Calcitonin Gene-related Peptide Immunoreactivity Research Articles

Evaluation of innervation in cleft affected oral mucosa

Orofacial clefts are one of the most common pathologies present at birth. The aim of the study was to evaluate the presence of PGP 9.5, substance P, VIP, CGRP, myelin and NFG in cleft affected mucosa of alveolar process, soft and hard palate, vomer and lip.
 Methods. Orofacial cleft affected mucosa tissue samples were obtained during surgical cleft correction from 21 children aged from 2 months to 9 years and 10 months. Prepared tissue sections were stained by immunohistochemistry for PGP 9.5, substance P, VIP, CGRP, myelin and NGF. The intensity of staining was graded semiquantitatively.
 Results. Mostly moderate number and numerous PGP 9.5, substance P, VIP, CGRP, myelin and NGF-containing nerve fibres and nerve fibre bundles and CGRP and NGF positive keratinocytes were detected. The most immunoreactive for PGP 9.5, myelin and NGF were nerve fibres, nerve fibre bundles and keratinocytes in lip mucosa tissue samples. Typically, PGP 9.5-containing nerve fibres were detected in subepithelium close to basal lamina, near blood vessels; thin PGP 9.5 positive nerve fibres were also observed between keratinocytes. Immunoreactivity of CGRP was higher than of substance P in nerve fibres and nerve fibre bundles. Statistically significant positive correlation was observed between all markers in subepithelium of all tissue samples.
 Conclusions. The proved statistically significant strong inter-correlation and the number of general neuropeptide-containing innervation (PGP 9.5), sensory nerves (CGRP and substance P), and parasympathetic nerves (VIP) is similar in orofacial cleft affected oral mucosa to such described in scientific literature healthy oral mucosa, except the alveolar process covering mucosa where the above innervation varies.
 Very strong and strong statistically significant positive correlations between the number of PGP 9.5, substance P, CGRP and VIP positive structures on the one hand, and NGF and myelin, on the other hand, indicate a connection between quality markers and common neuropeptide-containing, sensory and parasympathetic innervation in cleft affected oral tissue.
 The number of PGP 9.5, NGF and myelin immunoreactive neuronal structures in lip mucosa is higher than in the alveolar process, soft and hard palate and vomer mucosa, suggesting the significance of qualitative common innervation in this organ even in the cleft affected case.

Functional segregation within the pelvic nerve of male rats: a meso- and microscopic analysis.

The pelvic splanchnic nerves are essential for pelvic organ function and have been proposed as targets for neuromodulation. We have focused on the rodent homologue of these nerves, the pelvic nerves. Our goal was to define within the pelvic nerve the projections of organ‐specific sensory axons labelled by microinjection of neural tracer (cholera toxin, subunit B) into the bladder, urethra or rectum. We also examined the location of peptidergic sensory axons within the pelvic nerves to determine whether they aggregated separately from sacral preganglionic and paravertebral sympathetic postganglionic axons travelling in the same nerve. To address these aims, microscopy was performed on the major pelvic ganglion (MPG) with attached pelvic nerves, microdissected from young adult male Sprague–Dawley rats (6–8 weeks old) and processed as whole mounts for fluorescence immunohistochemistry. The pelvic nerves were typically composed of five discrete fascicles. Each fascicle contained peptidergic sensory, cholinergic preganglionic and noradrenergic postganglionic axons. Sensory axons innervating the lower urinary tract (LUT) consistently projected in specific fascicles within the pelvic nerves, whereas sensory axons innervating the rectum projected in a complementary group of fascicles. These discrete aggregations of organ‐specific sensory projections could be followed along the full length of the pelvic nerves. From the junction of the pelvic nerve with the MPG, sensory axons immunoreactive for calcitonin gene‐related peptide (CGRP) showed several distinct patterns of projection: some projected directly to the cavernous nerve, others projected directly across the surface of the MPG to the accessory nerves and a third class entered the MPG, encircling specific cholinergic neurons projecting to the LUT. A subpopulation of preganglionic inputs to noradrenergic MPG neurons also showed CGRP immunoreactivity. Together, these studies reveal new molecular and structural features of the pelvic nerves and suggest functional targets of sensory nerves in the MPG. These anatomical data will facilitate the design of experimental bioengineering strategies to specifically modulate each axon class.

