclude that an increased sensory afferent response is present in CIBP rats, and this is likely to reflect afferent recruitment from outside of the bone rather than increased intraosseous afferent activity.

Supplemental Digital Content is Available in the Text. Mast cells do not play a role as a direct nociceptor sensitiser during acute inflammatory pain and in mechanical hypersensitivity triggered by local nerve growth factor., The immune and sensory systems are known for their close proximity and interaction. Indeed, in a variety of pain states, a myriad of different immune cells are activated and recruited, playing a key role in neuronal sensitisation. During inflammatory pain it is thought that mast cells (MC) are one of the immune cell types involved in this process, but so far the evidence outlining their direct effect on neuronal cells remains unclear. To clarify whether MC are involved in inflammatory pain states, we used a transgenic mouse line (Mctp5Cre-iDTR) in which MC could be depleted in an inducible manner by administration of diphtheria toxin. Our results show that ablation of MC in male mice did not result in any change in mechanical and thermal hypersensitivity in the CFA model of inflammatory pain. Similarly, edema and temperature triggered by CFA inflammation at the injection site remained identical in MC depleted mice compared with their littermate controls. In addition, we show that Mctp5Cre-iDTR mice display normal levels of mechanical hypersensitivity after local injection of nerve growth factor (NGF), a factor well characterised to produce peripheral sensitisation and for being upregulated upon injury and inflammation. We also demonstrate that NGF treatment in vitro does not lead to an increased level of tumor necrosis factor-α in bone marrow-derived MC. Furthermore, our qRT-PCR data reveal that MC express negligible levels of NGF receptors, thereby explaining the lack of response to NGF. Together, our data suggest that MC do not play a direct role in peripheral sensitisation during inflammatory conditions.

Supplemental Digital Content is Available in the Text. Deletion of the potassium channel Kcns1 from peripheral sensory neurons alters acute and neuropathic pain processing. Compounds that enhance Kcns1 activity may provide analgesia., Voltage-gated potassium (Kv) channels are increasingly recognised as key regulators of nociceptive excitability. Kcns1 is one of the first potassium channels to be associated with neuronal hyperexcitability and mechanical sensitivity in the rat, as well as pain intensity and risk of developing chronic pain in humans. Here, we show that in mice, Kcns1 is predominantly expressed in the cell body and axons of myelinated sensory neurons positive for neurofilament-200, including Aδ-fiber nociceptors and low-threshold Aβ mechanoreceptors. In the spinal cord, Kcns1 was detected in laminae III to V of the dorsal horn where most sensory A fibers terminate, as well as large motoneurons of the ventral horn. To investigate Kcns1 function specifically in the periphery, we generated transgenic mice in which the gene is deleted in all sensory neurons but retained in the central nervous system. Kcns1 ablation resulted in a modest increase in basal mechanical pain, with no change in thermal pain processing. After neuropathic injury, Kcns1 KO mice exhibited exaggerated mechanical pain responses and hypersensitivity to both noxious and innocuous cold, consistent with increased A-fiber activity. Interestingly, Kcns1 deletion also improved locomotor performance in the rotarod test, indicative of augmented proprioceptive signalling. Our results suggest that restoring Kcns1 function in the periphery may be of some use in ameliorating mechanical and cold pain in chronic states.

Promoter-based genetic recombination (via, e.g., Cre-lox) is most useful when all cells of interest express a particular gene. The discovery that the actin-binding protein advillin is expressed in all somatic sensory neurons has been exploited repeatedly to drive DNA recombination therein, yet specificity of expression has not been well demonstrated. Here, we characterize advillin expression amongst sensory neurons and in several other neural and non-neural tissues. We first validate an advillin antibody against advillin knock-out tissue, advillin promoter-driven EGFP, and advillin mRNA expression. In the dorsal root ganglion (DRG), advillin is enriched in non-peptidergic nociceptors. We also show that advillin expression, and advillin promotor-driven EGFP and Cre-recombinase expression, occurs in multiple tissues including the dorsal habenula of the epithalamus, endocrine cells of the gut, Merkel cells in the skin, and most strikingly, throughout the autonomic nervous system (sympathetic, parasympathetic, and enteric neurons) in mice, rats, and non-human primates. In the mouse pelvic ganglion, advillin immunoreactivity is most intense in pairs of small neurons, and concentrated in spine-like structures on the axon initial segment contacted by sympathetic preganglionic axons. In autonomic targets (iris and blood vessels), advillin is distributed along cholinergic parasympathetic axons and in sympathetic varicosities. Developmentally, advillin expression is absent from sympathetics at postnatal day 4 but begins to emerge by day 7, accounting for previous reports (based on embryonic expression) of advillin’s specificity to sensory neurons. These results indicate that caution is warranted in interpreting previous studies in which advillin-driven genomic editing is either constitutive or performed after postnatal day 4.

