Elucidation of neuronal activity in mouse models of temporomandibular joint injury and inflammation by in vivo GCaMP Ca2+ imaging of intact trigeminal ganglion neurons.

ABSTRACT: Patients with temporomandibular disorders (TMDs) typically experience facial pain and discomfort or tenderness in the temporomandibular joint (TMJ), causing disability in daily life. Unfortunately, existing treatments for TMD are not always effective, creating a need for more advanced, mechanism-based therapies. In this study, we used in vivo GCaMP3 Ca2+ imaging of intact trigeminal ganglia (TG) to characterize functional activity of the TG neurons in vivo, specifically in mouse models of TMJ injury and inflammation. This system allows us to observe neuronal activity in intact anatomical, physiological, and clinical conditions and to assess neuronal function and response to various stimuli. We observed a significant increase in spontaneously and transiently activated neurons responding to mechanical, thermal, and chemical stimuli in the TG of mice with TMJ injection of complete Freund adjuvant or with forced mouth opening (FMO). An inhibitor of the calcitonin gene-related peptide receptor significantly attenuated FMO-induced facial hypersensitivity. In addition, we confirmed the attenuating effect of calcitonin gene-related peptide antagonist on FMO-induced sensitization by in vivo GCaMP3 Ca2+ imaging of intact TG. Our results contribute to unraveling the role and activity of TG neurons in the TMJ pain, bringing us closer to understanding the pathophysiological processes underlying TMJ pain after TMJ injury. Our study also illustrates the utility of in vivo GCaMP3 Ca2+ imaging of intact TG for studies aimed at developing more targeted and effective treatments for TMJ pain.

Genetic labeling of the nucleus of tractus solitarius neurons associated with electrical stimulation of the cervical or auricular vagus nerve in mice.

INTRODUCTION: Vagus nerve stimulation (VNS) is clinically useful for treating epilepsy, depression, and chronic pain. Currently, cervical VNS (cVNS) treatment is well-established, while auricular VNS (aVNS) is under development. Vagal stimulation regulates functions in diverse brain regions; therefore, it is critical to better understand how electrically-evoked vagal inputs following cVNS and aVNS engage with different brain regions. OBJECTIVE: As vagus inputs are predominantly transmitted to the nucleus of tractus solitarius (NTS), we directly compared the activation of NTS neurons by cVNS or aVNS and the brain regions directly projected by the activated NTS neurons in mice. METHODS: We adopted the targeted recombination in active populations method, which allows for the activity-dependent, tamoxifen-inducible expression of mCherry-a reporter protein-in neurons specifically associated with cVNS or aVNS. RESULTS: cVNS and aVNS induced comparable bilateral mCherry expressions in neurons within the NTS, especially in its caudal section (cNTS). However, the numbers of mCherry-expressing neurons within different subdivisions of cNTS was distinctive. In both cVNS and aVNS, anterogradely labeled mCherry-expressing axonal terminals were similarly observed across different areas of the forebrain, midbrain, and hindbrain. These terminals were enriched in the rostral ventromedial medulla, parabrachial nucleus, periaqueductal gray, thalamic nuclei, central amygdala, and the hypothalamus. Sex difference of cVNS- and aVNS-induced labeling of NTS neurons was modest. CONCLUSION: The central projections of mCherry-expressing cNTS terminals are comparable between aVNS and cVNS, suggesting that cVNS and aVNS activate distinct but largely overlapping projections into the brain through the cNTS.

Peripheral macrophages contribute to nociceptor priming in mice with chronic intermittent hypoxia.

Obstructive sleep apnea (OSA) is a prevalent sleep disorder that is associated with increased incidence of chronic musculoskeletal pain. We investigated the mechanism of this association in a mouse model of chronic intermittent hypoxia (CIH) that mimics the repetitive hypoxemias of OSA. After 14 days of CIH, both male and female mice exhibited behaviors indicative of persistent pain, with biochemical markers in the spinal cord dorsal horn and sensory neurons of the dorsal root ganglia consistent with hyperalgesic priming. CIH, but not sleep fragmentation alone, induced an increase in macrophage recruitment to peripheral sensory tissues (sciatic nerve and dorsal root ganglia), an increase in inflammatory cytokines in the circulation, and nociceptor sensitization. Peripheral macrophage ablation blocked CIH-induced hyperalgesic priming. The findings suggest that correcting the hypoxia or targeting macrophage signaling might suppress persistent pain in patients with OSA.