Contribution of nerves within osteochondral channels to osteoarthritis knee pain in humans and rats

Subchondral bone may contribute to knee osteoarthritis (OA) pain. Nerve growth factor (NGF) can stimulate nerve growth through TrkA. We aimed to identify how sensory nerve growth at the osteochondral junction in human and rat knees associates with OA pain. Eleven symptomatic chondropathy cases were selected from people undergoing total knee replacement for OA. Twelve asymptomatic chondropathy cases who had not presented with knee pain were selected post-mortem. OA was induced in rat knees by meniscal transection (MNX) and sham-operated rats were used as controls. Twice-daily oral doses (30mg/kg) of TrkA inhibitor (AR786) or vehicle were administered from before and up to 28 days after OA induction. Joints were analysed for macroscopic appearances of articular surfaces, OA histopathology and calcitonin gene-related peptide-immunoreactive (CGRP-IR) sensory nerves in medial tibial plateaux, and rats were assessed for pain behaviors. The percentage of osteochondral channels containing CGRP-IR nerves in symptomatic chondropathy was higher than in asymptomatic chondropathy (difference: 2.5% [95% CI: 1.1-3.7]), and in MNX-than in sham-operated rat knees (difference: 7.8% [95%CI: 1.7-15.0]). Osteochondral CGRP-IR innervation was significantly associated with pain behavior in rats. Treatment with AR786 prevented the increase in CGRP-IR nerves in osteochondral channels and reduced pain behavior in MNX-operated rats. Structural OA was not significantly affected by AR786 treatment. CGRP-IR sensory nerves within osteochondral channels are associated with pain in human and rat knee OA. Reduced pathological innervation of the osteochondral junction might contribute to analgesic effects of reduced NGF activity achieved by blocking TrkA.

Reactive dicarbonyl compounds cause Calcitonin Gene-Related Peptide release and synergize with inflammatory conditions in mouse skin and peritoneum

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene-Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

CGRP in rat mesenteric artery and vein - receptor expression, CGRP presence and potential roles

CGRP is a potent dilator of arteries and despite rich perivascular CGRP immunoreactivity in both arteries and veins the role of CGRP in veins remains unknown.The aim of the current study was to compare perivascular CGRP immunoreactivity and expression of CGRP receptor mRNA and CGRP receptor immunoreactivity in rat mesenteric arteries and veins. Furthermore, potential vasomotor effects of CGRP were explored in veins.Immunohistochemical studies reproduced rich perivascular CGRP innervation in arteries and in veins. Further, the presence of mRNA encoding the CGRP receptor subunits, CLR and RAMP1, were demonstrated in both arteries and veins using qPCR.Before comparing the vasoactive effects of CGRP in arteries and veins, we aimed to identify an experimental setting where vasomotor responses could be detected. Therefore, a length-tension study was performed in artery and vein segments. Whereas the arteries showed the characteristic monophasic curve with an IC/IC100 value of 0.9, surprisingly the veins showed a biphasic response with two corresponding IC/IC100 values of 0.7 and 0.9, respectively. There was no significant difference between fresh and cultured vasculature segments.To investigate whether a potential tension-dependent CGRP-induced dilation of veins caused the decline between the two IC/IC100 peaks, a second study was performed, with the CGRP receptor antagonist, BIBN4096BS (olcegepant) and the sensory nerve secretagogue, capsaicin. No significant vascular role of endogenous perivascular CGRP in mesenteric veins could be concluded, and a potential role of the rich perivascular CGRP and CGRP receptor abundancy in veins remains unknown.

Identification of spinal afferent nerve endings in the colonic mucosa and submucosa that communicate directly with the spinal cord: The gut-brain axis.

The major sensory nerve pathway between the colon and central nervous system (spinal cord and brain) that underlies the gut-brain axis, is via spinal afferent neurons, with cell bodies in dorsal root ganglia (DRG). Our aim was to identify the sensory nerve endings in the colon that arise from single colorectal-projecting DRG neurons. C57BL/6 mice were anesthetized and lumbosacral L6-S1 DRG injected with dextran biotin. Mice recovered for 7 days. The whole colon was then removed and stained to visualize single axons and nerve endings immunoreactive to calcitonin gene-related peptide (CGRP). Single axons arising from DRG were identified in the distal colon and their morphological features and CGRP immunoreactivity characterized. After entering the colon, single axons ramified rostrally or caudally along many rows of myenteric ganglia with little circumferential displacement, giving off varicose endings in multiple ganglia. Nerve endings arising from two classes of colorectal-projecting DRG neuron were identified. One class was peptidergic neurons that had nerve endings in circular muscle, myenteric ganglia, and submucosa. Another class of nonpeptidergic neurons innervated mucosal crypts, myenteric ganglia, and submucosa. Different morphological types of nerve endings which innervate different anatomical layers of colon can arise from the same axon and sensory neuron in DRG. These findings suggest single peptidergic and nonpeptidergic sensory neurons in DRG are potentially capable of detecting sensory stimuli from different anatomical layers of the colon, via different types of nerve endings.

