Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons.

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.

Nerve Growth Factor and Pain Mechanisms.

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.

Practical approaches to improving translatability and reproducibility in preclinical pain research.

Pain research continues to face the challenge of poor translatability of pre-clinical studies. In this short primer, we are summarizing the possible causes, with an emphasis on practical and constructive solutions. In particular, we stress the importance of increased heterogeneity in animal studies; formal or informal pre-registration to combat publication bias; and increased statistical training in order to help pre-clinical scientists appreciate the usefulness of available experimental design and reporting guidelines.

Changes in the transcriptional fingerprint of satellite glial cells following peripheral nerve injury.

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.

Neuroimmune interactions in chronic pain - An interdisciplinary perspective.

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.

HDAC4 is required for inflammation-associated thermal hypersensitivity.

Transcriptional alterations are characteristic of persistent pain states, but the key regulators remain elusive. HDAC4 is a transcriptional corepressor that has been linked to synaptic plasticity and neuronal excitability, mechanisms that may be involved in peripheral and central sensitization. Using a conditional knockout (cKO) strategy in mice, we sought to determine whether the loss of HDAC4 would have implications for sensory neuron transcription and nociception. HDAC4 was found to be largely unnecessary for transcriptional regulation of naïve sensory neurons but was essential for appropriate transcriptional responses after injury, with Calca and Trpv1 expression consistently down-regulated in HDAC4 cKO compared to levels in the littermate controls (0.2-0.44-fold change, n = 4 in 2 separate experiments). This down-regulation corresponded to reduced sensitivity to 100 nM capsaicin in vitro (IC50 = 230 ± 20 nM, 76 ± 4.4% wild-type capsaicin responders vs. 56.9 ± 4.7% HDAC4 cKO responders) and to reduced thermal hypersensitivity in the complete Freund's adjuvant (CFA) model of inflammatory pain (1.3-1.4-fold improvement over wild-type controls; n = 5-12, in 2 separate experiments). These data indicate that HDAC4 is a novel inflammatory pain mediator and may be a good therapeutic target, capable of orchestrating the regulation of multiple downstream effectors.

In vivo calcium imaging shows that satellite glial cells have increased activity in painful states.

Satellite glial cells are important for proper neuronal function of primary sensory neurons for which they provide homeostatic support. Most research on satellite glial cell function has been performed with in vitro studies, but recent advances in calcium imaging and transgenic mouse models have enabled this first in vivo study of single-cell satellite glial cell function in mouse models of inflammation and neuropathic pain. We found that in naïve conditions, satellite glial cells do not respond in a time-locked fashion to neuronal firing. In painful inflammatory and neuropathic states, we detected time-locked signals in a subset of satellite glial cells, but only with suprathreshold stimulation of the sciatic nerve. Surprisingly, therefore, we conclude that most calcium signals in satellite glial cells seem to develop at arbitrary intervals not directly linked to neuronal activity patterns. More in line with expectations, our experiments also revealed that the number of active satellite glial cells was increased under conditions of inflammation or nerve injury. This could reflect the increased requirement for homeostatic support across dorsal root ganglion neuron populations, which are more active during such painful states.

Spontaneous activity in peripheral sensory nerves: a systematic review.

ABSTRACT: In the peripheral nervous system, spontaneous activity in sensory neurons is considered to be one of the 2 main drivers of chronic pain states, alongside neuronal sensitization. Despite this, the precise nature and timing of this spontaneous activity in neuropathic pain is not well-established. Here, we have performed a systematic search and data extraction of existing electrophysiological literature to shed light on which fibre types have been shown to maintain spontaneous activity and over what time frame. We examined both in vivo recordings of preclinical models of neuropathic pain, as well as microneurography recordings in humans. Our analyses reveal that there is broad agreement on the presence of spontaneous activity in neuropathic pain conditions, even months after injury or years after onset of neuropathic symptoms in humans. However, because of the highly specialised nature of the electrophysiological methods used to measure spontaneous activity, there is also a high degree of variability and uncertainty around these results. Specifically, there are very few directly controlled experiments, with less directly comparable data between human and animals. Given that spontaneous peripheral neuron activity is considered to be a key mechanistic feature of chronic pain conditions, it may be beneficial to conduct further experiments in this space.

