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Background Amylin is a calcitonin gene-related peptide family member expressed by nociceptors. Amylin's expression is down-regulated following nerve damage, and studies suggested it affects nociception. We aimed at clarifying amylin's effects on chronic neuropathic pain and investigating its site of action. Methods Chronic neuropathic pain was induced in rats by spared nerve injury (SNI) surgery. Mechanical allodynia/hyperalgesia and cold allodynia/hyperalgesia were assessed by the von Frey, pinprick, acetone and cold plate behavioural tests, respectively. Amylin, amylin-receptor antagonist (AC187) or vehicle solutions were delivered chronically, by a subcutaneous (SC) mini-osmotic pump, or acutely, by SC or intrathecal (IT) injections. Cellular and fibre markers were used to detect spinal cord alterations in SNI rats after chronic amylin administration. Results Continuous subcutaneous amylin administration aggravated cold allodynia in SNI animals, possibly via amylin-receptors (AmyR) in supraspinal areas. Acute intrathecal administration of amylin attenuated mechanical hyperalgesia, whereas AC187 reduced mechanical allodynia, suggesting distinct roles of endogenous amylin and of pharmacological amylin doses when targeting spinal cord amylin receptors. Chronic amylin administration promoted c-Fos activation only in the dorsal horn neurons of SHAM animals, suggesting a distinctive role of amylin in the activation of the spinal neuronal circuitry under neuropathic and physiological conditions. ERK1/2 phosphorylation increased in the dorsal horn neurons of SNI rats chronically treated with amylin. This ERK1/2 cascade activation may be related to amylin's effect on the aggravation of cold allodynia in SNI rats. Conclusions Amylin's nociceptive effects seem to depend on the treatment duration and route of administration by acting at different levels of the nervous system. Significance Amylin modulated neuropathic pain by acting at different levels of the nervous system. Whereas supraspinal areas may be involved in amylin's induced pronociception, modulation of spinal cord amylin receptors by endogenous or pharmacological amylin doses triggers both pro- and antinociceptive effects.

BackgroundCalcitonin expression is a well-established marker for medullary thyroid carcinoma (MTC); yet the role of calcitonin receptor (CTR), its seven-transmembrane G-protein coupled receptor, remains to be established in C-cells derived thyroid tumors. The aim of this work was to investigate CTR expression in MTC and to correlate such expression with clinicopathological features in order to evaluate its possible role as a prognostic indicator of disease aggressiveness and outcome.MethodsCalcitonin receptor expression was analyzed in a series of 75 MTCs by immunohistochemistry, and by qPCR mRNA quantification in specimens from four patients. Statistical tests were used to evaluate the correlation between CTR expression and the clinicopathological and molecular characteristics of patients and tumors.ResultsCalcitonin receptor expression was detected in 62 out of 75 samples (82.7%), whereas 13 of the 75 samples (17.3%) were completely negative. CTR expression was significantly associated with expression of cytoplasmatic phosphatase and tensin homologue deleted on chromosome 10 and osteopontin, as well as with wild typeRET/RASgenes and absence of tumor stroma, suggesting that CTR expression do not associate with clinicopathological signs of worse prognosis.DiscussionCalcitonin receptor expression appears to be associated in MTC with more differentiated status of the neoplastic cells.

Purinergic receptors (P2XRs) have been widely associated with pain states mostly due to their involvement in neuron-glia communication. Interestingly, we have previously shown that satellite glial cells (SGC), surrounding dorsal root ganglia (DRG) neurons, become activated and proliferate during monoarthritis (MA) in the rat. Here, we demonstrate that P2X7R expression increases in ipsilateral DRG after 1 week of disease, while P2X3R immunoreactivity decreases. We have also reported a significant induction of the activating transcriptional factor 3 (ATF3) in MA. In this study, we show that ATF3 knocked down in DRG cell cultures does not affect the expression of P2X7R, P2X3R, or glial fibrillary acidic protein (GFAP). We suggest that P2X7R negatively regulates P2X3R, which, however, is unlikely mediated by ATF3. Interestingly, we found that ATF3 knockdown in vitro induced significant decreases in the heat shock protein 90 (HSP90) expression. Thus, we evaluated in vivo the involvement of HSP90 in MA and demonstrated that the HSP90 messenger RNA levels increase in ipsilateral DRG of inflamed animals. We also show that HSP90 is mostly found in a cleaved form in this condition. Moreover, administration of a HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), attenuated MA-induced mechanical allodynia in the first hours. The drug also reversed the HSP90 upregulation and cleavage. 17-DMAG seemed to attenuate glial activation and neuronal sensitization (as inferred by downregulation of GFAP and P2X3R in ipsilateral DRG) which might correlate with the observed pain alleviation. Our data indicate a role of HSP90 in MA pathophysiology, but further investigation is necessary to clarify the underlying mechanisms.