The Presence of Calcitonin Gene-Related Peptide and Its Receptors in Rat, Pig and Human Brain: Species Differences in Calcitonin Gene-Related Peptide Pharmacology

Aim: The aim of present study is to investigate the binding characteristics of non-peptide calcitonin gene-related peptide (CGRP) receptor antagonists (i.e., gepants) in the brain membranes of rat, pig and human. Methods: The interaction of available gepants with the CGRP receptor was studied in the brain membranes of 3 different species using a radioligand competitive binding assay. In addition, the distribution of CGRP and its receptor component receptor activity modifying protein 1 (RAMP1) in rat cerebellum and cortex was explored using immunohistochemistry. Results: All gepants, except SB268262, displaced 100% of the radioligand specific binding in the brain tissue of all 3 species and showed highest affinity for CGRP receptors in human brain as compared to rat and pig brain membranes. Furthermore, radioligand binding studies revealed the presence of higher CGRP receptor density in human cerebellum compared to human cortex. The morphology, size and density of CGRP immunoreactive cells suggest that all cerebral cortical neurons were positive for CGRP. Slender receptor immunoreactive fibres were found spanning through the entire cortex. CGRP immunoreactivity was displayed in the cell soma of cerebellar Purkinje cells and in large neurons in the medial cerebellar nucleus. RAMP1 was found on the surface of the Purkinje cells and in parallel fibres, indicating presence in the granule cell axons. Conclusion: Cerebellum and cerebral cortex are rich in CGRP and CGRP receptors, which can be antagonized by gepants. However, all gepants display higher affinity for human CGRP receptors as compared to rat and pig CGRP receptors. Furthermore, human cerebellum seems to express higher density of CGRP receptors.

Tachykinins modulate nociceptive responsiveness and sensitization: In vivo electrical characterization of primary sensory neurons in tachykinin knockout (Tac1 KO) mice.

Since the failure of specific substance P antagonists to induce analgesia, the role of tachykinins in the development of neuropathic pain states has been discounted. This conclusion was reached without studies on the role of tachykinins in normal patterns of primary afferents response and sensitization or the consequences of their absence on the modulation of primary mechanonociceptive afferents after injury. Nociceptive afferents from animals lacking tachykinins (Tac1 knockout) showed a disrupted pattern of activation to tonic suprathreshold mechanical stimulation. These nociceptors failed to encode the duration and magnitude of natural pronociceptive stimuli or to develop mechanical sensitization as consequence of this stimulation. Moreover, paw edema, hypersensitivity, and weight bearing were also reduced in Tac1 knockout mice 24 h after paw incision surgery. At this time, nociceptive afferents from these animals did not show the normal sensitization to mechanical stimulation or altered membrane electrical hyperexcitability as observed in wild-type animals. These changes occurred despite a similar increase in calcitonin gene-related peptide immunoreactivity in sensory neurons in Tac1 knockout and normal mice. Based on these observations, we conclude that tachykinins are critical modulators of primary nociceptive afferents, with a preeminent role in the electrical control of their excitability with sustained activation or injury.