PACAP38/mast-cell-specific receptor axis mediates repetitive stress-induced headache in mice.

BACKGROUND: Pain, an evolutionarily conserved warning system, lets us recognize threats and motivates us to adapt to those threats. Headache pain from migraine affects approximately 15% of the global population. However, the identity of any putative threat that migraine or headache warns us to avoid is unknown because migraine pathogenesis is poorly understood. Here, we show that a stress-induced increase in pituitary adenylate cyclase-activating polypeptide-38 (PACAP38), known as an initiator of allosteric load inducing unbalanced homeostasis, causes headache-like behaviour in male mice via mas-related G protein-coupled receptor B2 (MrgprB2) in mast cells. METHODS: The repetitive stress model and dural injection of PACAP38 were performed to induce headache behaviours. We assessed headache behaviours using the facial von Frey test and the grimace scale in wild-type and MrgprB2-deficient mice. We further examined the activities of trigeminal ganglion neurons using in vivo Pirt-GCaMP Ca2+ imaging of intact trigeminal ganglion (TG). RESULTS: Repetitive stress and dural injection of PACAP38 induced MrgprB2-dependent headache behaviours. Blood levels of PACAP38 were increased after repetitive stress. PACAP38/MrgprB2-induced mast cell degranulation sensitizes the trigeminovascular system in dura mater. Moreover, using in vivo intact TG Pirt-GCaMP Ca2+ imaging, we show that stress or/and elevation of PACAP38 sensitized the TG neurons via MrgprB2. MrgprB2-deficient mice showed no sensitization of TG neurons or mast cell activation. We found that repetitive stress and dural injection of PACAP38 induced headache behaviour through TNF-a and TRPV1 pathways. CONCLUSIONS: Our findings highlight the PACAP38-MrgprB2 pathway as a new target for the treatment of stress-related migraine headache. Furthermore, our results pertaining to stress interoception via the MrgprB2/PACAP38 axis suggests that migraine headache warns us of stress-induced homeostatic imbalance.

Forced mouth opening induces post-traumatic hyperalgesia and associated peripheral sensitization after temporomandibular joints injury in mice.

Temporomandibular disorder (TMD) is the most prevalent painful condition in the craniofacial area. The pathophysiology of TMD is not fully understood, and it is necessary to understand pathophysiology underlying painful TMD conditions to develop more effective treatment methods. Recent studies suggested that external or intrinsic trauma to TMJ is associated with chronic TMD in patients. Here, we investigated the effects of the TMJ trauma through forced-mouth opening (FMO) in mice to determine pain behaviors and peripheral sensitization of trigeminal nociceptors. FMO increased mechanical hyperalgesia assessed by von Frey test, spontaneous pain-like behaviors assessed by mouse grimace scale, and anxiety-like behaviors assessed by open-field test. In vivo GCaMP Ca 2+ imaging of intact trigeminal ganglia (TG) showed increased spontaneous Ca 2+ activity and mechanical hypersensitivity of TG neurons in the FMO compared to the sham group. Ca 2+ responses evoked by cold, heat, and capsaicin stimuli were also increased. FMO-induced hyperalgesia and neuronal hyperactivities were not sex dependent. TG neurons sensitized following FMO were primarily small to medium-sized nociceptive afferents. Consistently, most TMJ afferents in the TG were small-sized peptidergic neurons expressing calcitonin gene-related peptides, whereas nonpeptidergic TMJ afferents were relatively low. FMO-induced intraneural inflammation in the surrounding tissues of the TMJ indicates potentially novel mechanisms of peripheral sensitization following TMJ injury. These results suggest that the TMJ injury leads to persistent post-traumatic hyperalgesia associated with peripheral sensitization of trigeminal nociceptors.

