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.

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.

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.

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.

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.

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.

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.

Characterizing the conductance underlying depolarization-induced slow current in cerebellar Purkinje cells.

Brief strong depolarization of cerebellar Purkinje cells produces a slow inward cation current [depolarization-induced slow current (DISC)]. Previous work has shown that DISC is triggered by voltage-sensitive Ca influx in the Purkinje cell and is attenuated by blockers of vesicular loading and fusion. Here, we have sought to characterize the ion channel(s) underlying the DISC conductance. While the brief depolarizing steps that triggered DISC were associated with a large Ca transient, the onset of DISC current corresponded only with the Ca transient decay phase. Furthermore, substitution of external Na with the impermeant cation N-methyl-d-glucamine produced a complete and reversible block of DISC, suggesting that the DISC conductance was not Ca permeant. Transient receptor potential cation channel, subfamily M, members 4 (TRPM4) and 5 (TRPM5) are nonselective cation channels that are opened by Ca transients but do not flux Ca. They are expressed in Purkinje cells of the posterior cerebellum, where DISC is large, and, in these cells, DISC is strongly attenuated by nonselective blockers of TRPM4/5. However, measurement of DISC currents in Purkinje cells derived from TRPM4 null, TRPM5 null, and double null mice as well as wild-type mice with TRPM4 short hairpin RNA knockdown showed a partial attenuation with 35-46% of current remaining. Thus, while the DISC conductance is Ca triggered, Na permeant, and Ca impermeant, suggesting a role for TRPM4 and TRPM5, these ion channels are not absolutely required for DISC.

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.

Streptococcus mutans GS-5 antigen I/II stimulates cell survival in serum deprived-cultures through PI3K/Akt pathways.

The antigen I/II (AgI/II) protein is a major surface protein that mediates the attachment of Streptococcus mutans (S. mutans) to the saliva-coated pellicle. Numerous studies have investigated not only the mechanisms by which AgI/II signaling is transduced within cells, but have also attempted to use AgI/II-specific antibodies to treat dental caries and host immune responses. However, little information is available about the effects of AgI/II on basic cellular events in bone cells. In this study, we examined the effects of the His-tagged recombinant N-terminal half of the AgI/II protein (rAgI/II-N) generated from S. mutans GS-5 on the viability, proliferation, and cell cycle progression of primary calvarial osteoblasts. We also investigated the mechanisms involved in the rAgI/II-N-mediated survival of serum-starved osteoblasts. We found that rAgI/II treatment attenuated the serum deprivation-induced decrease in cell viability and proliferation of osteoblasts. rAgI/II-N also prevented the loss of mitochondrial membrane potential (MMP), alterations in levels of two key mitochondrial Bcl-2 family proteins, and the accumulation of numerous cells into the sub-G(1) phase that were observed in serum-starved osteoblasts. Pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), but not of extracellular signal-regulated kinase or Ras, blocked the rAgI/II-N-mediated protection against serum deprivation-induced cell death. Additional experiments revealed that the integrin α5ß1-mediated PI3K pathway is required for rAgI/II-N-mediated Akt phosphorylation in osteoblasts. Collectively, these results suggest that rAgI/II-N induces survival signals in serum-starved osteoblasts through integrin-induced PI3K/Akt signaling pathways.

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.

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.

Effects of gold nanorod concentration on the depth-related temperature increase during hyperthermic ablation.

The photothermal properties of gold nanorods (GNRs) provide an opportunity for the clinical application of highly efficient and tumor-specific photothermal therapy. For the effective hyperthermic ablation of tumor tissue using GNRs, it is essential to maintain a homogeneous therapeutic temperature in the target tissue during treatment. This study investigates whether the concentration of GNRs affects the distribution of the temperature increase during hyperthermal therapy. The investigation is conducted using polyacrylamide phantoms containing varying amounts of GNRs. In 0.1, 0.25, and 0.5 nM GNR-suspended phantoms, the change in temperature is relatively uniform along the depth of each phantom during laser irradiation at 2 W cm(-2) . In 1.0, 2.0, and 5.0 nM GNR-suspended phantoms, the rates of temperature increase in the deep regions of the phantoms decrease with increasing GNR concentration. At a laser irradiation of 5 W cm(-2) , the temperature of the GNR-suspended phantoms increases at a faster rate, whereas the range of GNR concentrations for maintaining the homogeneity of the temperature increase is not affected. This suggests that the concentration of GNRs is the major determinant of the depth-related temperature increase during hyperthermic ablation. Therefore, prior to the clinical application of hyperthermic ablation using GNRs, the concentration of GNRs has to be optimized to ensure a homogeneous distribution of therapeutic temperature in the targeted tissue.

Dendritic glutamate release produces autocrine activation of mGluR1 in cerebellar Purkinje cells.

In recent years, it has become clear that, in addition to conventional anterograde transmission, signaling in neural circuits can occur in a retrograde manner. This suggests the additional possibility that postsynaptic release of neurotransmitter might be able to act in an autocrine fashion. Here, we show that brief depolarization of a cerebellar Purkinje cell triggers a slow inward current. This depolarization-induced slow current (DISC) is attenuated by antagonists of mGluR1 or TRP channels. DISC is eliminated by a mixture of voltage-sensitive Ca2+ channel blockers and is mimicked by a brief climbing fiber burst. DISC is attenuated by an inhibitor of vesicular glutamate transporters or of vesicular fusion. These data suggest that Ca2+-dependent postsynaptic fusion of glutamate-loaded vesicles evokes a slow inward current produced by activation of postsynaptic mGluR1, thereby constituting a useful form of feedback regulation.

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.

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.

Dopamine signaling is required for depolarization-induced slow current in cerebellar Purkinje cells.

Brief strong depolarization of cerebellar Purkinje cells produces a slow inward cation current. This current, called depolarization-induced slow current (DISC), is triggered by Ca influx in the Purkinje cell and is attenuated by a blocker of vesicular fusion. Previous work in other brain regions, such as the substantia nigra and ventral tegmental area, has shown that dopamine can be released from dendrites to produce paracrine and autocrine signaling. Here, we test the hypothesis that postsynaptic release of dopamine and autocrine activation of dopamine receptors is involved in DISC. Light immunohistochemistry showed that D(3) dopamine receptors, vesicular monoamine transporter type 2 (VMAT2), and dopamine plasma membrane transporters (DATs) were all expressed in cerebellar Purkinje cells. However, their expression was strongest in the gyrus region of cerebellar lobules IX and X. Comparison of DISC across lobules revealed that it was weak in the anterior portions of the cerebellum (lobules II, V, and VI) and strong in lobules IX and X. DISC was blocked by dopamine receptor antagonists (haloperidol, clozapine, eticlopride, and SCH23390). Likewise, DISC was strongly attenuated by inhibitors of VMAT (reserpine and tetrabenazine) and DAT (GBR12909 and rimcazole). These drugs did not produce DISC attenuation through blockade of depolarization-evoked Purkinje cell Ca transients. Purkinje cells in cerebellar slices derived from DAT-null mice expressed DISC, but this DISC ran down at a significantly higher rate than littermate controls. Together, these results suggest that strong Purkinje cell depolarization produces Ca-dependent release of vesicular postsynaptic dopamine that then excites Purkinje cells in an autocrine manner.