Age-Dependent Anti-migraine Effects of Valproic Acid and Topiramate in Rats

Background: Valproic acid (VPA) and topiramate (TPM), initially developed as antiepileptics, are approved for migraine prophylaxis in adults but not children. The differences in their antimigraine mechanism(s) by age remain unclear.Methods: A migraine model induced by intra-cisternal (i.c.) capsaicin instillation in pediatric (4–5 weeks) and adult (8–9 weeks) rats was pretreated with VPA (30, 100 mg/kg) or TPM (10, 30, 100 mg/kg). Noxious meningeal stimulation by the irritant capsaicin triggered trigeminovascular system (TGVS) activation mimicking migraine condition, which were assessed peripherally by the depletion of calcitonin gene-related peptide (CGRP) in sensory nerve fibers of the dura mater, the increased CGRP immunoreactivity at trigeminal ganglia (TG) and centrally by the number of c-Fos-immunoreactive (c-Fos-ir) neurons in the trigeminocervical complex (TCC). Peripherally, CGRP released from dural sensory nerve terminals of TG triggered pain signal transmission in the primary afferent of trigeminal nerve, which in turn caused central sensitization of the TGVS due to TCC activation and hence contributed to migraine.Results: In the VPA-treated group, the central responsiveness expressed by reducing the number of c-Fos-ir neurons, which had been increased by i.c. capsaicin, was significant in pediatric, but not adult, rats. Inversely, VPA was effective in peripheral inhibition of elevated CGRP immunoreactivity in the TG and CGRP depletion in the dura mater of adult, but not pediatric, rats. In TPM group, the central responsiveness was significant in both adult and pediatric groups. Peripherally, TPM significantly inhibited capsaicin-induced CGRP expression of TG in adult, but not pediatric, rats. Interestingly, the capsaicin-induced depletion of CGRP in dura was significantly rescued by TPM at high doses in adults, but at low dose in pediatric group.Conclusion: These results suggest VPA exerted peripheral inhibition in adult, but central suppression in pediatric migraine-rats. In contrast, TPM involves both central and peripheral inhibition of migraine with an optimal therapeutic window in both ages. These findings may clarify the age-dependent anti-migraine mechanism of VPA and TPM, which may guide the development of new pediatric anti-migraine drugs in the future.

Anti-inflammatory role of methotrexate in adjuvant arthritis: effect on substance p and calcitonin gene-related Peptide in thymus and spleen.

To investigate the anti-inflammatory effect of methotrexate (MTX) in rats with adjuvant arthritis through its influence on the expression of proinflammatory neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) in immune organs, thymus and spleen. Phase-I pre-clinical trial. The Aga Khan University, Karachi, from July to December 2003. Adjuvant arthritis was induced in rats by inoculation with heat-killed mycobacteria. One group of arthritic rats (n=6) was treated with MTX (0.2 mg/kg body weight, subcutaneously) on every 4th day for a period of 18 weeks, while another group of arthritic rats (n=6) was treated with physiological saline served as control. At the end of experiment, animals were sacrificed and thymus and spleen were dissected and prepared for immunohistochemical analysis. The neuronal density of SP and CGRP immunoreactivity in thymus and spleen was assessed by semi-quantitative analysis. There was a marked reduction in hind paw swelling and inflammation in the MTX-treated rats after 18 weeks of treatment. Restoration of joint spaces (tibiotalar and subtalar) was seen after 9 weeks of MTX treatment. CGRP-positive nerve fibres were significantly reduced (p=0.0001) in thymus of rats treated with MTX compared to control rats. SP-positive nerve fibers were also found to be decreased in thymus of rats treated with MTX compared to controls, however, the decrease was not statistically significant. The neuronal density of SP and CGRP-immunoreactivity in spleen was not significantly different in MTX-treated and placebo-treated rats. In arthritic rats, MTX significantly reduced CGRP expression in thymus. Suppression of pro-inflammatory neuropeptides, such as CGRP and probably SP could be another mechanism by which MTX produces its anti-inflammatory effect in adjuvant arthritis.

The α6 subunit-containing GABAA receptor: A novel drug target for inhibition of trigeminal activation

