Animal Studies

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.

Neuropathic pain, or pain after nerve injury, is a disorder with a significant reliance on the signalling of cytokines such as IL-1β. However, quantifying the cytokine release repeatedly over time in vivo is technically challenging.

Cyclic nucleotide-gated (CNG) channels, which are directly activated by cAMP and cGMP, have long been known to play a key role in retinal and olfactory signal transduction. Emerging evidence indicates that CNG channels are also involved in signaling pathways important for pain processing. Here, we found that the expression of the channel subunits CNGA2, CNGA3, CNGA4 and CNGB1 in dorsal root ganglia, and of CNGA2 in the spinal cord, is transiently altered after peripheral nerve injury in mice. Specifically, we show using in situ hybridization and quantitative real-time RT-PCR that CNG channels containing the CNGB1b subunit are localized to populations of sensory neurons and predominantly excitatory interneurons in the spinal dorsal horn. In CNGB1 knockout (CNGB1) mice, neuropathic pain behavior is considerably attenuated whereas inflammatory pain behavior is normal. Finally, we provide evidence to support CNGB1 as a downstream mediator of cAMP signaling in pain pathways. Altogether, our data suggest that CNGB1-positive CNG channels specifically contribute to neuropathic pain processing after peripheral nerve injury.

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.

Neuropathic pain is an unneglectable pain condition with limited treatment options owing to its enigmatic underlying mechanisms. Long noncoding RNA small nucleolar RNA host gene 5 (SNHG5) is involved in the progression of a spectrum of human cancers. However, its role in neuropathic pain remains undiscovered. In the present study, we established a mouse spinal nerve ligation (SNL) model, and a significant upregulation of SNHG5 was observed. Then we knocked down SNHG5 level in mouse L5 dorsal root ganglion (DRG) by delivering specific short hairpin RNA against SNHG5 with adenovirus vehicle. Mouse paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in response to mechanical stimuli was increased after SNHG5 knockdown, accompanied with decreased protein levels of glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule 1 (IBA-1). Besides, SNHG5 directly modulated the expression of miR-154-5p, which was downregulated in SNL mice. MiR-154-5p inhibition abolished the effect of SNHG5 knockdown on mouse behavioral tests and GFAP and IBA-1 levels. In addition, we validated that C-X-C motif chemokine 13 (CXCL13) was a novel downstream target of miR-154-5p, and CXCL13 level was positively related to that of SNHG5 in SNL mice. In conclusion, our study demonstrated that SNHG5 knockdown alleviated neuropathic pain and inhibited the activation of astrocytes and microglia by targeting the miR-154-5p/CXCL13 axis, which might be a novel therapeutic target for neuropathic treatment clinically.

Although the anterior cingulate cortex (ACC) plays a vital role in neuropathic pain-related aversion, the underlying mechanisms haven't been fully studied. The mesolimbic dopamine system encodes reward and aversion, and participates in the exacerbation of chronic pain. Therefore, we investigated whether the ACC modulates aversion to neuropathic pain via control of the mesolimbic dopamine system, in a rat model of chronic constriction injury (CCI) to the sciatic nerve. Using anterograde and retrograde tracings, we confirmed that a subgroup of ACC neurons projected to the nucleus accumbens (NAc) and ventral tegmental area (VTA), which are two crucial nodes of the mesolimbic dopamine system. Combining electrophysiology in juvenile rats 7 days post-CCI, we found that the NAc/VTA-projecting neurons were hyperexcitable after CCI. Chemogenetic inhibition of these projections induced conditioned place preference in young adult rats 10-14 days post-CCI, without modulating the evoked pain threshold, whereas activation of these projections in sham rats mimicked aversive behavior. Furthermore, the function of the ACC projections was probably mediated by NAc D2-type medium spiny neurons and VTA GABAergic neurons. Taken together, our findings suggest that projections from the ACC to the NAc and VTA mediate neuropathic pain-related aversive behavior.

The goal of this study in anesthetized cats was to identify silent hypogastric nerve (HGN) afferent fibers that do not respond to bladder distention but become responsive after chemical irritation of the bladder. The HGN was split into multiple filaments small enough for recording action potentials from single or multiple afferent fibers. The bladder was distended by infusion of either saline or 0.5% acetic acid (AA) through a urethral catheter while recording intravesical pressure. A total of 90 HGN filaments from 17 cats responded to bladder distention with saline or AA. Three types of HGN afferents were identified. The first type was non-nociceptive mechano-sensitive that responded to bladder distention at normal physiological pressures (10-40 cmHO). The second type was nociceptive mechano-sensitive that only responded to high-pressure (50-80 cmHO) bladder distention with saline but responded to low-pressure bladder distention after sensitization with AA. The third type was chemo-sensitive nociceptive that was silent even during high-pressure bladder distention but after sensitization with AA did respond to low-pressure bladder distention. These results indicate that HGN afferents as well as pelvic nerve afferents may play a role in bladder nociception. The HGN afferent fibers that are silent during bladder distention at normal physiological pressures but become responsive after chemical irritation are important for understanding the possible pathophysiological mechanism underlying bladder allodynia in painful bladder syndrome.