Pain mechanismCalcitonin 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
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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