Calcitonin Gene-related Peptide In Neurons Research Articles

Sensory neuron transient receptor potential vanilloid-1 channel regulates angiogenesis through CGRP in vivo.

Angiogenesis plays a key role in bone regeneration. The role of neurons of peripheral nerves involved in angiogenesis of bone defects needs to be explored. The transient receptor potential vanilloid 1 (TRPV1), a nociceptor of noxious stimuli, is expressed on sensory neurons. Apart from nociception, little is known about the role of sensory innervation in angiogenesis. Calcitonin gene-related peptide (CGRP), a neuropeptide secreted by sensory nerve terminals, has been associated with vascular regeneration. We characterized the reinnervation of vessels in bone repair and assessed the impact of TRPV1-CGRP signaling on early vascularization. We investigated the pro-angiogenic effect of neuronal TRPV1 in the mouse model of femur defect. Micro-CT analysis with Microfil® reagent perfusion demonstrated neuronal TRPV1 activation enhanced angiogenesis by increasing vessel volume, number, and thickness. Meanwhile, TRPV1 activation upregulated the mRNA and protein expression of vascular endothelial growth factor A (VEGF-A), cell adhesion molecule-1 (CD31), and CGRP. Immunostaining revealed the co-localization of TRPV1 and CGRP in dorsal root ganglia (DRG) sensory neurons. By affecting neuronal TRPV1 channels, the release of neuronal and local CGRP was controlled. We demonstrated that TRPV1 influenced on blood vessel development by promoting CGRP release from sensory nerve terminals. Our results showed that neuronal TRPV1 played a crucial role in regulating angiogenesis during bone repair and provided important clinical implications for the development of novel therapeutic approaches for angiogenesis.

Activation of the BMP2-SMAD1-CGRP pathway in dorsal root ganglia contributes to bone cancer pain in a rat model

Peripheral nerve remodeling and sensitization are involved in cancer-related bone pain. As a member of the transforming growth factor-β class, bone morphogenetic protein 2 (BMP2) is recognized to have a role in the development of the neurological and skeletal systems. Our previous work showed that BMP2 is critical for bone cancer pain (BCP) sensitization. However, the mechanism remains unknown. In the current study, we demonstrated a substantial increase in BMP2 expression in the dorsal root ganglia (DRG) in a rat model of BCP. Knockdown of BMP2 expression ameliorated BCP in rats. Furthermore, the DRG neurons of rats with BCP expressed higher levels of calcitonin gene-related peptide (CGRP), and BCP was successfully suppressed by intrathecal injection of a CGRP receptor blocker (CGRP8-37). Downregulation of BMP2 expression reduced the expression of CGRP in the DRG of rats with BCP and relieved pain behavior. Moreover, we revealed that upregulation of CGRP expression in the DRG may be induced by activation of the BMPR/Smad1 signaling pathway. These findings suggest that BMP2 contributes to BCP by upregulating CGRP in DRG neurons via activating BMPR/Smad1 signaling pathway and that therapeutic targeting of the BMP2-Smad1-CGRP pathway may ameliorate BCP in the context of advanced cancer.

Effect of nerve growth factor on elderly degenerative knee osteoarthritis pain

To explore effect of nerve growth factor (NGF) antibody on knee osteoarthritis (KOA) pain model was evaluated by in vitro model. Thirty male SPF rats aged 28-week-old were divided into blank group (10 rats with anesthesia only). The other 20 rats were with monoiodoacetate (MIA) on the right knee joint to establish pain model of OA, and were randomly divided into control group (injected intraperitoneal injection of normal saline) and treatment group (injected anti-NGF) intraperitoneal after successful modeling, and 10 rats in each group. All rats were received retrograde injection of fluorogold (FG) into the right knee joint. Gait was assessed using catwalk gait analysis system before treatment, 1 and 2 weeks after treatment. Three weeks after treatment, right dorsal root ganglia (DRG) were excised on L4-L6 level, immunostained for calcitonin gene-related peptide (CGRP), and the number of DRGS was counted. In terms of gait analysis using cat track system, duty cycle, swing speed and print area ratio in control and treatment group were significantly reduced compared with blank group (P<0.05). Compared with control group, duty cycle and swing speed of treatment group were significantly improved (P<0.05), and there was no significant difference in print area ratio between treatment group and blank group (P>0.05). The number of FG-labeled DRG neurons in control group was significantly higher than that in treatment group and blank group (P<0.05). The expression of CGRP in control group was up-regulated, and differences were statistically significant compared with treatment group (P<0.05). Intraperitoneal injection of anti-NGF antibody inhibited gait injury and upregulation of CGRP in DRG neurons. The results suggest that anti-nerve growth factor therapy may be of value in treating knee pain. NGF may be an important target for the treatment of knee OA pain.

