Animal Studies

Neuropathic pain, which results from impairment of the somatosensory system, has affected about 8% population around the world and leads to considerable burdens for patients and world health care system. However, its underlying mechanisms remain poorly understood. In this study, we hypothesized that miR-24-3p was involved in the progression of neuropathic pain in CCI rat models. By measuring miR-24-3p expression in CCI rats, we found that miR-24-3p expression was increased in CCI rats, suggesting miR-24-3p might participate in neuropathic pain progression. Next, by conducting a serial in vitro and vivo experiments, we found that miR-24-3p regulated the Wnt5a/β-Catenin Signaling levels to promote neuropathic pain progression via targeting LPAR3 in CCI rats. Furthermore, we explored the upstream regulator of miR-24-3p by conducting bioinformatics analysis, we found that circular RNA cZRANB1 might sponge to miR-24-3p. Then we applied biotinylated RNA pull-down and luciferase reporter assays to assess the association between cZRANB1 and miR-24-3p. It was found that cZRANB1 mediated LPAR3 expression via sponging miR-24-3p. Collectively, our study suggests that cZRNAB1 regulated Wnt5a/β-Catenin Signaling expression via miR-24-3p/LPAR3 axis in CCI rat models.

There is increasing evidence that the toll-like receptor 4 (TLR4) signaling pathway contribute to development of hyperalgesia in the trigeminal system. The aim of the present study was to investigate the role of TLR4 in the trigeminal ganglion (TG) in facial hyperalgesia induced by injection of Lipopolysaccharide (LPS) or intraoral mucosal incision, which is an orofacial postoperative pain model, in male Wistar rats. The TLR4 antagonist (LPS-RS, 20 µg/10 µL) was administrated 30 min before LPS injection into the TG (10 µg/10 µL) or oral mucosa (10 µg/50 µL). In the postoperative pain model, rats were treated with LPS-RS (20 µg/10 µL) into the TG for three consecutive days after the incision. Facial heat and mechanical hyperalgesia were assessed hourly after LPS injection or intraoral incision. In addition, expression of NFκB was assessed in the TG on day 3 after intraoral incision. Our results showed that blockade of TLR4 in the TG attenuated facial heat and mechanical hyperalgesia induced by LPS or by mucosal incision, and that both conditions are associated to increase of phosphorylated NFκB in the TG. In conclusion, the present study suggests that activation of TLR4-NFκB signaling pathway in the TG contributes to the development of facial heat and mechanical hyperalgesia and may contribute to pain in inflammatory oral conditions.

This study aimed to evaluate whether the development and/or maintenance of chronic-latent muscle hyperalgesia is modulated by P2X3 receptors. We also evaluate the expression of P2X3 receptors and PKCε of dorsal root ganglions during these processes. A mouse model of chronic-latent muscle hyperalgesia, induced by carrageenan and evidenced by PGE, was used. Mechanical muscle hyperalgesia was measured by Randall-Selitto analgesimeter. The involvement of P2X3 receptors was analyzed by using the selective P2X3 receptors antagonist A-317491 by intramuscular or intrathecal injections. Expression of P2X3 and PKCε in dorsal root ganglion (L4-S1) were evaluated by Western blotting. Intrathecal blockade of P2X3 receptors previously to carrageenan prevented the development and maintenance of acute and chronic-latent muscle hyperalgesia, while intramuscular blockade of P2X3 receptors previously to carrageenan only reduced the acute muscle hyperalgesia and had no effect on chronic-latent muscle hyperalgesia. Intrathecal, but not intramuscular, blockade of P2X3 receptors immediately before PGE, in animals previously sensitized by carrageenan, reversed the chronic-latent muscle hyperalgesia. There was an increase in total and phosphorylated PKCε 48 h after the beginning of acute muscle hyperalgesia, and in P2X3 receptors at the period of chronic muscle hyperalgesia. P2X3 receptors expressed on spinal cord dorsal horn contribute to transition from acute to chronic muscle pain. We also suggest an interaction of PKCε and P2X3 receptors in this process. Therefore, we point out P2X3 receptors of the spinal cord dorsal horn as a pharmacological target to prevent the development or reverse the chronic muscle pain conditions.

