Dorsal neural tube-derived retinoic acid promotes the end of neural crest production and transition into a definitive roof plate. Here we analyze how this impacts the segregation of central and peripheral lineages, a process essential for tissue patterning and function. Localized in-ovo inhibition in quail embryos of retinoic acid activity followed by single cell transcriptomics unraveled a comprehensive list of differentially expressed genes relevant to these processes. Importantly, progenitors co-expressed neural crest, roof plate and dI1 interneuron markers indicating a failure in proper lineage segregation. Furthermore, separation between roof plate and dI1 interneurons is mediated by Notch activity downstream of retinoic acid, highlighting their critical role in establishing the roof plate-dI1 boundary. Within the peripheral branch, where absence of retinoic acid resulted in neural crest production and emigration extending into the roof plate stage, sensory progenitors failed to separate from melanocytes leading to formation of a common glia-melanocyte cell with aberrant migratory patterns. Together, the implementation of scRNA sequencing facilitated the discovery and characterization of a molecular mechanism responsible for the segregation of dorsal neural fates during development.

Friedreich ataxia (FA) is a rare neurodegenerative disease caused by decreased levels of the mitochondrial protein frataxin. Frataxin has been related in iron homeostasis, energy metabolism, and oxidative stress. Ferroptosis has recently been shown to be involved in FA cellular degeneration; however, its role in dorsal root ganglion (DRG) sensory neurons, the cells that are affected the most and the earliest, is mostly unknown. In this study, we used primary cultures of frataxin-deficient DRG neurons as well as DRG from the FXNI151F mouse model to study ferroptosis and its regulatory pathways. A lack of frataxin induced upregulation of transferrin receptor 1 and decreased ferritin and mitochondrial iron accumulation, a source of oxidative stress. However, there was impaired activation of NRF2, a key transcription factor involved in the antioxidant response pathway. Decreased total and nuclear NRF2 explains the downregulation of both SLC7A11 (a member of the system Xc, which transports cystine required for glutathione synthesis) and glutathione peroxidase 4, responsible for increased lipid peroxidation, the main markers of ferroptosis. Such dysregulation could be due to the increase in KEAP1 and the activation of GSK3β, which promote cytosolic localization and degradation of NRF2. Moreover, there was a deficiency in the LKB1/AMPK pathway, which would also impair NRF2 activity. AMPK acts as a positive regulator of NRF2 and it is activated by the upstream kinase LKB1. The levels of LKB1 were reduced when frataxin decreased, in agreement with reduced pAMPK (Thr172), the active form of AMPK. SIRT1, a known activator of LKB1, was also reduced when frataxin decreased. MT-6378, an AMPK activator, restored NRF2 levels, increased GPX4 levels and reduced lipid peroxidation. In conclusion, this study demonstrated that frataxin deficiency in DRG neurons disrupts iron homeostasis and the intricate regulation of molecular pathways affecting NRF2 activation and the cellular response to oxidative stress, leading to ferroptosis.

UNASSIGNED: Osteoarthritis (OA) is a painful degenerative joint disease and a leading source of years lived with disability globally due to inadequate treatment options. Neuroimmune interactions reportedly contribute to OA pain pathogenesis. Notably, in rodents, macrophages in the DRG are associated with onset of persistent OA pain. Our objective was to determine the effects of acute systemic macrophage depletion on pain-related behaviors and joint damage using surgical mouse models in both sexes.
UNASSIGNED: We depleted CSF1R+ macrophages by treating male macrophage Fas-induced apoptosis (MaFIA) transgenic mice 8- or 16-weeks post destabilization of the medial meniscus (DMM) with AP20187 or vehicle control (10 mg/kg i.p., 1x/day for 5 days), or treating female MaFIA mice 12 weeks post partial meniscectomy (PMX) with AP20187 or vehicle control. We measured pain-related behaviors 1-3 days before and after depletion, and, 3-4 days after the last injection we examined joint histopathology and performed flow cytometry of the dorsal root ganglia (DRGs). In a separate cohort of male 8-week DMM mice or age-matched naïve vehicle controls, we conducted DRG bulk RNA-sequencing analyses after the 5-day vehicle or AP20187 treatment.
UNASSIGNED: Eight- and 16-weeks post DMM in male mice, AP20187-induced macrophage depletion resulted in attenuated mechanical allodynia and knee hyperalgesia. Female mice showed alleviation of mechanical allodynia, knee hyperalgesia, and weight bearing deficits after macrophage depletion at 12 weeks post PMX. Macrophage depletion did not affect the degree of cartilage degeneration, osteophyte width, or synovitis in either sex. Flow cytometry of the DRG revealed that macrophages and neutrophils were reduced after AP20187 treatment. In addition, in the DRG, only MHCII+ M1-like macrophages were significantly decreased, while CD163+MHCII- M2-like macrophages were not affected in both sexes. DRG bulk RNA-seq revealed that Cxcl10 and Il1b were upregulated with DMM surgery compared to naïve mice, and downregulated in DMM after acute macrophage depletion.
UNASSIGNED: Acute systemic macrophage depletion reduced the levels of pro-inflammatory macrophages in the DRG and alleviated pain-related behaviors in established surgically induced OA in mice of both sexes, without affecting joint damage. Overall, these studies provide insight into immune cell regulation in the DRG during OA.