Folic acid relieves bone cancer pain by downregulating P2X2/3 receptors in rats

BackgroundBone cancer pain (BCP) remains a clinical challenge due to the limited and side effects of therapeutic methods. Folic acid has been known as an FDA approved dietary supplement and proved to have an analgesic effect in neuropathic pain. Here we investigate the role and mechanism of folic acid in bone cancer pain of a rat model. MethodsWalker 256 tumor cells were inoculated into the left tibia of rats to induce bone cancer pain model. Pain reflex were assessed by paw withdrawal threshold (PWT) response to Von Frey filaments and paw withdrawal latency (PWL) response to thermal stimulation. Folic acid was injected intraperitoneally to evaluate its analgesic effect in rats with bone cancer pain. Western blotting and qPCR were used to determine P2X2/3 receptor protein and mRNA levels in ipsilateral L4-6 dorsal root ganglion (DRG) and spinal dorsal horn (SDH). ResultsThe PWT and PWL of rats with bone cancer pain were obviously decreased compared to the naïve and sham rats. Interestingly, continuous folic acid treatment significantly increased the PWT and PWL of rats with bone cancer pain. P2X2 and P2X3 receptors were clearly upregulated at both mRNA and protein expression in L4-6 DRG and SDH of rats with bone cancer pain. P2X2 and P2X3 receptors were mainly localized with CGRP (calcitonin gene-related peptide) or IB4 (isolectin B4) positive neurons in L4-6 DRG of rats with bone cancer pain. Notably, continuous folic acid treatment significantly reduced the expression of P2X2 and P2X3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Finally, intrathecal injection of A317491 (a selective antagonist of P2X2/3 receptors) markedly elevated the PWT and PWL of rats with bone cancer pain. ConclusionThese results suggest that folic acid has an effective antinociceptive effect on bone cancer pain, which is mediated by downregulating P2X2/3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Folic acid may be a novel therapeutic strategy in cancer patients for pain relief.

Activation of Parabrachial Tachykinin 1 Neurons Counteracts Some Behaviors Mediated by Parabrachial Calcitonin Gene-related Peptide Neurons

Many threats activate parabrachial neurons expressing calcitonin gene-related peptide (CGRPPBN) which transmit alarm signals to forebrain regions. Most CGRPPBN neurons also express tachykinin 1 (Tac1), but there are also Tac1-expressing neurons in the PBN that do not express CGRP (Tac1+;CGRP− neurons). Chemogenetic or optogenetic activation of all Tac1PBN neurons in mice elicited many physiological/behavioral responses resembling the activation of CGRPPBN neurons, e.g., anorexia, jumping on a hot plate, avoidance of photostimulation; however, two key responses opposed activation of CGRPPBN neurons. Activating Tac1PBN neurons did not produce conditioned taste aversion and it elicited dynamic escape behaviors rather than freezing. Activating Tac1+;CGRP− neurons, using an intersectional genetic targeting approach, resembles activating all Tac1PBN neurons. These results reveal that activation of Tac1+;CGRP− neurons can suppress some functions attributed to the CGRPPBN neurons, which provides a mechanism to bias behavioral responses to threats.

NGF-Induced Upregulation of CGRP in Orofacial Pain Induced by Tooth Movement Is Dependent on Atp6v0a1 and Vesicle Release.

The nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) play a crucial role in the regulation of orofacial pain. It has been demonstrated that CGRP increases orofacial pain induced by NGF. V-type proton ATPase subunit an isoform 1 (Atp6v0a1) is involved in the exocytosis pathway, especially in vesicular transport in neurons. The objective was to examine the role of Atp6v0a1 in NGF-induced upregulation of CGRP in orofacial pain induced by experimental tooth movement. Orofacial pain was elicited by ligating closed-coil springs between incisors and molars in Sprague–Dawley rats. Gene and protein expression levels were determined through real-time polymerase chain reaction, immunostaining, and fluorescence in situ hybridization. Lentivirus vectors carrying Atp6v0a1 shRNA were used to knockdown the expression of Atp6v0a1 in TG and SH-SY5Y neurons. The release of vesicles in SH-SY5Y neurons was observed by using fluorescence dye FM1-43, and the release of CGRP was detected by Enzyme-Linked Immunosorbent Assy. Orofacial pain was evaluated through the rat grimace scale. Our results revealed that intraganglionic administration of NGF and Atp6v0a1 shRNA upregulated and downregulated CGRP in trigeminal ganglia (TG) and trigeminal subnucleus caudalis (Vc), respectively, and the orofacial pain was also exacerbated and alleviated, respectively, following administration of NGF and Atp6v0a1 shRNA. Besides, intraganglionic administration of NGF simultaneously caused the downregulation of Atp6v0a1 in TG. Moreover, the release of vesicles and CGRP in SH-SY5Y neurons was interfered by NGF and Atp6v0a1 shRNA. In conclusion, in the orofacial pain induced by experimental tooth movement, NGF induced the upregulation of CGRP in TG and Vc, and this process is dependent on Atp6v0a1 and vesicle release, suggesting that they are involved in the transmission of nociceptive information in orofacial pain.

Grape seed extract suppresses calcitonin gene-related peptide secretion and upregulates expression of GAD 65/67 and GABAB receptor in primary trigeminal ganglion cultures.

The trigeminal ganglion is implicated in the underlying pathology of migraine and temporomandibular joint disorders (TMD), which are orofacial pain conditions involving peripheral and central sensitization. The neuropeptide calcitonin gene-related peptide (CGRP) is synthesized in some trigeminal ganglion neurons, and its release promotes inflammation, peripheral and central sensitization, and pain signaling. Recent studies in preclinical migraine and TMD models provide evidence that dietary supplementation with grape seed extract (GSE) inhibits trigeminal pain signaling. The goal of this study was to investigate the cellular mechanisms by which GSE modulates primary trigeminal ganglion cultures. The effect of GSE on CGRP secretion was determined by radioimmunoassay. To determine if GSE effects involved modulation of CGRP or the GABAergic system, expression of CGRP, GAD 65 and 67, GABAA receptor, and GABAB1 and GABAB2 receptor subunits were investigated by immunocytochemistry. GSE significantly inhibited basal CGRP secretion but did not alter neuronal CGRP expression. GAD 65 and 67 expression levels in neurons were significantly increased in response to GSE. While GSE did not cause a change in the neuronal expression of GABAA, GSE significantly increased GABAB1 expression in neurons, satellite glial cells, and Schwann cells. GABAB2 expression was significantly elevated in satellite glia and Schwann cells. These findings support the notion that GSE inhibition of basal CGRP secretion involves increased neuronal GAD 65 and 67 and GABAB receptor expression. GSE repression of CGRP release coupled with increased GABAB1 and GABAB2 glial cell expression would be neuroprotective by suppressing neuronal and glial excitability in the trigeminal ganglion.

