Animal Studies

N-methyladenosine (mA) modification in RNA has been implicated in diverse biological processes. However, very little is currently known about its role in nociceptive modulation. Here, we found that the level of spinal mA modification was significantly increased in a mouse model of Complete Freund's Adjuvant (CFA)-induced chronic inflammatory pain, which was accompanied with the augmentation of methyltransferase-like 3 (METTL3) expression in the spinal cord. Knockdown of spinal METTL3 prevented and reversed CFA-induced pain behaviors and spinal neuronal sensitization. In contrast, overexpression of spinal METTL3 produced pain behaviors and neuronal sensitization in naive mice. Moreover, we found that METTL3 positively modulated the pri-miR-65-3p processing in a microprocessor protein DiGeorge critical region 8-dependent manner. Collectively, our findings reveal an important role of METTL3-mediated mA modification in nociceptive sensitization and provide a novel perspective on mA modification in the development of pathological pain.

Understanding how the nervous system bridges sensation and behavior requires the elucidation of complex neural and molecular networks. Forward genetic approaches, such as screens conducted in C. elegans, have successfully identified genes required to process natural sensory stimuli. However, functional redundancy within the underlying neural circuits, which are often organized with multiple parallel neural pathways, limits our ability to identify 'neural pathway-specific genes', i.e. genes that are essential for the function of some, but not all of these redundant neural pathways. To overcome this limitation, we developed a 'forward optogenetics' screening strategy in which natural stimuli are initially replaced by the selective optogenetic activation of a specific neural pathway. We used this strategy to address the function of the polymodal FLP nociceptors mediating avoidance of noxious thermal and mechanical stimuli. According to our expectations, we identified both mutations in 'general' avoidance genes that broadly impact avoidance responses to a variety of natural noxious stimuli (unc-4, unc-83, and eat-4) and mutations that produce a narrower impact, more restricted to the FLP pathway (syd-2, unc-14 and unc-68). Through a detailed follow-up analysis, we further showed that the Ryanodine receptor UNC-68 acts cell-autonomously in FLP to adjust heat-evoked calcium signals and aversive behaviors. As a whole, our work (i) reveals the importance of properly regulated ER calcium release for FLP function, (ii) provides new entry points for new nociception research and (iii) demonstrates the utility of our forward optogenetic strategy, which can easily be transposed to analyze other neural pathways.

Dysbiosis of gut microbiota is associated with many pathologies, yet host factors modulating microbiota remain unclear. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating condition of chronic pelvic pain often with co-morbid urinary dysfunction and anxiety/depression, and recent studies find fecal dysbiosis in IC/BPS patients. We identified the locus encoding acyloxyacyl hydrolase, Aoah, as a modulator of pelvic pain severity in a murine IC/BPS model. AOAH-deficient mice spontaneously develop rodent correlates of pelvic pain, increased responses to induced pelvic pain models, voiding dysfunction, and anxious/depressive behaviors. Here, we report that AOAH-deficient mice exhibit dysbiosis of GI microbiota. AOAH-deficient mice exhibit an enlarged cecum, a phenotype long associated with germ-free rodents, and a "leaky gut" phenotype. AOAH-deficient ceca showed altered gene expression consistent with inflammation, Wnt signaling, and urologic disease. 16S sequencing of stool revealed altered microbiota in AOAH-deficient mice, and GC-MS identified altered metabolomes. Co-housing AOAH-deficient mice with wild type mice resulted in converged microbiota and altered predicted metagenomes. Co-housing also abrogated the pelvic pain phenotype of AOAH-deficient mice, which was corroborated by oral gavage of AOAH-deficient mice with stool slurry of wild type mice. Converged microbiota also alleviated comorbid anxiety-like behavior in AOAH-deficient mice. Oral gavage of AOAH-deficient mice with anaerobes cultured from IC/BPS stool resulted in exacerbation of pelvic allodynia. Together, these data indicate that AOAH is a host determinant of normal gut microbiota, and dysbiosis associated with AOAH deficiency contributes to pelvic pain. These findings suggest that the gut microbiome is a potential therapeutic target for IC/BPS.