BACKGROUND: We discuss the diagnostic benefit of pulsed radiofrequency (PRF) of the dorsal root ganglion (DRG) in a case series of patients with different pathologies. We expand the diagnostic potential of DRG stimulation beyond paresthesia mapping by using DRG stimulation to help determine the role of the DRG in the patient\'s pain and narrow down the etiology. In some cases, DRG stimulation was also part of the treatment plan.
METHODS: Six patients underwent DRG radiofrequency as a diagnostic/therapeutic step before considering implantation of a DRG neurostimulator. First, patients underwent a basic bedside neurological evaluation. Next, an electrode was placed in the epidural space through the sacral hiatus or between vertebral laminae. Then, sensory stimulation was applied at 50 Hz and gradually increased from 0.1 V until the patient reported paresthesia or until a maximum intensity of 2 V was reached. Patients were asked to describe where the stimulation was felt and outline the anatomical area the paresthesia covered. Then a motor stimulation was applied at 2 Hz until muscle twitching was reported by the patient or observed by the physician.
RESULTS: The information obtained helped diagnose the type of lesion as principally preganglionic, ganglionic, or postganglionic. This information guided patient management.
CONCLUSIONS: PRF of the DRG can provide valuable diagnostic information and is a useful step before ganglionic electrode implantation. In all cases, PRF of the DRG provided valuable diagnostic information and guided management options.

Bone cancer pain (BCP) represents a prevalent symptom among cancer patients with bone metastases, yet its underlying mechanisms remain elusive. This study investigated the transcriptional regulation mechanism of Kv7(KCNQ)/M potassium channels in DRG neurons and its involvement in the development of BCP in rats. We show that HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes, which encode Kv7(KCNQ)/M potassium channels in dorsal root ganglion (DRG), contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Also, HDAC2 requires the formation of a corepressor complex with MeCP2 and Sin3A to execute transcriptional regulation of kcnq2/kcnq3 genes. Moreover, EREG is identified as an upstream signal molecule for HDAC2-mediated kcnq2/kcnq3 genes transcription repression. Activation of EREG/EGFR-ERK-Runx1 signaling, followed by the induction of HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes in DRG neurons, leads to neuronal hyperexcitability and pain hypersensitivity in tumor-bearing rats. Consequently, the activation of EREG/EGFR-ERK-Runx1 signaling, along with the subsequent transcriptional repression of kcnq2/kcnq3 genes by HDAC2 in DRG neurons, underlies the sensitization of DRG neurons and the pathogenesis of BCP in rats. These findings uncover a potentially targetable mechanism contributing to bone metastasis-associated pain in cancer patients.

BACKGROUND: Delivering optogenetic genes to the peripheral sensory nervous system provides an efficient approach to study and treat neurological disorders and offers the potential to reintroduce sensory feedback to prostheses users and those who have incurred other neuropathies. Adeno-associated viral (AAV) vectors are a common method of gene delivery due to efficiency of gene transfer and minimal toxicity. AAVs are capable of being designed to target specific tissues, with transduction efficacy determined through the combination of serotype and genetic promoter selection, as well as location of vector administration. The dorsal root ganglia (DRGs) are collections of cell bodies of sensory neurons which project from the periphery to the central nervous system (CNS). The anatomical make-up of DRGs make them an ideal injection location to target the somatosensory neurons in the peripheral nervous system (PNS).
METHODS: Previous studies have detailed methods of direct DRG injection in rats and dorsal horn injection in mice, however, due to the size and anatomical differences between rats and strains of mice, there is only one other published method for AAV injection into murine DRGs for transduction of peripheral sensory neurons using a different methodology.
UNASSIGNED: Here, we detail the necessary materials and methods required to inject AAVs into the L3 and L4 DRGs of mice, as well as how to harvest the sciatic nerve and L3/L4 DRGs for analysis. This methodology results in optogenetic expression in both the L3/L4 DRGs and sciatic nerve and can be adapted to inject any DRG.