Stimulation of CGRP-expressing neurons in the medial cerebellar nucleus induces light and touch sensitivity in mice

Calcitonin gene-related peptide (CGRP) is considered a major player in migraine pathophysiology. However, the location and mechanisms of CGRP actions in migraine are not clearly elucidated. One important question yet to be answered is: Does central CGRP signaling play a role in migraine? One candidate site is the cerebellum, which serves as a sensory and motor integration center and is activated in migraine patients. The cerebellum has the most CGRP binding sites in the central nervous system and a deep cerebellar nucleus, the medial nucleus (MN), expresses CGRP (MNCGRP). A previous study demonstrated that CGRP delivery into the cerebellum induced migraine-like behaviors. We hypothesized that stimulation of MNCGRP neurons might induce migraine-like behaviors. To test the hypothesis, we used an optogenetic strategy using CalcaCre/+ mice to drive Cre-dependent expression of channelrhodopsin-2 selectively in CGRP neurons in the cerebellar MN. A battery of behavioral tests was done to assess preclinical behaviors that are surrogates of migraine symptoms, including light aversion, cutaneous allodynia, and spontaneous pain when MNCGRP neurons were optically stimulated. Motor functions were also assessed. Optical stimulation of MNCGRP neurons decreased the time spent in the light, which was coupled to increased time spent resting in the dark, but not the light. These changes were only significant in female mice. Plantar tactile sensitivity was increased in the ipsilateral paws of both sexes, but contralateral paw data were less clear. There was no significant increase in anxiety-like behavior, spontaneous pain (squint), or changes in gait. These discoveries reveal that MNCGRP neurons may contribute to migraine-like sensory hypersensitivity to light and touch.

Promotion of corneal angiogenesis by sensory neuron-derived calcitonin gene-related peptide

The normal cornea has no blood vessels but has abundant innervation. There is emerging evidence that sensory nerves, originated from the trigeminal ganglion (TG) neurons, play a key role in corneal angiogenesis. In the current study, we examined the role of TG sensory neuron-derived calcitonin gene-related peptide (CGRP) in promoting corneal neovascularization (CNV). We found that CGRP was expressed in the TG and cultured TG neurons. In the cornea, minimal CGRP mRNA was detected and CGRP immunohistochemical staining was exclusively co-localized with corneal nerves, suggesting corneal nerves are likely the source of CGRP in the cornea. In response to intrastromal suture placement and neovascularization in the cornea, CGRP expression was increased in the TG. In addition, we showed that CGRP was potently pro-angiogenic, leading to vascular endothelial cell (VEC) proliferation, migration, and tube formation in vitro and corneal hemangiogenesis and lymphangiogenesis in vivo. In a co-culture system of TG neurons and VEC, blocking CGRP signaling in the conditioned media of TG neurons led to decreased VEC migration and tube formation. More importantly, subconjunctival injection of a CGRP antagonist CGRP8-37 reduced suture-induced corneal hemangiogenesis and lymphangiogenesis in vivo. Taken together, our data suggest that TG sensory neuron and corneal nerve-derived CGRP promotes corneal angiogenesis.

CGRP and the Calcitonin Receptor are Co-Expressed in Mouse, Rat and Human Trigeminal Ganglia Neurons.

The neuropeptide calcitonin gene-related peptide (CGRP) is expressed in the trigeminal ganglia, a key site in craniofacial pain and migraine. CGRP potently activates two receptors: the CGRP receptor and the AMY1 receptor. These receptors are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with either the calcitonin receptor-like receptor (CLR) to form the CGRP receptor or the calcitonin receptor (CTR) to form the AMY1 receptor. The expression of the CGRP receptor in trigeminal ganglia has been described in several studies; however, there is comparatively limited data available describing AMY1 receptor expression and in which cellular subtypes it is found. This research aimed to determine the relative distributions of the AMY1 receptor subunit, CTR, and CGRP in neurons or glia in rat, mouse and human trigeminal ganglia. Antibodies against CTR, CGRP and neuronal/glial cell markers were applied to trigeminal ganglia sections to investigate their distribution. CTR-like and CGRP-like immunoreactivity were observed in both discrete and overlapping populations of neurons. In rats and mice, 30–40% of trigeminal ganglia neurons displayed CTR-like immunoreactivity in their cell bodies, with approximately 78–80% of these also containing CGRP-like immunoreactivity. Although human cases were more variable, a similar overall pattern of CTR-like immunoreactivity to rodents was observed in the human trigeminal ganglia. CTR and CGRP appeared to be primarily colocalized in small to medium sized neurons, suggesting that colocalization of CTR and CGRP may occur in C-fiber neurons. CGRP-like or CTR-like immunoreactivity were not typically observed in glial cells. Western blotting confirmed that CTR was expressed in the trigeminal ganglia of all three species. These results confirm that CTR is expressed in trigeminal ganglia neurons. The identification of populations of neurons that express both CGRP and CTR suggests that CGRP could act in an autocrine manner through a CTR-based receptor, such as the AMY1 receptor. Overall, this suggests that a trigeminal ganglia CTR-based receptor may be activated during migraine and could therefore represent a potential target to develop treatments for craniofacial pain and migraine.