Genome editing techniques have facilitated significant advances in our understanding of fundamental biological processes, and the Cre-Lox system has been instrumental in these achievements. Driving Cre expression specifically in injured neurons has not been previously possible: we sought to address this limitation in mice using a Cre-ERT2 construct driven by a reliable indicator of axotomy, activating transcription factor 3 (ATF3). When crossed with reporter mice, a significant amount of recombination was achieved (without tamoxifen treatment) in peripherally-projecting sensory, sympathetic, and motoneurons after peripheral nerve crush in hemizygotes (65–80% by 16 d) and was absent in uninjured neurons. Importantly, injury-induced recombination did not occur in Schwann cells distal to the injury, and with a knock-out-validated antibody we verified an absence of ATF3 expression. Functional recovery following sciatic nerve crush in ATF3-deficient mice (both hemizygotes and homozygotes) was delayed, indicating previously unreported haploinsufficiency. In a proof-of-principle experiment, we crossed the ATF3-CreERT2 line with a floxed phosphatase and tensin homolog (PTEN) line and show significantly improved axonal regeneration, as well as more complete recovery of neuromuscular function. We also demonstrate the utility of the ATF3-CreERT2 hemizygous line by characterizing recombination after lateral spinal hemisection (C8/T1), which identified specific populations of ascending spinal cord neurons (including putative spinothalamic and spinocerebellar) and descending supraspinal neurons (rubrospinal, vestibulospinal, reticulospinal and hypothalamic). We anticipate these mice will be valuable in distinguishing axotomized from uninjured neurons of several different classes (e.g., via reporter expression), and in probing the function of any number of genes as they relate to neuronal injury and regeneration.

The microtubule-associated protein tau is implicated in various neurodegenerative diseases including Alzheimer’s disease, progressive supranuclear palsy and corticobasal degeneration, which are characterized by intracellular accumulation of hyperphosphorylated tau. Mutations in the tau gene MAPT cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In the human central nervous system, six tau isoforms are expressed, and imbalances in tau isoform ratios are associated with pathology. To date, few animal models of tauopathy allow for the potential influence of these protein isoforms, relying instead on cDNA-based transgene expression. Using the P1-derived artificial chromosome (PAC) technology, we created mouse lines expressing all six tau isoforms from the human MAPT locus, harbouring either the wild-type sequence or the disease-associated N296H mutation on an endogenous Mapt−/− background. Animals expressing N296H mutant tau recapitulated early key features of tauopathic disease, including a tau isoform imbalance and tau hyperphosphorylation in the absence of somatodendritic tau inclusions. Furthermore, N296H animals displayed behavioural anomalies such as hyperactivity, increased time in the open arms of the elevated plus maze and increased immobility during the tail suspension test. The mouse models described provide an excellent model to study the function of wild-type or mutant tau in a highly physiological setting.

Summary:Chronic pain is a common and devastating condition that induces well-characterized changes in neurons and microglia. One major unanswered question is why these changes should persist long after the precipitating injury has healed. Here, we suggest that some of the longer-lasting consequences of nerve injury may be hidden in the epigenome. Cell sorting and sequencing techniques were used to characterize the spinal cord immune response in a mouse model of chronic neuropathic pain. Infiltration of peripheral myeloid cells was found to be absent, and RNA sequencing (RNA-seq) of central microglia revealed transient gene expression changes in response to nerve ligation. Conversely, examination of microglial enhancers revealed persistent, post-injury alterations in close proximity to transcriptionally regulated genes. Enhancers are regions of open chromatin that define a cell’s transcription factor binding profile. We hypothesize that changes at enhancers may constitute a mechanism by which painful experiences are recorded at a molecular level.

There are many known risk factors for chronic pain conditions, yet the biological underpinnings that link these factors to abnormal processing of painful signals are only just beginning to be explored. This Review will discuss the potential mechanisms that have been proposed to underlie vulnerability and resilience toward developing chronic pain. Particular focus will be given to genetic and epigenetic processes, priming effects on a cellular level, and alterations in brain networks concerned with reward, motivation/learning and descending modulatory control. Although research in this area is still in its infancy, a better understanding of how pain vulnerability emerges has the potential to help identify individuals at risk and may open up new therapeutic avenues.

Summary Intrathecal delivery of histone deacetylase inhibitors ameliorates hypersensitivity in models of neuropathic pain. This effect may be mediated at the level of the spinal cord through inhibition of HDAC1 function., Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug–induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.