Elucidation of neuronal activity in mouse models of TMJ injury by in vivo GCaMP Ca 2+ imaging of intact trigeminal ganglion neurons.

Patients with temporomandibular disorders (TMD) typically experience facial pain and discomfort or tenderness in the temporomandibular joint (TMJ), causing disability in daily life. Unfortunately, existing treatments for TMD are not always effective, creating a need for more advanced, mechanism-based therapies. In this study, we used in vivo GCaMP3 Ca 2+ imaging of intact trigeminal ganglia (TG) to characterize functional activity of the TG neurons in vivo , specifically in TMJ animal models. This system allows us to observe neuronal activity in intact anatomical, physiological, and clinical conditions and to assess neuronal function and response to various stimuli. We observed a significant increase in spontaneously and transiently activated neurons responding to mechanical, thermal, and chemical stimuli in the TG of forced mouth open (FMO) mice. An inhibitor of the CGRP receptor significantly attenuated FMO-induced facial hypersensitivity. In addition, we confirmed the attenuating effect of CGRP antagonist on FMO-induced sensitization by in vivo GCaMP3 Ca 2+ imaging of intact TG. Our results contribute to unraveling the role and activity of TG neurons in the TMJ pain animal models of TMD, bringing us closer understanding the pathophysiological processes underlying TMD. Our study also illustrates the utility of in vivo GCaMP3 Ca 2+ imaging of intact TG for studies aimed at developing more targeted and effective treatments for TMD.

Mast-cell-specific receptor mediates alcohol-withdrawal-associated headache in male mice.

Rehabilitation from alcohol addiction or abuse is hampered by withdrawal symptoms including severe headaches, which often lead to rehabilitation failure. There is no appropriate therapeutic option available for alcohol-withdrawal-induced headaches. Here, we show the role of the mast-cell-specific receptor MrgprB2 in the development of alcohol-withdrawal-induced headache. Withdrawing alcohol from alcohol-acclimated mice induces headache behaviors, including facial allodynia, facial pain expressions, and reduced movement, which are symptoms often observed in humans. Those behaviors were absent in MrgprB2-deficient mice during alcohol withdrawal. We observed in vivo spontaneous activation and hypersensitization of trigeminal ganglia (TG) neurons in alcohol-withdrawal WT mice, but not in alcohol-withdrawal MrgprB2-deficient mice. Increased mast cell degranulation by alcohol withdrawal in dura mater was dependent on the presence of MrgprB2. The results indicate that alcohol withdrawal causes headache via MrgprB2 of mast cells in dura mater, suggesting that MrgprB2 is a potential target for treating alcohol-withdrawal-related headaches.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, Aß, Aδ, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

Increase of glutamate in satellite glial cells of the trigeminal ganglion in a rat model of craniofacial neuropathic pain.

Introduction: Satellite glial cells (SGCs) that envelop the cell bodies of neurons in sensory ganglia have been shown to both release glutamate, and be activated by glutamate in the context of nociceptive signaling. However, little is known about the subpopulations of SGCs that are activated following nerve injury and whether glutamate mechanisms in the SGCs are involved in the pathologic pain. Methods: To address this issue, we used light and electron microscopic immunohistochemistry to examine the change in the glutamate levels in the SGCs and the structural relationship between neighboring neurons in the trigeminal ganglion (TG) in a rat model of craniofacial neuropathic pain, CCI-ION. Results: Administration of ionomycin, ATP and Bz-ATP induced an increase of extracellular glutamate concentration in cultured trigeminal SGCs, indicating a release of glutamate from SGCs. The level of glutamate immunostaining in the SGCs that envelop neurons of all sizes in the TG was significantly higher in rats with CCI-ION than in control rats, suggesting that SGCs enveloping nociceptive as well as non-nociceptive mechanosensitive neurons are activated following nerve injury, and that the glutamate release from SGCs increases in pathologic pain state. Close appositions between substance-P (SP)-immunopositive (+) or calcitonin gene-related peptide (CGRP)+, likely nociceptive neurons, between Piezo1+, likely non-nociceptive, mechanosensitive neurons and SP+ or CGRP+ neurons, and between SGCs of neighboring neurons were frequently observed. Discussion: These findings suggest that glutamate in the trigeminal SGCs that envelop all types of neurons may play a role in the mechanisms of neuropathic pain, possibly via paracrine signaling.