Hypophagia induced by salmon calcitonin, but not by amylin, is partially driven by malaise and is mediated by CGRP neurons

ObjectiveThe behavioral mechanisms and the neuronal pathways mediated by amylin and its long-acting analog sCT (salmon calcitonin) are not fully understood and it is unclear to what extent sCT and amylin engage overlapping or distinct neuronal subpopulations to reduce food intake. We here hypothesize that amylin and sCT recruit different neuronal population to mediate their anorectic effects. MethodsViral approaches were used to inhibit calcitonin gene-related peptide (CGRP) lateral parabrachial nucleus (LPBN) neurons and assess their role in amylin’s and sCT’s ability to decrease food intake in mice. In addition, to test the involvement of LPBN CGRP neuropeptidergic signaling in the mediation of amylin and sCT’s effects, a LPBN site-specific knockdown was performed in rats. To deeper investigate whether the greater anorectic effect of sCT compared to amylin is due do the recruitment of additional neuronal pathways related to malaise multiple and distinct animal models tested whether amylin and sCT induce conditioned avoidance, nausea, emesis, and conditioned affective taste aversion. ResultsOur results indicate that permanent or transient inhibition of CGRP neurons in LPBN blunts sCT-, but not amylin-induced anorexia and neuronal activation. Importantly, sCT but not amylin induces behaviors indicative of malaise including conditioned affective aversion, nausea, emesis, and conditioned avoidance; the latter mediated by CGRPLPBN neurons. ConclusionsTogether, the present study highlights that although amylin and sCT comparably decrease food intake, sCT is distinctive from amylin in the activation of anorectic neuronal pathways associated with malaise.

Sensory nerves promote corneal inflammation resolution via CGRP mediated transformation of macrophages to the M2 phenotype through the PI3K/AKT signaling pathway

ObjectivesTo explore the role of the corneal sensory nerves in Pseudomonas aeruginosa (P. aeruginosa) keratitis, the synergistic effect between the sensory neurons and macrophages in calcitonin gene-related peptide (CGRP) release, and the functional mechanisms of CGRP-mediated transformation of macrophages to the M2 phenotype. MethodsCorneal nerve loss, macrophage recruitment, and CGRP expression were evaluated. To explore the synergistic effect between the sensory neurons and macrophages, RAW 264.7 cells were challenged with lipopolysaccharide (LPS), then trigeminal ganglion (TG) sensory neurons were isolated and co-incubated with macrophages, and CGRP expression was tested. To investigate the biological function of cornea neuron-initiated immune responses mediated by CGRP, BIBN 4096BS was used to inhibit CGRP in vivo and α-CGRP was used to simulate CGRP in vitro. The expressions of inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-10), M1 (CD80/CD86), M2 (CD163/CD206) macrophage markers, and signal transducers (PI3K/AKT) were detected. ResultsP. aeruginosa infection induced corneal nerve loss, macrophage recruitment, and CGRP up-expression. CGRP was co-localized with macrophages. Co-culture showed that sensory neurons and macrophages can mediate CGRP release. More CGRP was released when the two types of cells were combined to respond to LPS. BIBN 4096BS promoted pro-inflammatory cytokines and inhibited the anti-inflammatory cytokines and signal transducers, while, α-CGRP inhibited the pro-inflammatory cytokines and M1 markers and promoted the anti-inflammatory cytokine, M2 markers, and signal transducers. ConclusionsP. aeruginosa infection induces corneal sensory neuron activation, macrophage recruitment, and CGRP up-expression. The synergistic effect between the sensory neurons and macrophages promotes CGRP release. CGRP inhibits corneal inflammation and promotes the transformation of macrophages to the M2 phenotype through the PI3K/AKT signaling pathway.

