Elsevier

Neuroscience

Volume 148, Issue 1, 10 August 2007, Pages 250-265
Neuroscience

Pain mechanism
Calcitonin receptor-like receptor and receptor activity modifying protein 1 in the rat dorsal horn: Localization in glutamatergic presynaptic terminals containing opioids and adrenergic α2C receptors

https://doi.org/10.1016/j.neuroscience.2007.05.036Get rights and content

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 α2C receptors. Triple label studies demonstrated three-way colocalization of CRLR–VGLUT2–synaptophysin, CRLR–VGLUT2–opioids, and CRLR–opioids–α2C receptors. In conclusion, CRLR is located in glutamatergic presynaptic terminals in the dorsal horn that contain α2C 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.

Section snippets

Experimental procedures

Animal procedures were approved by the Institutional Animal Care and Use Committee of the Veteran Affairs Greater Los Angeles Healthcare System, and conform to U.S. National Institutes of Health guidelines. Efforts were made to minimize the number of animals and their suffering.

CRLR and RAMP1 are present in the superficial dorsal horn

CGRP receptors are heteromers of CRLR and RAMP1, so we used antibodies against these two proteins to localize these receptors in the rat spinal cord. The two antibodies used were extensively characterized in a previous study (Cottrell et al., 2005). CRLR immunoreactivity was abundant in the superficial dorsal horn (laminae I and II) and in the dorsolateral funiculus (Fig. 1A), consisting mostly of punctate staining. Very few cells were labeled for CRLR. RAMP1 immunoreactivity was also found in

Colocalization of CRLR and RAMP1

One critical issue in this study is whether the CRLR and RAMP1 proteins detected in our experiments dimerize to form CGRP receptors (McLatchie et al., 1998). A prerequisite for this is that these two proteins are found in the same cellular compartment in close proximity to each other. Although there were some clear instances of colocalization of CRLR and RAMP1 in dorsal horn puncta, many other puncta contained only one of these proteins. Overall, the colocalization of CRLR and RAMP1 was quite

Conclusion

To summarize, we used antisera specifically recognizing RAMP1 and CRLR to localize these components of the CGRP receptor in the rat spinal cord, and found they were present in presynaptic glutamatergic terminals that often contained opioids and α2C receptors. Because of incomplete colocalization between CRLR and RAMP1, some of the CRLR staining may reflect the presence of other CRLR-containing receptors. Their presynaptic location suggests that CGRP receptors may modulate neurotransmitter

Acknowledgments

Supported by NIDA grant 2-R01-DA012609 to J.C.M. and a grant from the Wellcome Trust to A.J.T. Confocal images were acquired at Carol Moss Spivak Cell Imaging Facility of the Brain Research Institute at UCLA, with the assistance of Dr. Matthew J. Schibler. We thank Dr. Javier Diez Guerra for his advice on colocalization measures. We would like to recognize the important contribution of the late Dr. Helen Wong, who prepared the CRLR and RAMP1 antibodies.

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