Activation of CD81+ skin ILC2s by cold-sensing TRPM8+ neuron-derived signals maintains cutaneous thermal homeostasis.

As the outermost barrier tissue of the body, the skin harbors a large number of innate lymphoid cells (ILCs) that help maintain local homeostasis in the face of changing environments. How skin-resident ILCs are regulated and function in local homeostatic maintenance is poorly understood. We here report the discovery of a cold-sensing neuron-initiated pathway that activates skin group 2 ILCs (ILC2s) to help maintain thermal homeostasis. In stearoyl-CoA desaturase 1 (SCD1) knockout mice whose skin is defective in heat maintenance, chronic cold stress induced excessive activation of CCR10-CD81+ST2+ skin ILC2s and associated inflammation. Mechanistically, stimulation of the cold-sensing receptor TRPM8 expressed in sensory neurons of the skin led to increased production of IL-18, which, in turn, activated skin ILC2s to promote thermogenesis. Our findings reveal a neuroimmune link that regulates activation of skin ILC2s to support thermal homeostasis and promotes skin inflammation after hyperactivation.

cytoNet: Spatiotemporal network analysis of cell communities.

We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNet's capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin α4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains.

Meclizine and metabotropic glutamate receptor agonists attenuate severe pain and Ca2+ activity of primary sensory neurons in chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) affects about 68% of patients undergoing chemotherapy, causing debilitating neuropathic pain and reducing quality of life. Cisplatin is a commonly used platinum-based chemotherapeutic drug known to cause CIPN, possibly by causing oxidative stress damage to primary sensory neurons. Metabotropic glutamate receptors (mGluRs) are widely hypothesized to be involved in pain processing and pain mitigation. Meclizine is an H1 histamine receptor antagonist known to have neuroprotective effects, including an anti-oxidative effect. Here, we used a mouse model of cisplatin-induced CIPN using male and female mice to test agonists of mGluR8 and group II mGluR as well as meclizine as interventions to reduce cisplatin-induced pain. We performed behavioral pain tests, and we imaged Ca2+ activity of the large population of DRG neurons in vivo For the latter, we used a genetically-encoded Ca2+ indicator, Pirt-GCaMP3, which enabled us to monitor different drug interventions at the level of the intact DRG neuronal ensemble. We found that CIPN increased spontaneous Ca2+ activity in DRG neurons, increased number of Ca2+ transients, and increased hyper-responses to mechanical, thermal, and chemical stimuli. We found that mechanical and thermal pain caused by CIPN was significantly attenuated by the mGluR8 agonist, (S)-3,4-DCPG, the group II mGluR agonist, LY379268, and the H1 histamine receptor antagonist, meclizine. DRG neuronal Ca2+ activity elevated by CIPN was attenuated by LY379268 and meclizine, but not by (S)-3,4-DCPG. Furthermore, meclizine and LY379268 attenuated cisplatin-induced weight loss. These results suggest that group II mGluR agonist, mGluR8 agonist, and meclizine are promising candidates as new treatment options for CIPN, and studies of their mechanisms are warranted.SIGNIFICANCE STATEMENTChemotherapy-induced peripheral neuropathy (CIPN) is a painful condition that affects most chemotherapy patients and persist several months or longer after treatment ends. Research on CIPN mechanism is extensive but has produced only few clinically useful treatments. Utilizing in vivo GCaMP Ca2+ imaging in live animals over 1800 neurons/DRG at once, we have characterized the effects of the chemotherapeutic drug, cisplatin and three treatments that decrease CIPN pain. Cisplatin increases sensory neuronal Ca2+ activity and develops various sensitization. Metabotropic glutamate receptor agonist, LY379268 or the H1 histamine receptor antagonist, meclizine decreases cisplatin's effects on neuronal Ca2+ activity and reduces pain hypersensitivity. Our results and experiments provide insights into cellular effects of cisplatin and drugs preventing CIPN pain.

