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Neurons of the peripheral nervous system are able to regenerate injured axons, a process requiring significant cellular resources to establish and maintain long-distance growth. Genetic activation of mTORC1, a potent regulator of cellular metabolism and protein translation, improves axon regeneration of peripheral neurons by an unresolved mechanism. To gain insight into this process, we activated mTORC1 signaling in mouse nociceptors via genetic deletion of its negative regulator Tsc2. Perinatal deletion of Tsc2 in nociceptors enhanced initial axon growth after sciatic nerve crush, however by three days post-injury axon elongation rate became similar to controls. mTORC1 inhibition prior to nerve injury was required to suppress the enhanced axon growth. Gene expression analysis in purified nociceptors revealed that Tsc2-deficient nociceptors had increased activity of regeneration-associated transcription factors (RATFs), including cJun and Atf3, in the absence of injury. Additionally, nociceptor deletion of Tsc2 activated satellite glial cells and macrophages in the dorsal root ganglia (DRG) in a similar manner to nerve injury. Surprisingly, these changes improved axon length but not percentage of initiating axons in dissociated cultures. The pro-regenerative environment in naïve DRG was recapitulated by AAV8-mediated deletion of Tsc2 in adult mice, suggesting that this phenotype does not result from a developmental effect. Consistently, AAV8-mediated Tsc2 deletion did not improve behavioral recovery after a sciatic nerve crush injury despite initially enhanced axon growth. Together, these data show that neuronal mTORC1 activation induces an incomplete pro-regenerative environment in the DRG that improves initial but not later axon growth after nerve injury. Long distance axon regeneration poses a significant hurdle to recovery following nervous system injury. Increased mTORC1 signaling improves axon regeneration, however the underlying mechanisms are incompletely understood. We activated neuronal mTORC1 signaling by genetically deleting Tsc2 in Nav1.8-positive neurons perinatally or by AAV8-mediated viral infection in adult mice and observed improved short- but not long-term axon regeneration after sciatic nerve injury. We suggest that Tsc2 deletion promotes initial but not later peripheral axon regeneration by upregulating expression of neuronal pro-regenerative genes and activating non-neuronal responses in the surrounding environment. Activating mTORC1 signaling in peripheral neurons may provide therapeutic benefit in circumstances with poor initial growth such as after spinal cord injury to the dorsal column or peripheral nerve repair.
We explored the ongoing question of whether placebo analgesia alters afferent nociceptive processing in a novel paradigm designed to minimize the role of response bias in placebo measurement. First, healthy adult participants received a standard heat placebo induction and conditioning procedure using a topical "analgesic" cream applied to one arm. During a subsequent placebo testing procedure, participants rated stimuli on the placebo-treated arm and untreated arm, using a task that minimized subjects' ability to guess the expected response, thus reducing experimenter demand. Retrospectively participants reported moderate analgesia effectiveness (mean=5.3/10), but for individual temperature ratings, only 2 subjects exhibited a perceptual placebo response >5 points. Next, these subjects completed a novel, exploratory task designed to measure changes in inter-arm in discriminative accuracy that would be expected from changes in afferent nociception. Both placebo responders (but no non-responders) showed reduced discriminative ability when the hotter stimulus occurred on the placebo arm, an effect consistent with alterations in nociceptive afferent flow and unlikely to be caused by response bias.
Radiotherapy-related pain is a common adverse reaction with a high incidence among cancer patients undergoing radiotherapy and remarkably reduces the quality of life. However, the mechanisms of ionizing radiation (IR)-induced pain are largely unknown. In present study, mice were treated with 20 Gy X-ray to establish IR-induced pain model. X-ray evoked a prolonged mechanical, heat and cold allodynia in mice. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) were significantly upregulated in lumbar dorsal root ganglion (DRG). The mechanical and heat allodynia could be transiently reverted by intrathecal injection of TRPV1 antagonist capsazepine and TRPA1 antagonist HC-030031. Additionally, the phosphorylated ERK and JNK in pain neural pathway were induced by X-ray treatment. Our findings indicated activation of TRPA1 and TRPV1 is essential for the development of X-ray-induced allodynia. Furthermore, our findings suggest targeting on TRPV1 and TRPA1 may be promising prevention strategies for X-ray-induced allodynia in clinical practice.