Novel treatments against migraine are an urgent medical requirement. The α6 subunit-containing GABAA receptors (α6GABAARs) are expressed in trigeminal ganglia (TG), the hub of the trigeminal vascular system (TGVS) that is involved in the pathogenesis of migraine. Here we reveal an unprecedented role of α6GABAARs in ameliorating TGVS activation using several pharmacological approaches in an animal model mimicking pathological changes in migraine. TGVS activation was induced by intra-cisternal (i.c.) instillation of capsaicin in Wistar rats. Centrally, i.c. capsaicin activated the trigeminal cervical complex (TCC) measured by the increased number of c-Fos-immunoreactive (c-Fos-ir) TCC neurons. Peripherally, it elevated calcitonin gene-related peptide immunoreactivity (CGRP-ir) in TG and depleted CGRP-ir in the dura mater. Pharmacological approaches included a recently identified α6GABAAR-selective positive allosteric modulator (PAM), the pyrazoloquinolinone Compound 6, two α6GABAAR-active PAMs (Ro15-4513 and loreclezole), an α6GABAAR-inactive benzodiazepine (diazepam), an α6GABAAR-selective antagonist (furosemide), and a clinically effective antimigraine agent (topiramate). We examined effects of these compounds on both central and peripheral TGVS responses induced by i.c. capsaicin. Compound 6 (3–10 mg/kg, i.p.) significantly attenuated the TCC neuronal activation and TG CGRP-ir elevation, and dural CGRP depletion induced by capsaicin. All these effects of Compound 6 were mimicked by topiramate, Ro15-4513 and loreclezole, but not by diazepam. The brain-impermeable furosemide antagonized the peripheral, but not central, effects of Compound 6. These results suggest that the α6GABAAR in TG is a novel drug target for TGVS activation and that α6GABAAR-selective PAMs have the potential to be developed as a novel pharmacotherapy for migraine.

Medullary neural circuit regeneration after trigeminal nerve transection.

The inferior alveolar nerve (IAN) comprises several types of sensory fibers. To clarify whether each type of primary afferent is regenerated comparably after injury, we developed a model of complete IAN transection (IANX) in mice. A retrograde tracer, fluoro-gold, injected into the mental skin was transferred to the cell bodies of a subset of isolectin B4 (IB4)-binding (non-peptidergic C) or CGRP-positive (peptidergic C) neurons at 2 weeks post-axotomy, indicating that the injured C afferents had regenerated anatomically. IANX led to a decrease of IB4-binding and CGRP immunoreactivity (IR) in the trigeminal ganglion (TG) and within the trigeminal spinal subnucleus caudalis (Vc) (i.e. terminals of the central branch of TG neurons). Two weeks after IANX, the reduction in IB4-binding activity and CGRP expression in the TG recovered to the control level; however, IB4-binding within the Vc did not, suggesting that central branch non-peptidergic neurons remained impaired. Two weeks after IANX, pinching or heat stimulus-induced extracellular signal-regulated kinase phosphorylation (pERK) was restored to the control level, but in the case of pinch stimulation the distribution pattern of pERK-IR cells was altered in the Vc. Taken together, our results support the possibility that peptidergic neurons regenerate more efficiently than non-peptidergic neurons after trigeminal nerve injury.

Innervation Changes Induced by Inflammation in the Murine Vagina

Vulvodynia is a prevalent chronic pain disorder associated with high medical costs and often ineffective treatments. The major pathological feature is proliferation of vaginal nerve fibers. This study aimed to develop a highly reproducible animal model to study neuroproliferation in the vagina and aid the identification of appropriately targeted treatments for conditions such as vulvodynia. Mild chronic inflammation was induced using microinjection of complete Freund’s adjuvant in the distal vagina of C57Bl/6 mice. Control mice received saline. Inflammation and innervation density were assessed at 7 and 28 days after a single administration or 14 days following repeated administration of complete Freund’s adjuvant or saline. Histochemistry and blinded-analysis of images were used to assess vaginal morphology (H & E) and abundance of macrophages (CD68-labeling), mast cells (toluidine blue staining, mast cell tryptase-immunoreactivity), blood vessels (αSMA-immunoreactivity) and nerve fibers immunoreactive for the pan-neuronal marker PGP9.5. Subpopulations of nerve fibers were identified using immunoreactivity for calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY). Single administration of complete Freund’s adjuvant resulted in vaginal swelling, macrophage infiltration, vascular proliferation and increased abundance of nerve fibers immunoreactive for CGRP, SP, VIP and/or PGP9.5 but not NPY, evident at seven days. Inflammation further increased following repeated administration of complete Freund’s adjuvant but nerve fiber proliferation did not. Nerve fiber proliferation continued to be evident at 28 days. The inter-individual differences within each treatment group were small, indicating that this model may be useful to study mechanisms underlying vaginal nerve fiber proliferation associated with inflammation.

Immunohistochemical characterization of brush cells in the rat larynx.