Nicotinamide Mononucleotide Prevents Cisplatin-Induced Mitochondrial Defects in Cortical Neurons Derived from Human Induced Pluripotent Stem Cells.

Background: Chemotherapy-induced cognitive impairment (CICI) is a neurotoxic side effect of chemotherapy that has yet to have an effective treatment. Objective: Using cisplatin, a platinum-based chemotherapy together with excitatory cortical neurons derived from human induced pluripotent cells (iPSCs) to model of CICI, our recent study demonstrated that dysregulation of brain NAD+ metabolism contributes to cisplatin-induced impairments in neurogenesis and cognitive function, which was prevented by administration of the NAD+ precursor, nicotinamide mononucleotide (NMN). However, it remains unclear how cisplatin causes neurogenic dysfunction and the mechanism by which NMN prevents cisplatin-induced cognitive impairment. Given that mitochondrial dysfunction is thought to play a prominent role in age-related neurodegenerative disease and chemotherapy-induced neurotoxicity, we sought to explore if NMN prevents chemotherapy-related neurotoxicity by attenuating cisplatin-induced mitochondrial damage. Results: We demonstrate that cisplatin induces neuronal DNA damage, increases generation of mitochondrial reactive oxygen species (ROS) and decreases ATP production, all of which are indicative of oxidative DNA damage and mitochondrial functional defects. Ultrastructural analysis revealed that cisplatin caused loss of cristae membrane integrity and matrix swelling in human cortical neurons. Notably, pretreatment with NMN prevents cisplatin-induced defects in mitochondria of human cortical neurons. Conclusion: Our results suggest that increased mitochondrial oxidative stress and functional defects play key roles in cisplatin-induced neurotoxicity. Thus, NMN may be an effective therapeutic strategy to prevent cisplatin-induced deleterious effects on mitochondria, making this organelle a key factor in amelioration of cisplatin-induced cognitive impairments.

Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system.

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3-L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3-L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Na+-dependent inactivation of vascular Na+/Ca2+ exchanger responsible for reduced peripheral blood flow in neuropathic pain model.

Reduced skin blood flow has been reported in neuropathic pain patients as well as various peripheral neuropathic pain model animals. We have previously shown that vasodilators, which improves reduced skin blood flow, correlatively alleviate neuropathic pain in chronic constriction injury (CCI) mice, a model of neuropathic pain from peripheral nerve injury. Here, we sought to elucidate the mechanism underlying the reduced skin blood flow in CCI rats. The skin blood flow of the ipsilateral plantar arteries was significantly reduced compared to that of the contralateral ones 4 weeks after loose ligation of the sciatic nerve. The contraction induced by noradrenaline, serotonin, and U46619, a thromboxane receptor agonist, in the isolated ipsilateral plantar arteries was significantly enhanced compared to that in the contralateral ones. KB-R7943, a Na+/Ca2+ exchanger (NCX) inhibitor, shifted the concentration-response curves of noradrenaline to the left in the contralateral arteries but had no effect on the ipsilateral side. There was no significant difference in concentration-response curves of noradrenaline between the ipsilateral and contralateral arteries in the presence of KB-R7943. Amiloride, a non-specific inhibitor of Na+ channels and transporters, comparably shifted concentration-response curves of noradrenaline to the left in both the contralateral and ipsilateral arteries. One hundred nM of noradrenaline induced intracellular Ca2+ elevation in the ipsilateral arteries, which was significantly larger than that induced by 300-nM noradrenaline in the contralateral arteries. These results suggest that reduced peripheral blood flow after nerve injury is due to Na+-dependent inactivation of NCX in the ipsilateral plantar arteries.

In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment.

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca2+ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca2+ response because of degeneration of primary sensory nerve fibers in the skin.SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca2+ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca2+ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca2+ activity and postoperative pain.

Acute and Chronic Pain from Facial Skin and Oral Mucosa: Unique Neurobiology and Challenging Treatment.