Cisplatin-based chemotherapy is a common treatment for paediatric cancer. Unfortunately, cisplatin treatment causes neuropathic pain, a highly prevalent adverse health related complication in adult childhood cancer survivors. Due to minimal understanding of this condition, there are currently no condition tailored analgesics available. Here we investigated an alteration in nociceptor maturation that results in neuronal sensitisation and manifestation of cisplatin induced survivorship pain in a TrkA dependent manner. Cisplatin was administered (i.p. 0.1 mg/kg Postnatal day 14 and 16) to neonatal male and female Wistar rats and nociceptive behavioural assays were performed. In vitro studies utilised isolated neonatal dorsal root ganglia sensory neurons treated with cisplatin (5 μg/ml) to elucidate impact upon nociceptor activation and neurite growth, in combination with TrkA inhibition (GW441756 10 nM and 100 nM). Cisplatin treated male and female neonatal Wistar rats developed a delayed but lasting mechanical and heat hypersensitivity. Cisplatin administration led to increased TrkA expression in dorsal root ganglia sensory neurons. Nerve growth factor (NGF) induced TrkA activation led to sensory neuritogenesis and nociceptor sensitisation, which could be prevented through pharmacological TrkA inhibition (GW441756 either s.c. 100 nM or i.p. 2 mg/kg). Administration of TrkA antagonist suppressed cisplatin induced TRPV1 mediated nociceptor sensitisation and prevented cisplatin induced neuropathic pain. These studies provide greater understanding of the underlying mechanisms that cause cisplatin induced childhood cancer survivorship pain and allowing identification of potential therapeutic targets.

Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.

Studies indicate that the lysine-specific demethylase 4A (KDM4A), acts as a key player in neuropathic pain, driving the process through its involvement in promoting neuroinflammation. Emerging evidence reveals that C-C Motif Chemokine Ligand 2 (CCL2) participates in neuroinflammation, which plays an important role in the development and maintenance of neuropathic pain. However, it remains unclear if KDM4A plays a role in regulating CCL2 in neuropathic pain. This study found that following spinal nerve transection (SNT) of the lumbar 5 nerve root in rats, the expression of KDM4A and CCL2 increased in the ipsilateral L4/5 dorsal root ganglia (DRG). Injecting KDM4A siRNA into the DRGs of rats post-SNT resulted in a higher paw withdrawal threshold (PWT) and paw-withdrawal latency (PWL) compared to the KDM4A scRNA group. In addition, prior microinjection of AAV-EGFP-KDM4A shRNA also alleviates the decrease in PWT and PWL caused by SNT. Correspondingly, microinjection of AAV-EGFP-KDM4A shRNA subsequent to SNT reduced the established mechanical and thermal hyperalgesia. Furthermore, AAV-EGFP-KDM4A shRNA injection decreased the expression of CCL2 in DRGs. ChIP-PCR analysis revealed that increased binding of p-STAT1 with the CCL2 promoter induced by SNT was inhibited by AAV-EGFP-KDM4A shRNA treatment. These findings suggest that KDM4A potentially influences neuropathic pain by regulating CCL2 expression in DRGs.

The cell intrinsic mechanisms directing peripheral nerve regeneration have remained largely understudied, thus limiting our understanding of these processes and constraining the advancement of novel clinical therapeutics. The use of primary adult rat dorsal root ganglion (DRG) neurons cultured in vitro is well established. Despite this, these cells can be challenging to culture and have so far not been amenable to robust transfection or live-cell imaging. The ability to transfect these cells with fluorescent plasmid constructs to label subcellular structures, combined with high resolution time-lapse imaging has the potential to provide invaluable insight into how peripheral neurons coordinate their regenerative response, and which specific cellular structures are involved in this process. Here we describe a protocol that facilitates transfection and subsequent live-imaging of adult rat DRG neurons.