Papers: 11 Apr 2020 - 17 Apr 2020

Itch and pain are distinct unpleasant sensations that can be triggered from the same receptive fields in the skin, raising the question of how pruriception and nociception are coded and discriminated. Here, we tested the multimodal capacity of peripheral first-order neurons, focusing on the genetically defined subpopulation of mouse C-fibers that express the chloroquine receptor MrgprA3. Using optogenetics, chemogenetics, and pharmacology, we assessed the behavioral effects of their selective stimulation in a wide variety of conditions. We show that metabotropic Gq-linked stimulation of these C-afferents, through activation of native MrgprA3 receptors or DREADDs, evokes stereotypical pruriceptive rather than nocifensive behaviors. In contrast, fast ionotropic stimulation of these same neurons through light-gated cation channels or native ATP-gated P2X3 channels predominantly evokes nocifensive rather than pruriceptive responses. We conclude that C-afferents display intrinsic multimodality, and we provide evidence that optogenetic and chemogenetic interventions on the same neuronal populations can drive distinct behavioral outputs.

The lateral parabrachial nucleus (lPBN) is a major target of spinal projection neurons conveying nociceptive input into supraspinal structures. However, the functional role of distinct lPBN efferents in diverse nocifensive responses have remained largely uncharacterized. Here we show that that the lPBN is required for escape behaviors and aversive learning to noxious stimulation. In addition, we find that two populations of efferent neurons from different regions of the lPBN collateralize to distinct targets. Activation of efferent projections to the ventromedial hypothalamus (VMH) or lateral periaqueductal gray (lPAG) drives escape behaviors, whereas activation of lPBN efferents to the bed nucleus stria terminalis (BNST) or central amygdala (CEA) generates an aversive memory. Finally, we provide evidence that dynorphin-expressing neurons, which span cytoarchitecturally distinct domains of the lPBN, are required for aversive learning.

While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: RelmαMgl1 snMacs in the epineurium and RelmαMgl1 snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.

TRPM8 is the main ion channel responsible for cold transduction in the somatosensory system. Nerve terminal availability of TRPM8 determines cold sensitivity, but how axonal secretory organelles control channel delivery remains poorly understood. Here we examine the distribution of TRPM8 and trafficking organelles in cold-sensitive peripheral axons and disrupt trafficking by targeting the ARF-GEF GBF1 pharmacologically or the small GTPase RAB6 by optogenetics. In axons of the sciatic nerve, inhibition of GBF1 interrupts TRPM8 trafficking and increases association with the trans-Golgi network, LAMP1, and Golgi satellites, which distribute profusely along the axonal shaft. Accordingly, both TRPM8-dependent ongoing activity and cold-evoked responses reversibly decline upon GBF1 inhibition in nerve endings of corneal cold thermoreceptors. Inhibition of RAB6, which also associates to Golgi satellites, decreases cold-induced responses in vivo. Our results support a non-conventional axonal trafficking mechanism controlling the availability of TRPM8 in axons and cold sensitivity in the peripheral nervous system.

Neonatal tissue damage induces long-term deficits in inhibitory synaptic transmission within the spinal superficial dorsal horn (SDH) that include a reduction in primary afferent-evoked, feedforward inhibition onto adult projection neurons. However, the subpopulations of mature GABAergic interneurons which are compromised by early life injury have yet to be identified. The present research illuminates the persistent effects of neonatal surgical injury on the function of inhibitory SDH interneurons derived from the prodynorphin (DYN) lineage, a population which synapses directly onto lamina I spinoparabrachial neurons and is known to suppress mechanical pain and itch in adults. The results demonstrate that hindpaw incision at postnatal day (P) 3 significantly decreased the strength of primary afferent-evoked glutamatergic drive onto DYN neurons within the adult mouse SDH, while increasing the appearance of afferent-evoked inhibition onto the same population. Neonatal injury also dampened the intrinsic membrane excitability of mature DYN neurons, and reduced their action potential discharge in response to sensory input, compared to naïve littermate controls. Furthermore, P3 incision decreased the efficacy of inhibitory DYN synapses onto adult spinoparabrachial neurons, which reflected a prolonged reduction in the probability of GABA release. Collectively, the data suggest that early life tissue damage may persistently constrain the ability of spinal DYN interneurons to limit ascending nociceptive transmission to the adult brain. This is predicted to contribute to the loss of feedforward inhibition onto mature projection neurons, and the 'priming' of nociceptive circuits in the developing spinal cord, following injuries during the neonatal period.Neonatal injury has lasting effects on pain processing in the adult CNS, including a reduction in feedforward inhibition onto ascending projection neurons in the spinal dorsal horn. While it is clear that spinal GABAergic interneurons are comprised of multiple subpopulations that play distinct roles in somatosensation, the identity of those interneurons which are compromised by tissue damage during early life remains unknown. Here we document persistent deficits in spinal inhibitory circuits involving dynorphin-lineage (DYN) interneurons previously implicated in gating mechanical pain and itch. Notably, neonatal injury reduced the strength of DYN inhibitory synapses onto mature lamina I spinoparabrachial neurons, a major output of the spinal nociceptive network, which could contribute to the priming of pain pathways by early tissue damage.