Oleoylethanolamide (OEA) is a gut-derived endogenous lipid that stimulates vagal fibers to induce satiety. Our previous work has shown that peripherally administered OEA activates c-fos transcription in the nucleus of the solitary tract (NST) and in the paraventricular nucleus (PVN), where it enhances oxytocin (OXY) expression. The anorexigenic action of OEA is prevented by the intracerebroventricular administration of a selective OXY receptor antagonist, suggesting a necessary role of OXYergic mediation of OEA's effect. The NST is the source of direct noradrenergic afferent input to hypothalamic OXY neurons, and therefore, we hypothesized that the activation of this pathway might mediate OEA effects on PVN neurons. To test this hypothesis, we subjected rats to intra-PVN administration of the toxin saporin (DSAP) conjugated to an antibody against dopamine-β-hydroxylase (DBH) to destroy hindbrain noradrenergic neurons. In these rats we evaluated the effects of OEA (10 mg/kg, ip) on feeding behavior, on c-Fos and OXY immunoreactivity in the PVN, and on OXY immunoreactivity in the posterior pituitary gland. We found that the DSAP lesion completely prevented OEA's effects on food intake, on Fos and OXY expression in the PVN, and on OXY immunoreactivity of the posterior pituitary gland; all effects were maintained in sham-operated rats. These results support the hypothesis that noradrenergic NST-PVN projections are involved in the activation of the hypothalamic OXY system, which mediates OEA's prosatiety action.

Background Amylin is a peptide from the calcitonin gene-related peptides (CGRP) family that is expressed by nociceptors. Amylin may modulate pain via a spinal action. Methods The effect of amylin's administration on the formalin test of acute and tonic pain was evaluated. Amylin's ability to modulate neuronal activity was analysed by c-Fos expression at the spinal cord lumbar 4–5 region (L4-5) and brain. Results Amylin subcutaneous administration 20 min prior, but not immediately before formalin, shortened the interphase and anticipated the beginning of the tonic pain phase. Amylin reduced the number of activated spinal cord neurons. Blockade of spinal amylin-receptors by prior L4-5 intrathecal administration of an amylin-receptor antagonist (AC187) attenuated these effects, whereas intrathecal BIBN4096 (CGRP-receptor antagonist) did not, proving that part of amylin's effects were spinally mediated via amylin-receptors. The locus coeruleus and other areas involved in descending modulation and affective responses to pain showed an increased number of activated neurons upon amylin subcutaneous administration, suggesting a role for supraspinal areas in some observed effects. L4-5 intrathecal injection of amylin or AC187 showed that both ligands attenuated tonic pain, but blockade of the amylin-receptor action by AC187 decreased further the number of paw jerks in this period. Conclusions Overall, data suggested that amylin modulates pain with an inflammatory component and the autoanalgesic/inhibitory mechanisms occurring in the interphase of the formalin test. Amylin might have affected the nociceptive system at different levels (spinal cord and brain), explaining the different effects observed according to the time of amylin injection. What does this study add? Amylin modulated formalin interphase and tonic pain behaviours probably by targeting spinal neurons and affecting supraspinal areas involved in affective and modulatory components of pain. Activation of spinal amylin-receptors may contribute to the initiation of inflammatory pain mechanisms.