Long-term potentiation (LTP) is an important molecular mechanism for chronic pain in the anterior cingulate cortex (ACC), a key cortical region for pain perception and emotional regulation. Inhibiting ACC LTP via various manipulations or pharmacological treatments blocks chronic pain. Long-term depression (LTD) is another form of synaptic plasticity in the ACC, which is also proved to be involved in the mechanisms of chronic pain. However, less is known about the interactive relationship between LTP and LTD in the ACC. Whether the synaptic depression could be induced after synaptic LTP in the ACC is not clear. In the present study, we used multi-channel field potential recording systems to study synaptic depression after LTP in the ACC of adult mice. We found that low frequency stimulus (LFS: 1 Hz, 15 min) inhibited theta burst stimulation (TBS)-induced LTP at 30 min after the induction of LTP. However, LFS failed to induce depression at 90 min after the induction of LTP. Furthermore, NMDA receptor antagonist AP-5 blocked the induction of synaptic depression after potentiation. The GluN2B-selective antagonist Ro25-6981 also inhibited the phenomenon in the ACC, while the GluN2A-selective antagonist NVP-AAM077 and the GluN2C/D-selective antagonist PPDA and UBP145 had no any significant effect. These results suggest that synaptic LTP can be depressed by LTD in a time dependent manner, and GluN2B-containing NMDA receptors play important roles in this form of synaptic depression.

Peripheral inflammatory and neuropathic pain are closely related to the activation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3) and transient receptor potential vanilloid 1 (TRPV1), but the interaction between P2X3 and TRPV1 in different types of pathological pain has rarely been reported. In this study, complete Freund's adjuvant (CFA)-induced inflammatory pain and spared nerve injury (SNI)-induced neuropathic pain models were established in adult rats. The interactions between P2X3 and TRPV1 in the dorsal root ganglion were observed by pharmacological, co-immunoprecipitation, immunofluorescence and whole-cell patch-clamp recording assays. TRPV1 was shown to promote the induction of spontaneous pain caused by P2X3 in the SNI model, but the induction of spontaneous pain behaviour by TRPV1 was not completely dependent on P2X3 . In both the CFA and SNI models, the activation of peripheral P2X3 enhanced the effect of TRPV1 on spontaneous pain, while the inhibition of peripheral TRPV1 reduced the induction of spontaneous pain by P2X3 in the CFA model. TRPV1 and P2X3 had inhibitory effects on each other in the inflammatory pain model. During neuropathic pain, P2X3 facilitated the function of TRPV1, while TRPV1 had an inhibitory effect on P2X3. These results suggest that the mutual effects of P2X3 and TRPV1 differ in cases of inflammatory and neuropathic pain in rats.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A (PLA) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

Naked mole-rats () have adaptations within their pain pathway that are beneficial to survival in large colonies within poorly ventilated burrow systems, with lower O and higher CO ambient levels than ground-level environments. These adaptations ultimately lead to a partial disruption of the C-fiber pain pathway, which enables naked mole-rats to not feel pain from the acidosis associated with CO accumulation. One hallmark of this disruption is that naked mole-rats do not express neuropeptides, such as Substance P and calcitonin gene-related peptide in their cutaneous C-fibers, effectively making the peptidergic pain pathway hypofunctional. One C-fiber pathway that remains unstudied in the naked mole-rat is the non-peptidergic, purinergic pathway, despite this being a key pathway for inflammatory pain. The current study aimed to establish the functionality of the purinergic pathway in naked mole-rats and the effectiveness of cannabinoids in attenuating pain through this pathway. Cannabinoids can manage chronic inflammatory pain in both humans and mouse models, and studies suggest a major downstream role for the purinergic receptor, P2X3, in this treatment. Here we used Ca-imaging of cultured dorsal root ganglion neurons and behavioral testing to demonstrate that the P2X3 pathway is functional in naked mole-rats. Additionally, formalin-induced inflammatory pain was reduced by the cannabinoid receptor agonist, WIN55 (inflammatory, but not acute phase) and the P2X3 receptor antagonist A-317491 (acute and inflammatory phases). This study establishes that the purinergic C-fiber pathway is present and functional in naked mole-rats and that cannabinoid-mediated analgesia occurs in this species.