Sex differences in the expression of calcitonin gene-related peptide receptor components in the spinal trigeminal nucleus

Abstract

Background and purpose: Calcitonin gene-related peptide (CGRP) plays an important role in migraine pathophysiology. CGRP acts primarily by activating a receptor composed of 3 proteins: calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP). We tested the hypothesis that sex differences exist in protein levels of two key components of this CGRP receptor: CLR and RCP.

Methods: We used specific antibodies to assess baseline protein levels of CLR and RCP in the spinal trigeminal nucleus caudalis (SpVc) and upper cervical spinal cord of both male and female rats. We also tested if manipulations that knock-down the expression of RCP in SpVc, using locally-mediated gene transfer of short hairpin RNA (shRNA), ameliorate pain in an animal model of intracranial migraine-like pain induced by chemical noxious stimulation of the meninges. To assess pain, we used tests of ongoing pain (rat face grimace test and freezing behavior) and tests of facial mechanical hypersensitivity and allodynia.

Results: There was no difference in CLR levels between male and female animals (p > 0.11) in SpVc and the upper cervical cord. However, female animals exhibited greater baseline levels of RCP (up to 3-fold higher) compared to males (p < 0.002). The knock-down of RCP expression in SpVc attenuated mechanical facial allodynia induced by chemical noxious stimulation of the meninges, but had little effect on ongoing pain behaviors in female and male animals.

Conclusions: RCP is an integral component of the CGRP receptor and may play a key role in mediating CGRP induced central sensitization after noxious stimulation of the meninges. RCP expression in the SpVc and upper cervical cord is sexually dimorphic, with higher levels of expression in females. This dimorphism may be related to the increased incidence of migraines in females-a hypothesis that should be tested in the future.

Keywords: ACSF, artificial cerebrospinal fluid; Allodynia; CGRP; CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like protein; Headache; IM, inflammatory mediators; Meninges; Migraine; RAMP1, receptor activity-modifying protein 1; RCP, receptor component protein; SpVc, spinal trigeminal nucleus caudalis; Trigeminal; shRNA, short hairpin RNA.

Fig. 1

Distribution of CLR and RCP in the SpVc and upper cervical spinal cord. (A) Western blots of CLR relative to β-actin, or β-tubulin in female (n = 6) and male (n = 6) rats (control) in cytoplasmic, (B) detergent soluble or (C) detergent resistant fractions of the homogenized tissues. Quantitation of protein levels is shown below each blot. In E–F, the same animals were used to assess changes in the RCP levels in the same fractions. Here and in all figures, error bars = Standard deviations (SD).

Fig. 2

IM application causes ongoing pain and facial allodynia. (A) Rat face grimace scores (RGS) assessed at baseline (1 week after cannula implantation surgery) and immediately after a single application of IM in male (n = 5) and female (n = 6) animals (means ± SD). (B) Freezing behavior determined in the same animals presented in A ((means ± SD). (C) Whisker plot of peri-orbital mechanical withdrawal thresholds assess at baseline (1 week after cannula implantation) and 30 and 60 min after the application of IM in male (n = 6) and female (n = 6) animals.

Fig. 3

RCP shRNA knocks-down RCP protein levels. Examples of western blot of homogenized brain tissues treated with a Lenti virus to express RCP shRNA. In control experiments scrambled RCP shRNA was expressed. RCP shRNA knocked-down RCP expression in all fractions tested: (A) Cytoplasmic, (B) Detergent soluble, and (C) Detergent resistant fractions.

Fig. 4

RCP shRNA blocks IM-induced allodynia but not ongoing pain. (A) RCP shRNA (n = 6) or control shRNA (n = 6) had no effect on RGS scores or (B) freezing behavior at baseline, or after the application of IM of female rats. Control-shRNA virus expressed a scrambled sequence of RCP shRNA. (C) Whisker plots of mechanical withdrawal thresholds induced by IM application in animals treated with RCP shRNA in SpVc (n = 6) and animals treated with control shRNA (n = 6). RCP shRNA blocked IM-induced facial allodynia. In male animals, (D) face grimace scores and (E) freezing behavior increased significantly after the injection of IM, even in animals treated with RCP shRNA. (F) Whisker plots of mechanical withdrawal thresholds induced by IM application in male animals treated with RCP shRNA in SpVc (n = 6) and animals treated with control shRNA (n = 6). As in females, RCP shRNA blocked IM-induced facial allodynia.

Fig. 5

RCP shRNA blocks acidic ACSF-induced allodynia but not ongoing pain. (A) RCP shRNA (n = 6) reduced RGS scores but not (B) freezing behavior of female rats receiving applications of acidic ACSF to the meninges. (C) Whisker plots of mechanical withdrawal thresholds induced by acidic ACSF application in animals treated with RCP shRNA in SpVc (n = 6) and animals treated with control shRNA (n = 6). RCP shRNA blocked acidic ACSF-induced facial allodynia. In male animals, (D) face grimace scores and (E) freezing behavior increased significantly after the injection of ACSF, even in animals treated with RCP shRNA. (F) Whisker plots of mechanical withdrawal thresholds induced by ACSF application in male animals treated with RCP shRNA in SpVc (n = 6) and animals treated with control shRNA (n = 6). As in females, RCP shRNA blocked IM-induced facial allodynia.