Dorsal horn excitatory interneurons that express gastrin-releasing peptide (GRP) are part of the circuit for pruritogen-evoked itch. They have been extensively studied in a transgenic line in which enhanced green fluorescent protein (eGFP) is expressed under control of the Grp gene. The GRP-eGFP cells are separate from several other neurochemically-defined excitatory interneuron populations, and correspond to a class previously defined as transient central cells. However, mRNA for GRP is widely distributed among excitatory interneurons in superficial dorsal horn. Here we show that although Grp mRNA is present in several transcriptomically-defined populations, eGFP is restricted to a discrete subset of cells in the GRP::eGFP mouse, some of which express the neuromedin receptor 2 and likely belong to a cluster defined as Glut8. We show that these cells receive much of their excitatory synaptic input from MrgA3/MrgD-expressing nociceptive/pruritoceptive afferents and C-low threshold mechanoreceptors. Although the cells were not innervated by pruritoceptors expressing brain natriuretic peptide (BNP) most of them contained mRNA for NPR1, the receptor for BNP. In contrast, these cells received only ~ 10% of their excitatory input from other interneurons. These findings demonstrate that the GRP-eGFP cells constitute a discrete population of excitatory interneurons with a characteristic pattern of synaptic input.

The nucleus accumbens shell (NAcSh) regulates emotional and motivational responses, a function mediated, in part, by integrating and prioritizing extensive glutamatergic projections from limbic and paralimbic brain regions. Each of these inputs is thought to encode unique aspects of emotional and motivational arousal. The projections do not operate alone, but rather are often activated simultaneously during motivated behaviors, during which they can interact and coordinate in shaping behavioral output. To understand the anatomical and physiological bases underlying these inter-projection interactions, the current study in mice of both sexes focused on how the basolateral amygdala projection to the NAcSh (BLAp) regulates, and is regulated by, projections from the medial prefrontal cortex (mPFCp) and paraventricular nucleus of the thalamus (PVTp). Using a dual-color SynaptoTag technique combined with a backfilling spine imaging strategy, we found that all three afferent projections primarily targeted the secondary dendrites of NAcSh medium spiny neurons, forming putative synapses. We detected a low percentage of BLAp contacts closely adjacent to mPFCp or PVTp presumed synapses, and, on some rare occasions, the BLAp formed heterosynaptic interactions with mPFCp or PVTp profiles or appeared to contact the same spines. Using dual-rhodopsin optogenetics, we detected signs of dendritic summation of BLAp with PVTp and mPFCp inputs. Furthermore, high-frequency activation of BLAp synchronous with the PVTp or mPFCp resulted in a transient enhancement of the PVTp, but not mPFCp, transmission. These results provide anatomical and functional indices that the BLAp interacts with the mPFCp and PVTp for informational processing within the NAcSh.The nucleus accumbens regulates emotional and motivational responses by integrating extensive glutamatergic projections, but the anatomical and physiological bases on which these projections integrate and interact remain underexplored. Here, we used dual-color synaptic markers combined with backfilling of nucleus accumbens medium spiny neurons to reveal some unique anatomical alignments of presumed synapses from the basolateral amygdala, medial prefrontal cortex, and paraventricular nucleus of thalamus. We also used dual-rhodopsin optogenetics in brain slices, which reveal a nonlinear interaction between some, but not all, projections. These results provide compelling anatomical and physiological mechanisms through which different glutamatergic projections to the nucleus accumbens, and possibly different aspects of emotional and motivational arousal, interact with each other for final behavioral output.

Stimulation of the occipital or trigeminal nerves has been successfully used to treat chronic refractory neurovascular headaches such as migraine or cluster headache, and painful neuropathies. Convergence of trigeminal and occipital sensory afferents in the 'trigeminocervical complex' (TCC) from cutaneous, muscular, dural, and visceral sources is a key mechanism for the input-induced central sensitization that may underlie the altered nociception. Both excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) mechanisms are involved in modulating nociception in the spinal and medullary dorsal horn neurons, but the mechanisms by which nerve stimulation effects occur are unclear. This study was aimed at investigating the acute effects of electrical stimulation of the greater occipital nerve (GON) on the responses of neurons in the TCC to the mechanical stimulation of the vibrissal pad.

There are currently no rapid, operant pain behaviors in rodents that use a self-report to directly engage higher-order brain circuitry. We have developed a pain detection assay consisting of a lick behavior in response to optogenetic activation of predominantly nociceptive peripheral afferent nerve fibers in head-restrained transgenic mice expressing ChR2 in TRPV1 containing neurons. TRPV1-ChR2-EYFP mice (n = 5) were trained to provide lick reports to the detection of light-evoked nociceptive stimulation to the hind paw. Using simultaneous video recording, we demonstrate that the learned lick behavior may prove more pertinent in investigating brain driven pain processes than the reflex behavior. Within sessions, the response bias of transgenic mice changed with respect to lick behavior but not reflex behavior. Furthermore, response similarity between the lick and reflex behaviors diverged near perceptual threshold. Our nociceptive lick-report detection assay will enable a host of investigations into the millisecond, single cell, neural dynamics underlying pain processing in the central nervous system of awake behaving animals.