In mice, spared nerve injury replicates symptoms of human neuropathic pain and induces upregulation of many genes in somatosensory neurons. Here we used single cell transcriptomics to probe the effects of partial infraorbital transection of the trigeminal nerve at the cellular level. Uninjured neurons were unaffected by transection of major nerve branches, segregating into many different classes. In marked contrast, axotomy rapidly transformed damaged neurons into just two new and closely-related classes where almost all original identity was lost. Remarkably, sensory neurons also adopted this transcriptomic state following various minor peripheral injuries. By genetically marking injured neurons, we showed that the injury-induced transformation was reversible, with damaged cells slowly reacquiring normal gene expression profiles. Thus, our data expose transcriptomic plasticity, previously thought of as a driver of chronic pain, as a programed response to many types of injury and a potential mechanism for regulating sensation during wound healing.
The spinal dorsal horn is the first relay structure coding for pain transmission and modulation. Previous anatomical and electrophysiological studies have examined spinal dorsal horn circuitry, functional studies of circuit connections and network activity. Further work is required to understand spinal cord sensory information processing that underlies pathological neuropathic pain states. Our previous studies suggest that peripheral nerve injury enhances presynaptic excitatory input onto spinal superficial dorsal horn neurons, which in turn contributes to pathologic nociception. The potential changes in local postsynaptic circuits in the dorsal horn that lead to pathologically heightened behavioral responses to pain remain largely unexplored. We combined whole cell electrophysiological recordings with laser scanning photostimulation (LSPS) to test whether the spinal nerve ligation (SNL) mouse model of neuropathic pain leads to alterations in the functional connectivity of spinal cord circuits including lamina II excitatory interneurons. Here we show that SNL enhances excitation and decreases inhibition to lamina II excitatory interneurons along with their increased glutamate-evoked excitability. The enhanced excitatory postsynaptic input and connectivity evoked by SNL eventually return to normal levels concurrently with the resolution of the neuropathic pain states. The physiological pattern highly correlates with mouse pain behaviors following SNL, supporting a neurophysiological mechanism of central sensitization and neuropathic pain that is functionally localized to the spinal dorsal horn. Together, these data support that SNL induces functional changes in synaptic input and connectivity to lamina II excitatory interneurons that code for pain perception, and thus provide new insights into the mechanism and locus of pain hypersensitivity. Neuropathic pain presumably results from alterations in neuronal circuits that process nociception. This form of pain is often maladaptive. The contribution of circuit connections and detailed local spinal cord circuits underlying neuropathic pain are not well understood. Here, we apply laser-scanning photostimulation (LSPS) combined with whole cell recordings to investigate local circuit connectivity onto the lamina II interneurons during and after recovery following spinal nerve ligation that causes pathological neuropathic pain. The present study sheds light on local circuit organization in spinal dorsal horn and shows that reciprocal changes occur in local excitatory interneurons during both peak and after the gradual normalization of neuropathic pain. This elucidates nociceptive processing changes during and after neuropathic pain conditions and suggests new treatments.
TRPM8 is the primary detector of environmental cold and an important target for treating pathological cold hypersensitivity. Here, we present cryo-EM structures of TRPM8 in ligand-free, antagonist- or calcium-bound forms, revealing how robust conformational changes give rise to two non-conducting states, closed and desensitized. We describe a malleable ligand-binding pocket that accommodates drugs of diverse chemical structures, and delineate the ion permeation pathway, including the contribution of lipids to pore architecture. Furthermore, we show that direct calcium binding mediates stimulus-evoked desensitization, clarifying this important mechanism of sensory adaptation. We observe large rearrangements within the S4-S5 linker that reposition the S1-S4 and pore domains relative to the TRP helix, leading us to propose a distinct model for modulation of TRPM8 and possibly other TRP channels.