Circulating amylin inhibits food intake via activation of the area postrema (AP). The aim of this study was to identify the neurochemical phenotype of the neurons mediating amylin's hypophagic action by immunohistochemical and feeding studies in rats. Expression of c-Fos protein was used as a marker for neuronal activation and dopamine-beta-hydroxylase (DBH), the enzyme-catalyzing noradrenaline synthesis, as a marker for noradrenergic neurons. We found that approximately 50% of amylin-activated AP neurons are noradrenergic. To clarify the functional role of these neurons in amylin's effect on eating, noradrenaline-containing neurons in the AP were lesioned using a saporin conjugated to an antibody against DBH. Amylin (5 or 20 microg/kg s.c.)-induced anorexia was observed in sham-lesioned rats with both amylin doses. Rats with a lesion of > 50% of the noradrenaline neurons were unresponsive to the low dose of amylin (5 microg/kg) and only displayed a reduction in food intake 60 min after injection of the high amylin dose (20 microg/kg). In a terminal experiment, the same rats received amylin (20 microg/kg) or saline. The AP and nucleus of the solitary tract (NTS) were stained for DBH to assess noradrenaline lesion success and for c-Fos expression to evaluate amylin-induced neuronal activation. In contrast to sham-lesioned animals, noradrenaline-lesioned rats did not show a significant increase in amylin-induced c-Fos expression in the AP and NTS. We conclude that the noradrenergic neurons in the AP mediate at least part of amylin's hypophagic effect.

The ability of the pancreatic hormone amylin to inhibit food intake relies on a direct activation of the area postrema (AP). This activation is synaptically transmitted to the nucleus of the solitary tract (NTS), the lateral parabrachial nucleus (LPB), the central amygdaloid nucleus (Ce) and the lateral bed nucleus of stria terminalis (BSTL). Interestingly, neurons of the rostro-dorsal lateral hypothalamic area (dLHA), which are activated during fasting, are inhibited by peripheral amylin, although they lack amylin receptors. Using the retrograde tracer cholera toxin-B (Ctb) we analyzed whether the dLHA receives neuronal projections from amylin-activated brain areas. The anterograde tracer biotinylated dextran-amine (BDA) was used to confirm the projections and to identify further neuronal pathways potentially involved in amylin signaling. We identified dense projections from the amylin activated neurons in the LPB and sparse projections from the NTS to the dLHA. LPB fiber efferents were found in close proximity to dLHA nuclei activated by 24h of fasting. The AP and the Ce showed no projections to the dLHA. Dense efferents were also observed from the LPB to other hypothalamic areas, namely to the ventromedial, dorsomedial, paraventricular and arcuate nuclei. This study provides neuroanatomical evidence that among the amylin activated areas, the LPB provides the strongest input to the dLHA, thus it may mediate the amylin-induced inhibition of the dLHA.

The role of mu-opioid receptors (MORs) in the inflammatory pain processing mechanisms within the ventrobasal complex of the thalamus (VB) is not well understood. This study investigated the effect of modulating MOR activity upon nociception, by stereotaxically injecting specific ligands in the VB. Nociceptive behaviour was evaluated in two established animal models of inflammatory pain, by using the formalin (acute and tonic pain) and the ankle-bend (chronic monoarthritic pain) tests. Control (saline intra-VB injection) formalin-injected rats showed acute and tonic pain-related behaviours. In contrast, intrathalamic administration of [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate (DAMGO), a MOR-specific agonist, induced a statistically significant decrease of all tonic phase pain-related behaviours assessed until 30–35 min after formalin hind paw injection. In the acute phase only the number of paw-jerks was affected. In monoarthritic rats, there was a noticeable antinociceptive effect with approximately 40 min of duration, as denoted by the reduced ankle-bend scores observed after DAMGO injection. Intra-VB injection of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a specific MOR antagonist, or of CTOP followed, 10 min after, by DAMGO had no effects in either formalin or ankle-bend tests. Data show that DAMGO-induced MOR activation in the VB has an antinociceptive effect in the formalin test as well as in chronic pain observed in MA rats, suggesting an important and specific role for MORs in the VB processing of inflammatory pain.

