Genetic enhancement of calcitonin gene-related Peptide-induced central sensitization to mechanical stimuli in mice

Abstract

Calcitonin gene-related peptide (CGRP) is a key player in migraine. To address the role of CGRP in mechanical allodynia, which is a common feature of migraine, we used CGRP-sensitized transgenic mice. These mice have elevated nervous-system expression of the human receptor activity-modifying protein-1 (hRAMP1) subunit of the CGRP receptor. Under baseline conditions, the nestin/hRAMP1 mice and control littermates had similar hindpaw withdrawal thresholds to von Frey filaments. The effect of CGRP was tested using a filament that elicited a withdrawal response on 20% of its presentations. Following intrathecal injection of 1 nmol CGRP in the nestin/hRAMP1 mice, the response frequency was 80% within 30 minutes. The antagonist CGRP(8-37) blocked the increased response. In control littermates, a 5-fold higher dose of CGRP was required to elicit a similar response. In contrast to intrathecal injection, peripheral CGRP did not increase the mechanical responses. Intraplantar injection of capsaicin was used to test the efficacy of endogenous CGRP. Capsaicin increased mechanical responses in the nestin/hRAMP1 and control mice, although a higher dose was required in controls. In contrast to control mice, there was also a contralateral paw response in nestin/hRAMP1 mice, which is consistent with central sensitization.

Perspective: In this study we show central CGRP-induced mechanical allodynia that is enhanced by overexpression of RAMP1 in nervous system. These data suggest that hypersensitivity to CGRP could be a potential mechanism underlying central sensitization in migraine and point to CGRP-receptor antagonists as a possible therapy for other pain disorders.

Figure 1. RAMP1 mRNA levels in the spinal cord

(A) Schematic of the CGRP receptor. The calcitonin-like receptor (CLR), receptor activity modifying protein-1 (RAMP1), and receptor component protein (RCP) are shown in the plasma membrane. CGRP binding requires both CLR and RAMP1. (B) Levels of mouse (m), human (h), and total RAMP1 mRNA in the lumbar spinal cord were measured by Q-PCR. RNA was isolated and amplified in duplicate from nestin/hRAMP1 mice and nestin-cre control littermates. The copies of mouse and human RAMP1 were determined by a standard curve and normalized to GAPDH. The numbers of mice are indicated in parentheses and the mean ± SEM are given, asterisk indicates p<0.05 between nestin/hRAMP1 and control.

Figure 2. Baseline mechanical responses are similar in nestin/hRAMP1 mice and littermate controls

The response frequencies (percentage of responses to 5 filament applications averaged for both hindpaws) are shown for von Frey filaments E to J that have increasing forces of 1.5, 3, 5, 9.8, 13.7 and 19.6 mNewtons, as indicated. The numbers of mice are indicated in parentheses. Symbols are the mean ± SEM.

Figure 3. Intrathecal CGRP-evoked mechanical allodynia

All panels show the response frequency to 5 applications of the F filament following intrathecal injection of CGRP, CGRP(8-37), or PBS. (A) Injection of 1 or 5 nmol CGRP or PBS in control mice. (B) Injection of 1 nmol CGRP, PBS, or 1 nmol CGRP + 5 nmol CGRP(8-37) in nestin/hRAMP1 mice. Injection of 5 nmol CGRP(8-37) or PBS in nestin/hRAMP1 mice. PBS data from panels B is repeated in panels C for comparison purposes. The numbers of mice are indicated in parentheses. Symbols are the mean ± SEM. Asterisk indicates p<0.05 between CGRP and PBS or CGRP(8-37) and PBS at the indicated time points.

Figure 4. Intraplantar CGRP does not increase mechanical nociception

All panels show the response frequency to 5 applications of the F filament following intraplantar injection of 1.3 nmol CGRP or PBS. (A) Responses in the ipsilateral paw in control and (B) nestin/hRAMP1 mice. (C) Responses in the contralateral paw in control and (D) nestin/hRAMP1 mice. The numbers of mice are indicated in parentheses. Symbols are the mean ± SEM.

Figure 5. Sensitized response of the nestin/hRAMP1 mice to intraplantar capsaicin

All panels show the response frequency to 5 applications of the F filament following intraplantar injection of capsaicin. (A) Responses in the ipsilateral paw in control and nestin/hRAMP1 mice following injection of 0.01% capsaicin. (B) Responses in the contralateral paw in control and nestin/hRAMP1 mice following injection of 0.01% capsaicin. (C) Responses in the ipsilateral paw in control and nestin/hRAMP1 mice following injection of 0.001% capsaicin. (D) Responses in the contralateral paw in control and nestin/hRAMP1 mice following injection of 0.001% capsaicin. The numbers of mice are indicated in parentheses. Symbols are the mean ± SEM. Asterisk indicates p<0.05 between nestin/hRAMP1 and control at the indicated time points.