The study reported here investigated the role of the central vascular endothelial growth factor-A (VEGF-A) pathway in the development of trigeminal neuropathic pain following nerve injury. A Sprague-Dawley rat model of trigeminal neuropathic pain was produced using malpositioned dental implants. The left mandibular second molar was extracted under anesthesia and replaced with a miniature dental implant to induce injury to the inferior alveolar nerve. The inferior alveolar nerve injury produced a significant upregulation of astrocytic VEGF-A expression in the medullary dorsal horn. The nerve injury-induced mechanical allodynia was inhibited by an intracisternal infusion of VEGF-A164 antibody. Although both VEGF-A Receptor 1 (VEGF-A R1; colocalized with the blood-brain barrier) and VEGF-A Receptor 2 (VEGF-A R2; colocalized with astrocytes) participated in the development of trigeminal neuropathic pain following nerve injury, only the intracisternal infusion of a VEGF-A R1 antibody, and not that of a VEGF-A R2 antibody, inhibited the increased blood-brain barrier (BBB) permeability produced by nerve injury. Finally, we confirmed the participation of the central VEGF-A pathway in the development of trigeminal neuropathic pain by reducing VEGF-A expression using VEGF-A164 siRNA. This suppression of VEGF-A produced significant prolonged anti-allodynic effects. These results suggest that the central VEGF-A pathway plays a key role in the development of trigeminal neuropathic pain following nerve injury through two separate pathways: VEGF-A R1 and VEGF-A R2. Hence, a blockade of the central VEGF-A pathway provides a new therapeutic avenue for the treatment of trigeminal neuropathic pain.
Identification of genetic variants that influence susceptibility to pain is key to identifying molecular mechanisms and targets for effective and safe therapeutic alternatives to opioids. To identify genes and variants associated with persistent pain, we measured late-phase response to formalin injection in 275 male and female Diversity Outbred mice genotyped for over 70,000 single nucleotide polymorphisms. One quantitative trait locus reached genome-wide significance on chromosome 1 with a support interval of 3.1 Mb. This locus, Nociq4 (nociceptive sensitivity quantitative trait locus 4; MGI: 5661503), harbors the well-known pain gene Trpa1 (transient receptor potential cation channel, subfamily A, member 1). Trpa1 is a cation channel known to play an important role in acute and chronic pain in both humans and mice. Analysis of Diversity Outbred founder strain allele effects revealed a significant effect of the CAST/EiJ allele at Trpa1, with CAST/EiJ carrier mice showing an early, but not late, response to formalin relative to carriers of the 7 other inbred founder alleles (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). We characterized possible functional consequences of sequence variants in Trpa1 by assessing channel conductance, TRPA1-TRPV1 interactions, and isoform expression. The phenotypic differences observed in CAST/EiJ relative to C57BL/6J carriers were best explained by Trpa1 isoform expression differences, implicating a splice junction variant as the causal functional variant. This study demonstrates the utility of advanced, high-precision genetic mapping populations in resolving specific molecular mechanisms of variation in pain sensitivity.
We investigated the calcium dynamics of dorsal root ganglion (DRG) neurons using transgenic mice to target expression of the genetically encoded calcium indicator (GECI), GCaMP6s, to a subset of neurons containing parvalbumin (PV), a calcium-binding protein present in proprioceptors and low-threshold mechanoreceptors. This study provides the first analysis of GECI calcium transient parameters from large-diameter DRG neurons. Our approach generated calcium transients of consistent shape and time-course, with quantifiable characteristics. Four parameters of calcium transients were determined to vary independently from each other and thus are likely influenced by different calcium-regulating mechanisms: peak amplitude, rise time (RT), decay time, and recovery time. Pooled analysis of 188 neurons demonstrated unimodal distributions, providing evidence that PV+ DRG neurons regulate calcium similarly as a population despite their differences in size, electrical properties, and functional sensitivities. Calcium transients increased in size with elevated extracellular calcium, longer trains of action potentials, and higher stimulation frequencies. RT and decay time increased with the addition of the selective sarco/endoplasmic reticulum calcium ATPases (SERCA) blocker, thapsigargin (TG), while peak amplitude and recovery time remained the same. When elevating bath pH to 8.8 to block plasma-membrane calcium ATPases (PMCA), all measured parameters significantly increased. These results illustrate that GECI calcium transients provide sufficient resolution to detect changes in electrical activity and intracellular calcium concentration, as well as discern information about the activity of specific subclasses of calcium regulatory mechanisms.