Morara, S. et al. Calcitonin gene-related peptide receptor expression in the neurons and glia of developing rat cerebellum: an autoradiographic and immunohistochemical analysis. Neuroscience 100, 381-391

Istituto Neuroscienze e Bioimmagini CNR, 20131, Milano, Italy.
Neuroscience (Impact Factor: 3.36). 02/2000; 100(2):381-91. DOI: 10.1016/S0306-4522(00)00276-1
Source: PubMed


Quantitative autoradiography (using [125I]human alpha-calcitonin gene-related peptide as a ligand) and immunofluorescence (using monoclonal antibodies directed against a purified receptor) followed by confocal analysis were applied to analyse the distribution and cellular localization of the calcitonin gene-related peptide receptor in the rat cerebellum during development. From late embryonic days to the end of the second postnatal week, during the time window of calcitonin gene-related peptide expression in climbing fibers, high levels of calcitonin gene-related peptide binding sites were found in the white matter, where immunolabeling was present in oligodendrocytes. Lower levels were found in the cerebellar cortex, where receptor immunolabeling was found in Bergmann glia in a presumptive cell surface location and, during the second postnatal week, also in the cytoplasm of Purkinje cells. From the end of the second postnatal week to adulthood, when calcitonin gene-related peptide is no longer present in climbing fibers, the number of calcitonin gene-related peptide binding sites increased in the molecular layer, where not only Bergmann glia but also Purkinje cell distal dendritic branchlets were immunolabeled in a presumptive cell surface location. Concomitantly, the number of calcitonin gene-related peptide binding sites sharply decreased in the white matter. The developmental expression of the calcitonin gene-related peptide receptor and the previously described proliferating/differentiating effects of the peptide on glial cells suggest that calcitonin gene-related peptide and its receptor may promote a coordinated development of cerebellar glial cells, an effect driven mainly by the calcitonin gene-related peptide released by climbing fibers. As a result of glia-neuron interactions, an indirect effect on the differentiation of the cerebellar neuronal circuitry is also likely to occur.