A randomised controlled trial of the effect of intra-articular lidocaine on pain scores in inflammatory arthritis.

ABSTRACT: Chronic pain in inflammatory arthritis (IA) reflects a complex interplay between active disease in a peripheral joint and central pronociceptive mechanisms. Because intra-articular lidocaine may be used to abolish joint-specific peripheral input to the central nervous system, we aimed to validate its use as a clinical tool to identify those patients with IA whose pain likely incorporates centrally mediated mechanisms. We began by investigating whether there was a placebo response of intra-articular injection in patients with IA 1:1 randomised to receive intra-articular lidocaine or control (0.9% saline). After, in a larger patient cohort not randomized to placebo vs lidocaine groups, we tested whether patients with IA could be stratified into 2 cohorts based on their response to intra-articular lidocaine according to markers of centrally mediated pain. To this end, we evaluated postlidocaine pain numerical rating scale (NRS) scores alongside baseline painDETECT, fibromyalgia criteria fulfillment, and quantitative sensory testing outcomes. Numerical rating scale scores were collected at baseline and 3-, 5-, and 10-minutes postinjection. Firstly, the placebo effect of intra-articular injection was low: compared to baseline, the mean pain NRS score 5-minutes postinjection was reduced by 3.5 points in the lidocaine group vs 1.2 points in the control group. Secondly, postlidocaine NRS scores were significantly higher in those with a high (>18) baseline painDETECT score, fibromyalgia, and low-pressure pain threshold at the trapezius (P = 0.002, P = 0.001, P = 0.005, respectively). Persistent high pain after intra-articular lidocaine injection could be used as an indicator of pronociceptive mechanisms that are centrally mediated, informing centrally targeted analgesic strategies.

Assessing spontaneous sensory neuron activity using in vivo calcium imaging.

ABSTRACT: Heightened spontaneous activity in sensory neurons is often reported in individuals living with chronic pain. It is possible to study this activity in rodents using electrophysiology, but these experiments require great skill and can be prone to bias. Here, we have examined whether in vivo calcium imaging with GCaMP6s can be used as an alternative approach. We show that spontaneously active calcium transients can be visualised in the fourth lumbar dorsal root ganglion (L4 DRG) through in vivo imaging in a mouse model of inflammatory pain. Application of lidocaine to the nerve, between the inflamed site and the DRG, silenced spontaneous firing and revealed the true baseline level of calcium for spontaneously active neurons. We used these data to train a machine learning algorithm to predict when a neuron is spontaneously active. We show that our algorithm is accurate in 2 different models of pain: intraplantar complete Freund adjuvant and antigen-induced arthritis, with accuracies of 90.0% ±1.2 and 85.9% ±2.1, respectively, assessed against visual inspection by an experienced observer. The algorithm can also detect neuronal activity in imaging experiments generated in a different laboratory using a different microscope configuration (accuracy = 94.0% ±2.2). We conclude that in vivo calcium imaging can be used to assess spontaneous activity in sensory neurons and provide a Google Colaboratory Notebook to allow anyone easy access to our novel analysis tool, for the assessment of spontaneous neuronal activity in their own imaging setups.

Cerebrospinal fluid immune cells appear similar across neuropathic and non-neuropathic pain conditions.

Background: Microglia have been implicated in the pathophysiology of neuropathic pain. Here, we sought to investigate whether cerebrospinal fluid (CSF) might be used as a proxy-measure of microglial activation in human participants. Methods: We preformed fluorescence-activated cell sorting (FACS) of CSF immune cell populations derived from individuals who experienced pain with neuropathic features. We sorted CD4+, CD8+ T cells and monocytes and analyzed their transcriptome using RNA sequencing. We also performed Cellular Indexing of Transcriptomes and Epitopes (CITE) sequencing to characterize the expression of all CSF immune cells in a patient with postherpetic neuralgia and in a patient with neuropathic pain after failed back surgery. Results: Immune cell numbers and phenotypes were not obviously different between individuals regardless of the etiology of their pain. This was true when examining our own dataset, as well as when comparing it to previously published single-cell RNA sequencing data of human CSF. In all instances, CSF monocytes showed expression of myeloid cell markers commonly associated with microglia ( P2RY12, TMEM119 and OLFML3), which will make it difficult to ascertain the origin of CSF proteins: do they derive directly from circulating CSF monocytes or could some originate in spinal cord microglia in the parenchyma? Conclusions: We conclude that it will not be straightforward to use CSF as a biomarker for microglial function in humans.

Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons.

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify 3 clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 & ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.

Tau protein is required for amyloid {beta}-induced impairment of hippocampal long-term potentiation.

Amyloid ß (Aß) 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 Aß-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 Aß-induced impairment of LTP. Moreover, we show that Aß increases tau phosphorylation and that a specific inhibitor of the tau kinase glycogen synthase kinase 3 blocks the increased tau phosphorylation induced by Aß and prevents Aß-induced impairment of LTP in wild-type mice. Together, these findings show that tau protein is required for Aß to impair synaptic plasticity in the hippocampus and suggest that the Aß-induced impairment of LTP is mediated by tau phosphorylation. We conclude that preventing the interaction between Aß and tau could be a promising strategy for treating cognitive impairment in MCI and early AD.

Fibroblasts: the neglected cell type in peripheral sensitisation and chronic pain? A review based on a systematic search of the literature.

Chronic pain and its underlying biological mechanisms have been studied for many decades, with a myriad of molecules, receptors and cell types known to contribute to abnormal pain sensations. Besides an obvious role for neurons, immune cells like microglia, macrophages and T cells are also important drivers of persistent pain. While neuroinflammation has therefore been widely studied in pain research, there is one cell type that appears to be rather neglected in this context: the humble fibroblast. Fibroblasts may seem unassuming but actually play a major part in regulating immune cell function and driving chronic inflammation. Here, our aim was to determine the breadth and quality of research that implicates fibroblasts in chronic pain conditions and models. OBJECTIVES: We set out to analyse the current literature on this topic-using systematic screening and data extraction methods to obtain a balanced view on what has been published. METHODS: We categorised the articles we included-stratifying them according to what was investigated, the estimated quality of results and any common conclusions. RESULTS: We found that there has been surprisingly little research in this area: 134 articles met our inclusion criteria, only a tiny minority of which directly investigated interactions between fibroblasts and peripheral neurons. CONCLUSIONS: Fibroblasts are a ubiquitous cell type and a prominent source of many proalgesic mediators in a wide variety of tissues. We think that they deserve a more central role in pain research and propose a new, testable model of how fibroblasts might drive peripheral neuron sensitisation.

Examination of the contribution of Nav1.7 to axonal propagation in nociceptors.

ABSTRACT: Nav1.7 is a promising drug target for the treatment of pain. However, there is a mismatch between the analgesia produced by Nav1.7 loss-of-function and the peripherally restricted Nav1.7 inhibitors, which may reflect a lack of understanding of the function of Nav1.7 in the transmission of nociceptive information. In the periphery, the role of Nav1.7 in transduction at nociceptive peripheral terminals has been comprehensively examined, but its role in axonal propagation in these neurons is less clearly defined. In this study, we examined the contribution of Nav1.7 to axonal propagation in nociceptors using sodium channel blockers in in vivo electrophysiological and calcium imaging recordings in mice. Using the sodium channel blocker tetrodotoxin (TTX) (1-10 µM) to inhibit Nav1.7 and other tetrodotoxin-sensitive sodium channels along the sciatic nerve, we first showed that around two-thirds of nociceptive L4 dorsal root ganglion neurons innervating the skin, but a lower proportion innervating the muscle (45%), are blocked by TTX. By contrast, nearly all large-sized cutaneous afferents (95%-100%) were blocked by axonal TTX. Many cutaneous nociceptors resistant to TTX were polymodal (57%) and capsaicin sensitive (57%). Next, we applied PF-05198007 (300 nM-1 µM) to the sciatic nerve between stimulating and recording sites to selectively block axonal Nav1.7 channels. One hundred to three hundred nanomolar PF-05198007 blocked propagation in 63% of C-fiber sensory neurons, whereas similar concentrations produced minimal block (5%) in rapidly conducting A-fiber neurons. We conclude that Nav1.7 is essential for axonal propagation in around two-thirds of nociceptive cutaneous C-fiber neurons and a lower proportion (≤45%) of nociceptive neurons innervating muscle.