The immunohistochemical characteristics of brush cells in the laryngeal mucosa were examined using immunohistochemistry for various immunohistochemical cell markers including villin at the light and electron microscopic levels. Cells that were immunoreactive to villin were barrel-shaped with thick cytoplasmic processes extending toward the lumen of the laryngeal cavity. Immunoelectron microscopic observations revealed thick and short microvilli with long rootlets of microfilaments. Numerous small clear vesicles and small finger-like cytoplasmic processes were observed in the apical process and lateral membrane, respectively. Double immunofluorescence showed villin-immunoreactive cells were not immunoreactive for the markers of solitary chemosensory cells, GNAT3 and phospholipase C, β2-subunit (PLCβ2), or for that of neuroendocrine cells, synaptosome-associated protein 25kD. Furthermore, immunoreactivities for cytokeratin 18 (CK18) and doublecortin like-kinase 1 in the perinuclear cytoplasm of villin-immunoreactive cells. However, some CK18-immunoreactive cells were immunoreactive to GNAT3 but not to villin. Regarding sensory innervation, only a few intraepithelial nerve endings with P2X3, SP, or CGRP immunoreactivity attached to villin-immunoreactive cells. In the present study, brush cells in the rat laryngeal mucosa were classified by immunoreactivity for villin, and were independent of other non-ciliated epithelial cells such as solitary chemosensory cells and neuroendocrine cells.

Synaptic activation of putative sensory neurons by hexamethonium-sensitive nerve pathways in mouse colon.

The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene-related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modeling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator fluo 4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5-10 mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300 μM) and by low-calcium Krebs solution. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways. NEW & NOTEWORTHY Myenteric neurons in isolated mouse colon were recorded using calcium imaging and then neurochemically defined. Short-latency calcium transients were detected in >90% of calcitonin gene-related peptide-immunoreactive neurons to electrical stimulation of hexamethonium-sensitive pathways. Putative sensory Dogiel type II calcitonin gene-related peptide-immunoreactive myenteric neurons may receive widespread fast synaptic inputs in mouse colon.

The role of the transient receptor potential ankyrin type-1 (TRPA1) channel in migraine pain: evaluation in an animal model

BackgroundClinical and experimental studies have pointed to the possible involvement of the transient receptor potential ankyrin type-1 (TRPA1) channels in migraine pain. In this study, we aimed to further investigate the role of these channels in an animal model of migraine using a novel TRPA1 antagonist, ADM_12, as a probe.MethodsThe effects of ADM_12 on nitroglycerin-induced hyperalgesia at the trigeminal level were investigated in male rats using the quantification of nocifensive behavior in the orofacial formalin test. The expression levels of the genes coding for c-Fos, TRPA1, calcitonin gene-related peptide (CGRP) and substance P (SP) in peripheral and central areas relevant for migraine pain were analyzed. CGRP and SP protein immunoreactivity was also evaluated in trigeminal nucleus caudalis (TNC).ResultsIn rats bearing nitroglycerin-induced hyperalgesia, ADM_12 showed an anti-hyperalgesic effect in the second phase of the orofacial formalin test. This effect was associated to a significant inhibition of nitroglycerin-induced increase in c-Fos, TRPA1 and neuropeptides mRNA levels in medulla-pons area, in the cervical spinal cord and in the trigeminal ganglion. No differences between groups were seen as regards CGRP and SP protein expression in the TNC.ConclusionsThese findings support a critical involvement of TRPA1 channels in the pathophysiology of migraine, and show their active role in counteracting hyperalgesia at the trigeminal level.

Distribution of CGRP and CGRP receptor components in the rat brain.

Calcitonin gene-related peptide and its receptor, consisting of receptor activity-modifying protein 1 and calcitonin receptor-like receptor, are of considerable interest because of the role they play in migraine and recently developed migraine therapies. To better understand the function of this neuropeptide, we used immunohistochemistry to determine a detailed distribution of calcitonin gene-related peptide, receptor activity-modifying protein 1 and calcitonin receptor-like receptor in the rat brain in a region of 0.5-1.5 mm lateral to the midline. We found calcitonin gene-related peptide immunoreactivity in most of the neurons of the cerebral cortex, hippocampus, cerebellum, thalamic nuclei, hypothalamic nuclei and brainstem nuclei. In contrast, receptor activity-modifying protein 1 and calcitonin receptor-like receptor immunoreactivity were found almost exclusively in the neuronal processes in the investigated regions. Overall, the degree of expression of calcitonin gene-related peptide and calcitonin gene-related peptide receptor components in the central nervous system is astonishingly complex and suggestive of many different brain functions, including a possible role in migraine. However, currently, the presence of calcitonin gene-related peptide and the nature of its receptors throughout the brain is an enigma yet to be solved.