Many Alzheimer's disease (AD) patients suffer from persistent neuropathic pain (NP), which is mediated, at least partially, but microglia. Nevertheless, the exact underlying mechanism is unknown. Moreover, a clinically translatable approach through modulating microglia for treating AD-associated NP is not available. Here, in a doxycycline-induced mouse model (rTg4510) for AD, we showed development of NP. We found that the total number of microglia in the CA3 region was not increased, but polarized to pro-inflammatory M1-like phenotype, with concomitant increases in production and secretion of pro-inflammatory cytokines. To examine whether this microglia polarization plays an essential role in the AD-associated NP, we generated an adeno-associated virus (AAV) serotype PHP.B (capable of crossing the blood-brain barrier) carrying shRNA for DNA methyltransferase 1 (DNMT1) under a microglia-specific TMEM119 promoter (AAV-pTMEM119-shDNMT1), which specifically targeted microglia and induced a M2-like polarization in vitro and in vivo in doxycycline-treated rTg4510 mice. Intravenous infusion of AAV-pTMEM119-shDNMT1 induced M2-polarization of microglia and attenuated both AD-associated behavior impairment but also NP in the doxycycline-treated rTg4510 mice. Thus, our data suggest that AD-associated NP may be treated through M2-polarization of microglia.

We recently showed that the mesolimbic dopaminergic system was tonically suppressed during chronic pain by enhanced corticotropin releasing factor (CRF) signaling within the dorsolateral bed nucleus of the stria terminalis (dlBNST), and that inhibition of intra-dlBNST CRF signaling restored the mesolimbic dopaminergic system function. Specifically, bilateral intra-dlBNST injections of the CRF type 1 receptor antagonist NBI27914 increased intra-nucleus accumbens dopamine release and induced reward-related behaviors in rats with chronic pain. Here, we used a conditioned place preference (CPP) test to explore whether intra-dlBNST injections of neuropeptide Y (NPY) restored the mesolimbic reward system function in chronic pain rats, because we previously showed that NPY had an effect opposite to that of CRF in dlBNST neurons. Specifically, CRF depolarized type II dlBNST neurons whereas NPY hyperpolarized them. However, unexpectedly, intra-dlBNST NPY injections had no effect on CPP test outcomes. Then, we compared the effects of NPY on the membrane potentials of type II dlBNST neurons of sham-operated control rats and those of chronic pain animals. Whole-cell patch-clamp electrophysiology revealed that NPY hyperpolarized type II dlBNST neurons in the sham-operated group. By contrast, in the chronic pain group, NPY did not hyperpolarize, but rather depolarized, type II dlBNST neurons. These results indicate that NPY no longer hyperpolarize type II dlBNST neurons in rats with chronic pain, therefore it does not reverse the excitatory effects of CRF. This may be why intra-dlBNST injections of NPY into chronic pain rats did not exhibit a rewarding effect in the CPP test, whereas intra-dlBNST injections of NBI27914 did. This is the first study to demonstrate a chronic pain-induced neuroplastic change in NPY signaling in the dlBNST. Such a change may be involved in the dysfunction of the mesolimbic reward system under the chronic pain condition.

Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

Emerging evidence has implicated poly-(ADP-ribose) polymerase 1 (PARP-1), a transcriptional coregulator, in a variety of inflammatory diseases. In the current study, the role of PARP-1 in neuropathic pain and the underlying mechanisms were investigated. Neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rates. Western blotting, qRT-PCR, immunohistochemistry, chromatin immunoprecipitation (ChIP), and Co-IP assays were performed to elucidate the mechanisms. The results showed that SNL resulted in a significant increase in the expression and activation of PARP-1 in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn, which occurred on day one, reached peak on day 7, and persisted more than 2 weeks after surgery. Double immunofluorescence staining revealed that PARP-1 was expressed exclusively in DRG A-type and C-type neurons. In the spinal cord, PARP-1 mainly colocalized with the neuronal marker NeuN and the astrocytic marker GFAP specifically in the superficial lamina. Prior intrathecal (i.t.) injection of PJ-34, a PARPs inhibitor, or Tiq-A, a specific PARP-1 inhibitor, dose-dependently prevented the reductions in PWT and PWL following SNL. Established neuropathic pain-like hypersensitivity was also attenuated with i.t. injection of PJ-34 and Tiq-A starting on day 7 following SNL, a timepoint at which neuropathic pain was fully established. SNL-induced mechanical allodynia and thermal hyperalgesia were also alleviated by i.t. injection of PARP-1 siRNA following a reduction in PARP-1 expression in the dorsal horn. Moreover, the SNL-induced increases in TNF-α protein and mRNA in the dorsal horn and DRG were dramatically suppressed by i.t. injection of Tiq-A or PARP-1 siRNA. The i.t. lipopolysaccharide (LPS)-induced increase in the production of TNF-α in the dorsal horn was also inhibited by prior to i.t. injection of PARP-1 siRNA. Results of ChIP assay showed that SNL-induced PARP-1 activation promoted the binding of NF-κB p65 with the TNF-α promoter in the dorsal horn and that PARP-1 inhibition reduced this binding and suppressed TNF-α expression. Co-IP assay revealed that SNL caused a significant increase in the level of histone H1 poly(ADP)-ribosylation. Together, these results indicate that PARP-1-regulated TNF-α expression in the DRG and spinal dorsal horn following SNL contributes to the development and maintenance of neuropathic pain. Targeting PARP-1 might be a promising therapeutic strategy for the treatment of the chronic pain.

Recent studies have revealed gender differences in cold perception, and pointed to a possible direct action of testosterone (TST) on the cold-activated TRPM8 (Transient Receptor Potential Melastatin Member 8) channel. However, the mechanisms by which TST influences TRPM8-mediated sensory functions remain elusive. Here, we show that TST inhibits TRPM8-mediated mild-cold perception through the noncanonical engagement of the Androgen Receptor (AR). Castration of both male rats and mice increases sensitivity to mild cold, and this effect depends on the presence of intact TRPM8 and AR. TST in nanomolar concentrations suppresses whole-cell TRPM8-mediated currents and single-channel activity in native dorsal root ganglion (DRG) neurons and HEK293 cells co-expressing recombinant TRPM8 and AR, but not TRPM8 alone. AR cloned from rat DRGs shows no difference from standard AR. However, biochemical assays and confocal imaging reveal the presence of AR on the cell surface and its interaction with TRPM8 in response to TST, leading to an inhibition of channel activity.

Myofascial pain syndrome (MPS) is a type of skeletal pain identified by myofascial trigger points (MTrPs). The formation of MTrPs is linked to muscle damage. The fibroblast growth factor receptor (FGFR1) has been found to cause pain sensitivity while repairing tissue damage. The aim of the current study was to explore the mechanism of FGFR1 in MTrPs. We used a RayBio human phosphorylation array kit to measure p-FGFR1 levels in human control subjects and patients with MTrPs. P-FGFR1 was upregulated in the patients with MTrPs. Then a rat model of myofascial pain syndrome was established by a blunt strike on the left gastrocnemius muscles (GM) and eccentric-exercise for 8 weeks with 4 weeks of recovery. After establishing the myofascial pain syndrome model, the morphology of the GM changed, and the differently augmented sizes of round fibres (contracture knots) in the transverse section and fusiform shapes in the longitudinal section were clearly seen in the rats with myofascial pain. The expression of p-FGFR1 was upregulated on the peripheral nerves and dorsal root ganglion (DRG) neurons in the MTrPs group. The spinal Fos protein expression was increased in the MTrPs group. Additionally, the mechanical pain threshold was reduced, and the expression of FGF2, p-FGFR1, PI3K-p110γ, and p-AKT increased in the MTrPs group. PD173074 increased the mechanical pain threshold of the MTrPs group, and inhibited the expression of p-FGFR1, PI3K-p110γ, and p-AKT. Moreover, LY294002 increased the mechanical pain threshold of the MTrPs group. These findings suggest that FGFR1 may regulate myofascial pain in rats through the PI3K/AKT pathway.

Behavioral alterations, like mechanical and thermal hyperalgesia, and modulation of biomarkers in the peripheral and central nervous systems (CNS) are markers of chronic pain. Transcranial direct current stimulation (tDCS) with exercise is a promising therapy for pain due to its neuromodulatory capacity.