Comparative transcriptional analysis of satellite glial cell injury response.

Background: Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNA sequencing (scRNAseq) technologies has enabled a new approach to investigate SGCs. Methods: In this study we used scRNAseq to investigate SGCs from mice subjected to sciatic nerve injury. We used a meta-analysis approach to compare the injury response with that found in other published datasets.  Furthermore, we also used scRNAseq to investigate how cells from the dorsal root ganglion (DRG) change after 3 days in culture. Results: From our meta-analysis of the injured conditions, we find that SGCs share a common signature of 18 regulated genes following sciatic nerve crush or sciatic nerve ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state while others start resembling Schwann cell-like precursors. Conclusion: By using integrated analyses of new and previously published scRNAseq datasets, this study provides a consensus view of which genes are most robustly changed in SGCs after injury. Our results are available via the Broad Institute Single Cell Portal, so that readers can explore and search for genes of interest.

Early life inflammation is associated with spinal cord excitability and nociceptive sensitivity in human infants.

Immune function and sensitivity to pain are closely related, but the association between early life inflammation and sensory nervous system development is poorly understood-especially in humans. Here, in term-born infants, we measure brain activity and reflex withdrawal activity (using EEG and EMG) and behavioural and physiological activity (using the PIPP-R score) to assess the impact of suspected early-onset neonatal infection on tactile- and noxious-evoked responses. We present evidence that neonatal inflammation (assessed by measuring C-reactive protein levels) is associated with increased spinal cord excitability and evoked brain activity following both tactile and noxious stimulation. There are early indications that this hyperalgesia could be maintained post-inflammation, supporting pre-clinical reports of early-life immune dysfunction influencing pain sensitivity in adults.

TRPC5 and the path towards analgesic drug development.

A recent study by Sadler et al. highlights transient receptor potential canonical 5 (TRPC5) as a potential target for treating pain conditions. This article discusses their findings in the context of analgesic drug development, an urgent pursuit required to combat the opioid crisis and help millions of people with chronic pain.

Probing the peripheral immune response in mouse models of oxaliplatin-induced peripheral neuropathy highlights their limited translatability.

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of various chemotherapeutic agents, including oxaliplatin. It is highly prevalent amongst cancer patients, causing sensory abnormalities and pain. Unfortunately, as the underlying mechanisms remain poorly understood, effective therapeutics are lacking. Neuro-immune interactions have been highlighted as potential contributors to the development and maintenance of CIPN, however, whether this is the case in oxaliplatin-induced peripheral neuropathy (OIPN) is yet to be fully established. Methods: In this study we used flow cytometry to examine the peripheral immune response of male C57BL/6 mice following both single and repeated oxaliplatin administration. In animals exposed to repeated dosing, we also undertook mechanical and thermal behavioural assays to investigate how oxaliplatin alters phenotype, and conducted RT-qPCR experiments on bone marrow derived macrophages in order to further inspect the effects of oxaliplatin on immune cells. Results: In contrast to other reports, we failed to observe substantial changes in overall leukocyte, lymphocyte or myeloid cell numbers in dorsal root ganglia, sciatic nerves or inguinal lymph nodes. We did however note subtle, tissue-dependant alterations in several myeloid subpopulations following repeated dosing. These included a significant reduction in MHCII antigen presenting cells in the sciatic nerve and an increase in infiltrating cell types into the inguinal lymph nodes. Though repeated oxaliplatin administration had a systemic effect, we were unable to detect a pain-like behavioural phenotype in response to either cold or mechanical stimuli. Consequently, we cannot comment on whether the observed myeloid changes are associated with OIPN. Conclusions: Our discussion puts these results into the wider context of the field, advocating for greater transparency in reporting, alignment in experimental design and the introduction of more clinically relevant models. Only through joint concerted effort can we hope to increase our understanding of the underlying mechanisms of CIPN, including any immune contributions.