Calcitonin gene related peptide \u03b1 is dispensable for many danger-related motivational responses

Calcitonin gene related peptide (CGRP) expressing neurons in the parabrachial nucleus have been shown to encode danger. Through projections to the amygdala and other forebrain structures, they regulate food intake and trigger adaptive behaviors in response to threats like inflammation, intoxication, tumors and pain. Despite the fact that this danger-encoding neuronal population has been defined based on its CGRP expression, it is not clear if CGRP is critical for its function. It is also not clear if CGRP in other neuronal structures is involved in danger-encoding. To examine the role of CGRP in danger-related motivational responses, we used male and female mice lacking αCGRP, which is the main form of CGRP in the brain. These mice had no, or only very weak, CGRP expression. Despite this, they did not behave differently compared to wildtype mice when they were tested for a battery of danger-related responses known to be mediated by CGRP neurons in the parabrachial nucleus. Mice lacking αCGRP and wildtype mice showed similar inflammation-induced anorexia, conditioned taste aversion, aversion to thermal pain and pain-induced escape behavior, although it should be pointed out that the study was not powered to detect any possible differences that were minor or sex-specific. Collectively, our findings suggest that αCGRP is not necessary for many threat-related responses, including some that are known to be mediated by CGRP neurons in the parabrachial nucleus.

Satb2 neurons in the parabrachial nucleus mediate taste perception

The neural circuitry mediating taste has been mapped out from the periphery to the cortex, but genetic identity of taste-responsive neurons has remained elusive. Here, we describe a population of neurons in the gustatory region of the parabrachial nucleus that express the transcription factor Satb2 and project to taste-associated regions, including the gustatory thalamus and insular cortex. Using calcium imaging in awake, freely licking mice, we show that Satb2 neurons respond to the five basic taste modalities. Optogenetic activation of these neurons enhances taste preferences, whereas chronic inactivation decreases the magnitude of taste preferences in both brief- and long-access taste tests. Simultaneous inactivation of Satb2 and calcitonin gene-related peptide neurons in the PBN abolishes responses to aversive tastes. These data suggest that taste information in the parabrachial nucleus is conveyed by multiple populations of neurons, including both Satb2 and calcitonin gene-related peptide neurons.

Neuronal calcitonin gene-related peptide promotes prostate tumor growth in the bone microenvironment

Advanced stage of prostate cancer cells preferentially metastasizes to varying bones of prostate cancer patients, resulting in incurable disease with poor prognosis and limited therapeutical treatment options. Calcitonin gene-related peptide (CGRP), a neuropeptide produced by prostate gland, is known to play a pivotal role in facilitating tumor growth and metastasis of numerous human cancers. In this study, we aim to investigate the clinical relevance of CGRP in prostate cancer patients and the effects of CGRP and CGRP antagonists on prostate tumor growth in the mouse model. The prostate tumor-bearing mice were received either CGRP or CGRP antagonist treatment, and the tumor growth was monitored by quantification of luminescence intensities. We found that the CGRP+ nerve fiber density and serum CGRP levels were substantially upregulated in the bone or serum specimens from advanced prostate cancer patients as well as in prostate tumor-bearing mice. Administration of CGRP promoted, whereas treatment of CGRP antagonists inhibited prostate tumor growth in the femurs of mice. In addition, CGRP treatment activated extracellular signal-regulated kinases (ERKs)/ Signal transducer and activator of transcription 3 (STAT3) signaling in prostate cancer cells. Targeting CGRP may serve as a potential therapeutic strategy for advanced prostate cancer patients.