Amyloid β (Aβ) and tau protein are both implicated in memory impairment, mild cognitive impairment (MCI), and early Alzheimer's disease (AD), but whether and how they interact is unknown. Consequently, we asked whether tau protein is required for the robust phenomenon of Aβ-induced impairment of hippocampal long-term potentiation (LTP), a widely accepted cellular model of memory. We used wild-type mice and mice with a genetic knock-out of tau protein and recorded field potentials in an acute slice preparation. We demonstrate that the absence of tau protein prevents Aβ-induced impairment of LTP. Moreover, we show that Aβ increases tau phosphorylation and that a specific inhibitor of the tau kinase glycogen synthase kinase 3 blocks the increased tau phosphorylation induced by Aβ and prevents Aβ-induced impairment of LTP in wild-type mice. Together, these findings show that tau protein is required for Aβ to impair synaptic plasticity in the hippocampus and suggest that the Aβ-induced impairment of LTP is mediated by tau phosphorylation. We conclude that preventing the interaction between Aβ and tau could be a promising strategy for treating cognitive impairment in MCI and early AD.

Tauopathies, characterized by the dysfunction and aggregation of the microtubule-associated protein tau (MAPT), represent some of the most devastating neurodegenerative disorders afflicting the elderly, including Alzheimer's disease and progressive supranuclear palsy. Here we review the range of Mapt knock-out and MAPT transgenic mouse models which have proven successful at providing insights into the molecular mechanisms of neurodegenerative disease. In this overview we highlight several themes, including the insights such models provide into the cellular and molecular mechanisms of tauopathy, the direct relationship between neuropathology and behaviour, and the use of mouse models to help provide a platform for testing novel therapies. Mouse models have helped clarify the relationship between pathological forms of tau, cell death, and the emergence of disease, as well as the interaction between tau and other disease-associated molecules, such as the A beta peptide. Finally, we discuss potential future MAPT genomic DNA models to investigate the importance of alternative splicing of the MAPT locus and its role in sporadic tauopathies.

RATIONALE: Although tasks assessing the role of dopamine in effort-reward decisions are similar to those concerned with the role of serotonin in impulsive choice in that both require analysis of the costs and benefits of possible actions, they have never been directly compared. OBJECTIVES: This study investigated the involvement of serotonin and dopamine in two cost-benefit paradigms, one in which the cost was delay and the other in which it was physical effort. METHODS: Sixteen rats were trained on a T-maze task in which they chose between high and low reward arms. In one version, the high reward arm was obstructed by a barrier, in the other, delivery of the high reward was delayed by 15 s. Serotonin and dopamine function were manipulated using systemic pCPA and haloperidol injections, respectively. RESULTS: Haloperidol-treated rats were less inclined either to exert more effort or to countenance a delay for a higher reward. pCPA had no effect on the performance of the rats on the effortful task, but significantly increased the rats' preference for an immediate but smaller reward. All animals (drug treated and controls) chose the high reward arm on the majority of trials when the delay or effort costs were matched in both high and low reward arms. CONCLUSION: A dissociation was found between the neurotransmitter systems involved in different types of cost-benefit decision making. While dopaminergic systems were required for decisions about both effort and delay, serotonergic systems were only needed for the latter.

Background Tamoxifen (TAM) is an important cancer therapeutic and an experimental tool for effecting genetic recombination using the inducible Cre-Lox technique. Despite its widespread use in the clinic and laboratory, we know little about its effects on the nervous system. This is of significant concern because TAM, via unknown mechanisms, induces cognitive impairment in humans. A hallmark of cellular stress is induction of Activating Transcription Factor 3 (Atf3), and so to determine whether TAM induces cellular stress in the adult nervous system, we generated a knock-in mouse in which Atf3 promoter activity drives transcription of TAM-dependent Cre recombinase (Cre-ERT2); when crossed with tdtomato reporter mice, Atf3 induction results in robust and permanent genetic labeling of cells in which it is up-regulated even transiently. Results We found that granular neurons of the olfactory bulb and dentate gyrus, vascular cells and ependymal cells throughout the brain, and peripheral sensory neurons expressed tdtomato in response to TAM treatment. We also show that TAM induced Atf3 up-regulation through inhibition of cholesterol epoxide hydrolase (ChEH): reporter expression was mitigated by delivery in vitamin E-rich wheat germ oil (vitamin E depletes ChEH substrates), and was partially mimicked by a ChEH-specific inhibitor. Conclusions This work demonstrates that TAM stresses cells of the adult central and peripheral nervous systems and highlights concerns about clinical and experimental use of TAM. We propose TAM administration in vitamin E-rich vehicles such as wheat germ oil as a simple remedy.