The ventrobasal complex of the thalamus (VB) participates in the transmission and modulation of noxious information. Recent data suggested that GABA(B) receptors in the VB might be involved in the modulation of neuronal activity in response to chronic noxious input. However, in acute inflammatory pain, the role of GABA(B) receptors in the VB remains unknown. The formalin test of nociception was performed in rats stereotaxically injected in the VB contralateral to the formalin-injected paw, with saline (controls), baclofen (0.5 and 0.875 microg), a specific GABA(B) receptor agonist or CGP35348 (25 microg), a GABA(B) receptor antagonist. Control animals exhibited phase 1 (acute pain) and phase 2 (tonic pain) nociception-related activities as previously described. The higher dose of baclofen induced a significant decrease of all pain-related behaviors in both phases of the test and had no observable effects on the animals' motor function, while the lower dose could not reduce the total pain-related activities. Injection of CGP35348 prior to baclofen reduced the antinociceptive effect caused by baclofen during phase 2 in the paw-jerks and in total pain-related activities. CGP35348 alone had antinociceptive effects in both phases, though less pronounced than baclofen 0.875 microg in the total pain-related activities during phase 2. Data demonstrate that both the blockade and the activation of GABA(B) receptors in the VB of rats induce antinociception in acute and tonic pain. An important role for GABA(B) receptors on the thalamic processing of nociceptive input in the VB is suggested.

Background Experimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase. Methods Osteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3-L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection. Results GFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception. Conclusion Collagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord. Inhibition of glial cell activation by fluorocitrate decreases these osteoarthritis-associated nociceptive behaviours. These results suggest that glial cell activation may play a role in the development of chronic pain in this experimental model of osteoarthritis.

Peripheral amylin inhibits eating via the area postrema (AP). Because amylin activates the extracellular-signal regulated kinase 1/2 (ERK) pathway in some tissues, and because ERK1/2 phosphorylation (pERK) leads to acute neuronal responses, we postulated that it may be involved in amylin's eating inhibitory effect. Amylin-induced ERK phosphorylation (pERK) was investigated by immunohistochemistry in brain sections containing the AP. pERK-positive AP neurons were double-stained for the calcitonin 1a/b receptor, which is part of the functional amylin-receptor. AP sections were also phenotyped using dopamine-β-hydroxylase (DBH) as a marker of noradrenergic neurons. The effect of fourth ventricular administration of the ERK cascade blocker U0126 on amylin's eating inhibitory action was tested in feeding trials. The number of pERK-positive neurons in the AP was highest ∼10–15 min after amylin treatment; the effect appeared to be dose-dependent (5–20 μg/kg amylin). A portion of pERK-positive neurons in the AP carried the amylin-receptor and 22% of the pERK-positive neurons were noradrenergic. Pretreatment of rats with U0126 decreased the number of pERK-positive neurons in the AP after amylin injection. U0126 also attenuated the ability of amylin to reduce eating, at least when the animals had been fasted 24 h prior to the feeding trial. Overall, our results suggest that amylin directly stimulates pERK in AP neurons in a time- and dose-dependent manner. Part of the AP neurons displaying pERK were noradrenergic. At least under fasting conditions, pERK was shown to be a necessary part in the signaling cascade mediating amylin's anorectic effect.

Treatment with the bacterial endotoxin lipopolysaccharide (LPS) is a commonly used model to induce disease-related anorexia. Following LPS treatment inducible nitric oxide synthase (iNOS) is expressed in the hypothalamic arcuate nucleus (ARC), where nitric oxide (NO) inhibits orexigenic neurons. Intracellular STAT signaling is triggered by inflammatory stimuli and has been linked to the transcriptional regulation of iNOS. We evaluated whether pharmacological blockade of iNOS by the specific inhibitor 1400W attenuates LPS-induced anorexia. Furthermore, we hypothesized that the tolerance to the anorectic effect occurring after repeated LPS treatment is paralleled by a blunted STAT3 phosphorylation in the ARC. Rats treated with a subcutaneous injection of 1400W (10 mg/kg) showed an attenuated anorectic LPS response relative to control rats receiving only LPS (100 µg/kg; i.p.). Similarly, iNOS blockade attenuated LPS-induced adipsia, hyperthermia, inactivity and the concomitant drop in energy expenditure. While single LPS treatment increased STAT3 phosphorylation in the ARC, rats treated repeatedly with LPS showed no anorectic response and also no STAT3 phosphorylation in the ARC after the second and third LPS injections, respectively. Hence, pSTAT3 signaling in the ARC might be part of the intracellular cascades translating pro-inflammatory stimuli into suppression of food intake. The current findings substantiate a role of iNOS dependent NO formation in disease-related anorexia.