The oral cavity is a portal into the digestive system, which exhibits unique sensory properties. Like facial skin, the oral mucosa needs to be exquisitely sensitive and selective, in order to detect harmful toxins versus edible food. Chemosensation and somatosensation by multiple receptors, including transient receptor potential channels, are well-developed to meet these needs. In contrast to facial skin, however, the oral mucosa rarely exhibits itch responses. Like the gut, the oral cavity performs mechanical and chemical digestion. Therefore, the oral mucosa needs to be insensitive, to some degree, in order to endure noxious irritation. Persistent pain from the oral mucosa is often due to ulcers, involving both tissue injury and infection. Trigeminal nerve injury and trigeminal neuralgia produce intractable pain in the orofacial skin and the oral mucosa, through mechanisms distinct from those seen in the spinal area, which is particularly difficult to predict or treat. The diagnosis and treatment of idiopathic chronic pain, such as atypical odontalgia (idiopathic painful trigeminal neuropathy or post-traumatic trigeminal neuropathy) and burning mouth syndrome, remain especially challenging. The central integration of gustatory inputs might modulate chronic oral and facial pain. A lack of pain in chronic inflammation inside the oral cavity, such as chronic periodontitis, involves the specialized functioning of oral bacteria. A more detailed understanding of the unique neurobiology of pain from the orofacial skin and the oral mucosa should help us develop novel methods for better treating persistent orofacial pain.

Visualization of Peripheral Neuron Sensitization in a Surgical Mouse Model of Osteoarthritis by In Vivo Calcium Imaging.

OBJECTIVE: To develop a method for analyzing sensory neuron responses to mechanical stimuli in vivo, and to evaluate whether these neuronal responses change after destabilization of the medial meniscus (DMM). METHODS: DMM or sham surgery was performed in 10-week-old male C57BL/6 wild-type or Pirt-GCaMP3+/- mice. All experiments were performed 8 weeks after surgery. Knee and hind paw hyperalgesia were assessed in wild-type mice. The retrograde label DiI was injected into the ipsilateral knee to quantify the number of knee-innervating neurons in the L4 dorsal root ganglion (DRG) in wild-type mice. In vivo calcium imaging was performed on the ipsilateral L4 DRG of Pirt-GCaMP3+/- mice as mechanical stimuli (paw pinch, knee pinch, or knee twist) were applied to the ipsilateral hind limb. RESULTS: Eight weeks after surgery, mice subjected to DMM had more hyperalgesia in the knee and hind paw compared to mice subjected to sham surgery. Intraarticular injection of DiI labeled similar numbers of neurons in the L4 DRG of mice subjected to sham surgery and mice subjected to DMM. Increased numbers of sensory neurons responded to all 3 mechanical stimuli in mice subjected to DMM, as assessed by in vivo calcium imaging. The majority of responses in mice subjected to sham surgery and mice subjected to DMM were in small to medium-sized neurons, consistent with the size of nociceptors. The magnitude of responses was similar between mice subjected to sham surgery and mice subjected to DMM. CONCLUSION: Our findings indicate that increased numbers of small to medium-sized DRG neurons respond to mechanical stimuli 8 weeks after DMM surgery, suggesting that nociceptors have become sensitized by lowering the response threshold.

Coupled Activation of Primary Sensory Neurons Contributes to Chronic Pain.

Primary sensory neurons in the DRG play an essential role in initiating pain by detecting painful stimuli in the periphery. Tissue injury can sensitize DRG neurons, causing heightened pain sensitivity, often leading to chronic pain. Despite the functional importance, how DRG neurons function at a population level is unclear due to the lack of suitable tools. Here we developed an imaging technique that allowed us to simultaneously monitor the activities of >1,600 neurons/DRG in live mice and discovered a striking neuronal coupling phenomenon that adjacent neurons tend to activate together following tissue injury. This coupled activation occurs among various neurons and is mediated by an injury-induced upregulation of gap junctions in glial cells surrounding DRG neurons. Blocking gap junctions attenuated neuronal coupling and mechanical hyperalgesia. Therefore, neuronal coupling represents a new form of neuronal plasticity in the DRG and contributes to pain hypersensitivity by "hijacking" neighboring neurons through gap junctions.