title: Calcitonin receptor-like receptor and receptor activity modifying protein 1 in the rat dorsal horn: localization in glutamatergic presynaptic terminals containing opioids and adrenergic alpha2C receptors.
abstract: Calcitonin gene-related peptide (CGRP) is abundant in the central terminals of primary afferents. However, the function of CGRP receptors in the spinal cord remains unclear. CGRP receptors are heterodimers of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1). We studied the localization of CRLR and RAMP1 in the rat dorsal horn using well-characterized antibodies against them, which labeled numerous puncta in laminae I-II. In addition, RAMP1 was found in cell bodies, forming patches at the cell surface. The CRLR- and RAMP1-immunoreactive puncta were further characterized using double and triple labeling. Colocalization was quantified in confocal stacks using Imaris software. CRLR did not colocalize with primary afferent markers, indicating that these puncta were not primary afferent terminals. CRLR- and RAMP1-immunoreactive puncta contained synaptophysin and vesicular glutamate transporter-2 (VGLUT2), showing that they were glutamatergic presynaptic terminals. Electron microscopic immunohistochemistry confirmed that CRLR immunoreactivity was present in axonal boutons that were not in synaptic glomeruli. Using tyramide signal amplification for double labeling with the CRLR and RAMP1 antibodies, we found some clear instances of colocalization of CRLR with RAMP1 in puncta, but their overall colocalization was low. In particular, CRLR was absent from RAMP1-containing cells. Many of the puncta stained for CRLR and RAMP1 were labeled by anti-opioid and anti-enkephalin antibodies. CRLR and, to a lesser extent, RAMP1 also colocalized with adrenergic alpha(2C) receptors. Triple label studies demonstrated three-way colocalization of CRLR-VGLUT2-synaptophysin, CRLR-VGLUT2-opioids, and CRLR-opioids-alpha(2C) receptors. In conclusion, CRLR is located in glutamatergic presynaptic terminals in the dorsal horn that contain alpha(2C) adrenergic receptors and opioids. Some of these terminals contain RAMP1, which may form CGRP receptors with CRLR, but in others CRLR may form other receptors, possibly by dimerizing with RAMP2 or RAMP3. These findings suggest that CGRP or adrenomedullin receptors modulate opioid release in the dorsal horn.
authors.affiliation: Center for Neurovisceral Sciences and Women's Health and CURE: Department of Medicine, David Geffen School of Medicine at UCLA and Veteran Affairs Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA. marvizon@ucla.edu
authors.forename: J C G
authors.initials: JC
authors.lastname: Marvizón
authors.forename: O A
authors.initials: OA
authors.lastname: Pérez
authors.forename: B
authors.initials: B
authors.lastname: Song
authors.forename: W
authors.initials: W
authors.lastname: Chen
authors.forename: N W
authors.initials: NW
authors.lastname: Bunnett
authors.forename: E F
authors.initials: EF
authors.lastname: Grady
authors.forename: A J
authors.initials: AJ
authors.lastname: Todd
mesh_terms: D000344:Afferent Pathways / Q000378:metabolism / Q000648:ultrastructure; D000701:Analgesics, Opioid / Q000378:metabolism*; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D058286:Calcitonin Receptor-Like Protein; D018698:Glutamic Acid / Q000378:metabolism; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D016253:Microscopy, Immunoelectron; D009619:Nociceptors / Q000378:metabolism; D020671:Posterior Horn Cells / Q000378:metabolism* / Q000648:ultrastructure; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D018003:Receptors, Calcitonin / Q000378:metabolism*; D013126:Spinal Nerve Roots / Q000378:metabolism / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology; D016708:Synaptophysin / Q000032:analysis / Q000378:metabolism; D050599:Vesicular Glutamate Transport Protein 2 / Q000032:analysis / Q000378:metabolism
chemical_list: C547776:Adra2c protein, rat; D000701:Analgesics, Opioid; D015415:Biomarkers; D058286:Calcitonin Receptor-Like Protein; C545580:Calcrl protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C545508:Ramp1 protein, rat; C545509:Ramp2 protein, rat; C545510:Ramp3 protein, rat; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018341:Receptors, Adrenergic, alpha-2; D018003:Receptors, Calcitonin; C493475:Slc17a6 protein, rat; D016708:Synaptophysin; D050599:Vesicular Glutamate Transport Protein 2; D018698:Glutamic Acid; D015740:Calcitonin Gene-Related Peptide