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    • "Please cite this article as: Figueira, L., Israel, A., Role of cerebellar adrenomedullin in blood pressure regulation, Neuropeptides (2015), http:// suggesting that CGRP1 and its receptor may promote a coordinated development of cerebellar glial cells, an effect driven mainly by the CGRP released by climbing fibers (Morara et al., 2000). Similar results has been described in the periphery, indeed Hwang et al. (2007) found that in rats of 3, 12 and 20 months old, there were age-related increases in lung AM gene expression and peptide levels, as well as the mRNA levels of CRLR and RAMPs. "
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    ABSTRACT: Adrenomedullin (AM) and their receptor components, calcitonin-receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP1, RMP2 and RAMP3) are widely expressed in the central nervous system, including cerebellum. We have shown that AM binding sites are altered in cerebellum during hypertension, suggesting a role for cerebellar adrenomedullinergic system in blood pressure regulation. To further evaluate the role of AM in cerebellum, we assessed the expression of AM, RAMP1, RAMP2, RAMP3 and CRLR in the cerebellar vermis of 8 and 16week old spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. In addition, the effect of microinjection of AM into rat cerebellar vermis on arterial blood pressure (BP) was determined. Animals were sacrificed by decapitation and cerebellar vermis was dissected for quantification of AM, CRLR, RAMP1, RAMP2 and RAMP3 expression using western blot analysis. Another group of male, 16week old SHR and WKY rats was anesthetized, and a cannula was implanted in the cerebellar vermis. Following recovery AM (0.02 to 200pmol/5μL) or vehicle was injected into cerebellar vermis. BP was determined, before and after treatments, by non-invasive plethysmography. In addition, to establish the receptor subtype involved in AM action in vivo, animals received microinjections of AM22-52 (200pmol/5μL), an AM1 receptor antagonist, or the CGRP1 receptor antagonist, CGRP8-37 (200pmol/5μL) into the cerebellar vermis, administered simultaneously with AM or vehicle microinjection. Cannulation was verified post mortem with the in situ injection of a dye solution. Our findings demonstrated that the expression of CRLR, RAMP1 and RAMP3 was higher in cerebellum of SHR rats, while AM and RAMP2 expression was lower than those of WKY rats, both in 8 and 16week old rats. In vivo microinjection of AM into the cerebellar vermis caused a profound, dose dependent, hypotensive effect in SHR but not in normotensive WKY rats. Coinjections of a putative AM receptor antagonist, AM22-52 abolished the decreases in mean arterial pressure (MAP) evoked by AM, showing that AM acts through its AM1 receptor in the vermis to reduce MAP. These findings demonstrate a dysregulation of cerebellar AM-system during hypertension, and suggest that cerebellar AM plays an important role in the regulation of BP. Likewise; they constitute a novel mechanism of BP control which has not been described so far. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Neuropeptides 07/2015; 54. DOI:10.1016/j.npep.2015.07.003 · 2.64 Impact Factor
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    • "Mice were sacrificed by perfusion with 4% paraformaldehyde following deep general anesthesia with urethane, and the brains were embedded in sucrose, frozen and cut at the cryostat. Spinal cord sections for histology and immunofluorescence examination were prepared as previously described (Morara et al., 2000). In brief, following a short perfusion with 4% paraformaldehyde, the dissected spinal cords were postfixed for 30 min in the same fixative, soaked overnight in 20% sucrose at 4 °C, frozen, cut at cryostat in the coronal plane at 10 μm thickness and collected on gelatin-coated slides. "
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    ABSTRACT: Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null>heterozygote>wild type. In wild type mice, CGRP delivery into the cerebrospinal fluid (CSF) 1) reduced chronic EAE (C-EAE) signs, 2) inhibited microglia activation (revealed by quantitative shape analysis), and 3) did not alter GFAP expression, cell density, lymphocyte infiltration, and peripheral lymphocyte production of IFN-gamma, TNF-alpha, IL-17, IL-2, and IL-4. RCP (probe for receptor involvement) was expressed in white matter microglia, astrocytes, oligodendrocytes, and vascular-endothelial cells: in EAE, also in infiltrating lymphocytes. In relapsing-remitting EAE (R-EAE) RCP increased during relapse, without correlation with lymphocyte density. RCP nuclear localization (stimulated by CGRP in vitro) was I) increased in microglia and decreased in astrocytes (R-EAE), and II) increased in microglia by CGRP CSF delivery (C-EAE). Calcitonin like receptor was rarely localized in nuclei of control and relapse mice. CGRP increased in motoneurons. In conclusion, CGRP can inhibit microglia activation in vivo in EAE. CGRP and its receptor may represent novel protective factors in EAE, apparently acting through the differential cell-specific intracellular translocation of RCP.
    Journal of neuroimmunology 03/2014; 271(1-2). DOI:10.1016/j.jneuroim.2014.03.008 · 2.47 Impact Factor
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    • "The discrepancies of the CGRP-receptor components localization in the cerebellar glial cells could be due to several factors. In the early studies of Morara and colleagues (Morara et al. 2000, 1998), they used an antibody against the CGRP receptor. At that time the CGRP receptor was not well characterized [Hay et al. 2008]. "
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    ABSTRACT: Migraine is considered a neurovascular disease affecting more than 10% of the general population. Currently available drugs for the acute treatment of migraine are vasoconstrictors, which have limitations in their therapeutic use. The calcitonin gene-related peptide (CGRP) has a key role in migraine, where levels of CGRP are increased during acute migraine attacks. CGRP is expressed throughout the central and peripheral nervous system, consistent with control of vasodilatation and transmission of nociceptive information. In migraine, CGRP is released from the trigeminal system. At peripheral synapses CGRP results in vasodilatation via receptors on the smooth muscle cells. At central synapses, CGRP acts postjunctionally on second-order neurons to transmit pain centrally via the brainstem and midbrain to higher cortical pain regions. The recently developed CGRP-receptor antagonists have demonstrated clinical efficacy in the treatment of acute migraine attacks. A remaining question is their site of action. The CGRP-receptor components (calcitonin receptor-like receptor, receptor activity modifying protein 1 and receptor component protein) are found to colocalize in the smooth muscle cells of intracranial arteries and in large-sized neurons in the trigeminal ganglion. The CGRP receptor has also been localized within parts of the brain and the brainstem. The aim of this paper is to review recent localization studies of CGRP and its receptor components within the nervous system and to discuss whether these sites could be possible targets for the CGRP-receptor antagonists.
    Therapeutic Advances in Neurological Disorders 11/2010; 3(6):369-78. DOI:10.1177/1756285610388343 · 3.14 Impact Factor
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