Plasticity of Select Primary Afferent Projections to the Dorsal Horn after a Lumbosacral Ventral Root Avulsion Injury and Root Replantation in Rats.

Injuries to the conus medullaris and cauda equina portions of the spinal cord result in neurological impairments, including paralysis, autonomic dysfunction, and pain. In experimental studies, earlier investigations have shown that a lumbosacral ventral root avulsion (VRA) injury results in allodynia, which may be ameliorated by surgical replantation of the avulsed ventral roots. Here, we investigated the long-term effects of an L6 + S1 VRA injury on the plasticity of three populations of afferent projections to the dorsal horn in rats. At 8 weeks after a unilateral L6 + S1 VRA injury, quantitative morphological studies of the adjacent L5 dorsal horn showed reduced immunoreactivity (IR) for the vesicular glutamate transporter, VGLUT1 and isolectin B4 (IB4) binding, whereas IR for calcitonin gene-related peptide (CGRP) was unchanged. The IR for VGLUT1 and CGRP as well as IB4 binding was at control levels in the L5 dorsal horn at 8 weeks following an acute surgical replantation of the avulsed L6 + S1 ventral roots. Quantitative morphological studies of the L5 dorsal root ganglia (DRGs) showed unchanged neuronal numbers for both the VRA and replanted series compared to shams. The portions of L5 DRG neurons expressing IR for VGLUT1 and CGRP, and IB4 binding were also the same between the VRA, replanted, and sham-operated groups. We conclude that the L5 dorsal horn shows selective plasticity for VGLUT1 and IB4 primary afferent projections after an L6 + S1 VRA injury and surgical repair.

Distribution of CGRP and its receptor components CLR and RAMP1 in the rat retina

Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide with several functions including vasodilation, the perception of painful stimuli, and inflammation. The CGRP receptor consists of two main components; calcitonin-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). While there is a growing recognition that CGRP plays a key role in migraine, the function of CGRP in the retina has not been fully established. This study aims to investigate the distribution of CGRP and its two receptor components in the rat retina, visually by immunohistochemistry and quantitatively using flow cytometry. CGRP immunoreactivity was found in the Müller cells while CLR/RAMP1 was located in the nerve fiber layer. Furthermore, since almost all RAMP1 immunoreactive cells co-express CLR, we propose that RAMP1 expression in the retina reflects functional CGRP receptors.

Opioids and opioid receptors orchestrate wound repair

We have previously shown that topical opioids including morphine and its congeners promote healing of full thickness ischemic wounds in rats. We examined the contribution of mu opioid receptor (MOPr)-mediated healing of full thickness ischemic wounds using MOPr and delta or kappa opioid receptor knockout (KO) mice. Wound closure in the early (day 5) as well as later phases was delayed in topical morphine or PBS-treated MOPr-KO mice compared with reciprocal treatments of wounds in wild-type (WT) mice. MOPr expression was significantly upregulated at 30min in the wound margins and colocalized with wound margins and vasculature in the epidermal and dermal layers of the skin. We next examined whether neuropeptide expression was involved in the mechanism of MOPr-mediated wound closure. Substance P (SP) and calcitonin gene-related peptide immunoreactivity (ir) was significantly increased in the skin of MOPr-KO mice as compared with WT mice. Neuropeptide-ir was increased significantly in PBS-treated wounds of MOPr and WT mice, but morphine treatment reduced neuropeptide immunoreactivity in both as compared with PBS. Wounding of keratinocytes led to the release of opioid peptide beta-endorphin (β-END) in conditioned medium, which stimulated the proliferation of endothelial cells. MOPr-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, CTOP) and nonselective OPr antagonist naloxone-inhibited endothelial proliferation induced by wounded keratinocyte-conditioned medium. In addition, accelerated wound area closure invitro by morphine was suppressed by methylnaltrexone, a nonselective OPr antagonist with high affinity for MOPr. Morphine and its congeners stimulated the proliferation of endothelial cells from WT mice but not those from MOPr-KO mice. Furthermore, morphine-induced mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation in endothelial cells was significantly decreased in MOPr-KO mice as compared with WT mice. Collectively, these data suggest that MOPr plays a critical role in the proliferation phase with the formation of granulation tissue during wound healing.