Amylin is secreted by pancreatic beta-cells and is believed to be a physiological signal of satiation. Amylin's effect on eating has been shown to be mediated via a direct action at the area postrema (AP) via amylin receptors that are heterodimers of the calcitonin receptor core protein with a receptor activity modifying protein. Peripheral amylin leads to accumulation of cyclic guanosine monophosphate, phosphorylated extracellular-signal regulated kinase 1/2 and c-Fos protein in AP neurons. The particular amylin-activated AP neurons mediating its anorexigenic action seem to be noradrenergic. The central pathways mediating amylin's effects have been characterized by lesioning and tracing studies, identifying important connections from the AP to the nucleus of the solitary tract and lateral parabrachial nucleus. Amylin was shown to interact, probably at the brainstem, with other signals involved in the short term control of food intake, namely cholecystokinin, glucagon-like peptide 1 and peptide YY. Amylin also interacts with the adiposity signal leptin; this interaction, which is thought to involve the hypothalamus, may have important implications for the development of new and improved hormonal obesity treatments. In conclusion, amylin actions on food intake seem to reside primarily within the brainstem, and the associated mechanisms are starting to be unraveled. The paper represents an invited review by a symposium, award winner or keynote speaker at the Society for the Study of Ingestive Behavior [SSIB] Annual Meeting in Portland, July 2009.

Peripheral amylin inhibits food intake via activation of the area postrema (AP). 59% of amylin-activated AP neurons are noradrenergic (NA), i.e., they express dopamine-beta-hydroxylase (DBH). Here, we wanted to testwhether APNAneuronsmediate amylin’s anorectic effect.We performed a specific lesion of AP NA neurons using a saporin conjugated to an antibody against DBH (DSAP). IgG-saporin was used in sham controls. After 2–3 weeks necessary for neuronal degeneration, we tested the rats for the effect of amylin (5 or 20_g/kg BW, s.c.) to reduce food intake. In a terminal experiment, the rats received amylin (20_g/kg) or saline; brain sections with the AP and nucleus of the solitary tract (NTS) were stained for DBH to assess lesion success and for c-Fos expression to evaluate amylin-induced neuronal activation. DBH staining revealed that 10 DSAP-injected rats had NA lesion equal to or above 50%, defined as successful; 6 had lesions below 50%. Daily food intake and body weight gain did not differ between lesioned and sham groups. Amylin-induced anorexia was observed in sham rats with both amylin doses, while rats with a successful lesion had no significant reduction in eating after either amylin dose. Rats with lesions below 50% only ate less after the higher amylin dose. In contrast to sham-lesioned animals, successfully NA-lesioned rats did not show amylin-induced c-Fos expression in the AP and NTS. These results provide first evidence for a functional role of NA neurons in the AP in the mediation of amylin’s anorectic effect.

gamma-Aminobutyric acid type B (GABAB) receptors are involved in the modulation of neuronal activity in response to chronic noxious input. However, the effect of their activation in chronic inflammatory pain in relay thalamic nuclei such as the ventrobasal complex (VB) is not known. In this study, experimental groups of 2, 4, and 14 days monoarthritic (MA) rats were injected with saline (controls) or baclofen (0.875 microg), a specific GABAB receptor agonist, in the VB contralateral to the inflamed joint, and the ankle-bend test was performed. Ankle-bend scores in control animals were near the maximum and were rather constant throughout the entire experimental period, indicating severe nociception. The same was observed in 2 days MA rats injected with baclofen. In the 4 days MA group, the response to baclofen injection was inconsistent among different animals, whereas, in 14 days MA rats, baclofen caused clear antinociceptive effects. Additionally, a 0.5 microg dose of baclofen was tested in 14 days MA rats, but no effect was observed, whereas a 1.25 mug dose produced visible side effects. Baclofen injections that did not target the VB but reached neighboring nuclei were ineffective in reducing nociception. Data demonstrate that the activation of the GABAB receptors by baclofen in the VB of MA rats leads to a decrease of nociception. Moreover, the response depends on the time course of the disease, suggesting the occurrence of different excitatory states of thalamic VB neurons. In conclusion, GABAB receptors in the VB play an important role in chronic inflammatory pain processing.