Noradrenaline signaling in the LPBN mediates amylin's and salmon calcitonin's hypophagic effect in male rats

The LPBN (lateral parabrachial nucleus) plays an important role in feeding control. CGRP (calcitonin gene-related peptide) LPBN neurons activation mediates the anorectic effects of different gut-derived peptides, including amylin. Amylin and its long acting analog sCT (salmon calcitonin) exert their anorectic actions primarily by directly activating neurons located in the area postrema (AP). A large proportion of projections from the AP and the adjacent nucleus of the solitary tractNTS to the LPBN, are noradrenergic (NA), and amylin-activated NAAP neurons are critical in mediating amylin's hypophagic effects. Here, we determine the functional role of NAAP amylin activated neurons to activate CGRP and non-CGRP LPBN neurons. To this end, NA was specifically depleted in the rat LPBN through a stereotaxic microinfusion of 6-OHDA, a neurotoxic agent that destroys NA terminals. While amylin (50μg/kg) and sCT (5μg/kg) reduced eating in sham-lesioned rats, no reduction in feeding occurred in NA-depleted animals. Further, the amylin-induced c-Fos response in the LPBN and c-Fos/CGRP colocalization were reduced in NA-depleted animals compared to controls. We conclude that AP→LPBN NA signaling, through the activation of LPBN CGRP neurons, mediates part of amylin's hypophagic effect.

Cytokines Modulate the Expression of Calcitonin‐Gene‐Related‐Peptide and Substance P in Primary Rat DRG Neuron‐glia Co‐cultures

In the peripheral nervous system, dorsal root ganglion (DRG) neurons are the primary sensory neuron responsible for the transmission of temperature, touch, proprioception and pain. Recently, there has been increased interest in the role of neuro‐immune interactions in peripherally‐mediated neuropathic pain. Activated immune cells that infiltrate DRG release a mixture of inflammatory mediators that play a role in the development and maintenance of pain. However, the relationship between immune cells, pro‐inflammatory mediators and sensory neurons in the development and maintenance of pain are not completely understood. In this study, we investigated the effects of the cytokines interferon gamma (IFNγ) and tumor necrosis factor‐alpha (TNFα), and the chemokine fractalkine on the expression of calcitonin‐gene‐related‐peptide (CGRP) and substance P (SP) in DRG neuron‐glia co‐cultures. IFNγ decreased CGRP and substance P expression while TNFα increased CGRP expression, preferentially in small and medium size neurons. IFNγ and TNFα, both also increased nitric oxide (NO) production in a concentration‐dependent manner, at concentrations that affected CGRP and SP expression. However, inhibitors of nitric oxide synthase (L‐NAME) and of IKKB/NFkB (TPCA‐1) did not alter TNFα effects on CGRP expression. The data from this study demonstrates that cytokines and chemokines differentially modulate the expression of CGRP and SP in DRG neurons. Understanding the regulation of these two key inflammatory neuropeptides may provide novel insights regarding the development of new and effective therapeutics for neuropathic pain.Support or Funding InformationThis work was supported by NIH (grant # R21 AG065908).

Transient receptor potential melastatin-3 in the rat sensory ganglia of the trigeminal, glossopharyngeal and vagus nerves

Transient receptor potential melastatin-3 (TRPM3) is a nonselective cation channel, has permeability of Ca2+, and probably participates in thermosensitive nociception. In this study, immunohistochemistry for TRPM3 was conducted in the rat trigeminal, glossopharyngeal and vagal sensory ganglia. TRPM3-immunoreactivity was expressed by half of sensory neurons in the trigeminal (TG), petrosal (PG) and jugular ganglia (JG), and by about 80% of sensory neurons in the nodose ganglion (NG). They mostly had small to medium-sized cell bodies. A trichrome immunofluorescence method showed co-existence of TRPM3 with TRP vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP). Approximately 70% of TRPM3-immunoreactive (-IR) neurons contained TRPV1-immunoreactivity in all the examined ganglia. More than 40% of TRPM3-IR neurons exhibited CGRP-immunoreactivity in the TG, PG and JG. Only a few sensory neurons co-expressed TRPM3- and CGRP-immunoreactivity in the NG. In addition, more than 40% of TRPM3-IR neurons bound to isolectin B4 in all the examined ganglia. By combination of retrograde tracing method and immunohistochemistry, half of TG neurons innervating the facial skin and incisive papilla expressed TRPM3-immunoreactivity whereas approximately 20% of those innervating the tooth pulp contained TRPM3-immunoreactivity. Co-expression of TRPM3-immunoreactivity with TRPV1- or CGRP-immunoreactivity was common among cutaneous and papillary TG neurons but not among pulpal TG neurons. More than 60% of PG and JG neurons innervating the external ear canal skin and circumvallate papilla contained TRPM3-immunoreactivity. Co-expression of TRPM3 with TRPV1 or CGRP was common among PG and JG neurons innervating the external ear canal skin. However, a smaller number of TRPM3-IR neurons co-expressing TRPV1- or CGRP-immunoreactivity innervate the circumvallate papilla in the PG. The present study suggests that expression of TRPM3 and its co-existence with TRPV1 and CGRP in sensory neurons depend on the variety of their peripheral targets in the trigeminal, glossopharyngeal and vagal nervous systems.

Dynamic changes in CGRP, PACAP, and PACAP receptors in the trigeminovascular system of a novel repetitive electrical stimulation rat model: Relevant to migraine.

Migraine is the seventh most disabling disorder globally, with prevalence of 11.7% worldwide. One of the prevailing mechanisms is the activation of the trigeminovascular system, and calcitonin gene-related peptide (CGRP) is an important therapeutic target for migraine in this system. Recent studies suggested an emerging role of pituitary adenylate cyclase-activating peptide (PACAP) in migraine. However, the relation between CGRP and PACAP and the role of PACAP in migraine remain undefined. In this study, we established a novel repetitive (one, three, and seven days) electrical stimulation model by stimulating dura mater in conscious rats. Then, we determined expression patterns in the trigeminal ganglion and the trigeminal nucleus caudalis of the trigeminovascular system. Electrical stimulation decreased facial mechanical thresholds, and the order of sensitivity was as follows: vibrissal pad >inner canthus >outer canthus (P < 0.001). The electrical stimulation group exhibited head-turning and head-flicks (P < 0.05) nociceptive behaviors. Importantly, electrical stimulation increased the expressions of CGRP, PACAP, and the PACAP-preferring type 1 (PAC1) receptor in both trigeminal ganglion and trigeminal nucleus caudalis (P < 0.05). The expressions of two vasoactive intestinal peptide (VIP)-shared type 2 (VPAC1 and VPAC2) receptors were increased in the trigeminal ganglion, whereas in the trigeminal nucleus caudalis, their increases were peaked on Day 3 and then decreased by Day 7. PACAP was colocalized with NEUronal Nuclei (NeuN), PAC1, and CGRP in both trigeminal ganglion and the trigeminal nucleus caudalis. Our results demonstrate that the repetitive electrical stimulation model can simulate the allodynia during the migraine chronification, and PACAP plays a role in the pathogenesis of migraine potentially via PAC1 receptor.

Parabrachial CGRP Neurons Establish and Sustain Aversive Taste Memories

Food aversions develop when the taste of a novel food is associated with sickness, which often occurs after food poisoning or chemotherapy treatment. We identified calcitonin-gene-related peptide (CGRP) neurons in the parabrachial nucleus (PBN) as sufficient and necessary for establishing a conditioned taste aversion (CTA). Photoactivating projections from CGRPPBN neurons to either the central nucleus of the amygdala or the bed nucleus of the stria terminalis can also induce robust CTA. CGRPPBN neurons undergo plasticity following CTA, and inactivation of either Arc or Grin1 (genes involved in memory consolidation) prevents establishment of a strong CTA. Calcium imaging reveals that the novel food re-activates CGRPPBN neurons after conditioning. Inhibition of these neurons or inactivation of the Grin1 gene after conditioning attenuates CTA expression. Our results indicate that CGRPPBN neurons not only play a key role for learning food aversions but also contribute to the maintenance and expression of those memories.