Critical role of calcitonin gene-related peptide receptors in cortical spreading depression

Abstract

Cortical spreading depression (CSD) is a key pathogenetic step in migraine with aura. Dysfunctions of voltage-dependent and receptor-operated channels have been implicated in the generation of CSD and in the pathophysiology of migraine. Although a known correlation exists between migraine and release of the calcitonin gene-related peptide (CGRP), the possibility that CGRP is involved in CSD has not been examined in detail. We analyzed the pharmacological mechanisms underlying CSD and investigated the possibility that endogenous CGRP contributes to this phenomenon. CSD was analyzed in rat neocortical slices by imaging of the intrinsic optical signal. CSD was measured as the percentage of the maximal surface of a cortical slice covered by the propagation of intrinsic optical signal changes during an induction episode. Reproducible CSD episodes were induced through repetitive elevations of extracellular potassium concentration. AMPA glutamate receptor antagonism did not inhibit CSD, whereas NMDA receptor antagonism did inhibit CSD. Blockade of voltage-dependent sodium channels by TTX also reduced CSD. CSD was also decreased by the antiepileptic drug topiramate, but not by carbamazepine. Interestingly, endogenous CGRP was released in the cortical tissue in a calcium-dependent manner during CSD, and three different CGRP receptor antagonists had a dose-dependent inhibitory effect on CSD, suggesting a critical role of CGRP in this phenomenon. Our findings show that both glutamate NMDA receptors and voltage-dependent sodium channels play roles in CSD. They also demonstrate that CGRP antagonism reduces CSD, supporting the possible use of drugs targeting central CGRP receptors as antimigraine agents.

Fig. 1.

Reproducible effects of high potassium-induced CSD. (A) Representation of the acquisition system for CSD analysis. The IOS emitted by the slice are acquired by a CCD camera and transferred to a personal computer. A KCl application delivered by the perfusion system can trigger a CSD episode that originates near the KCl input. (B) A neocortical slice showing the effect of KCl application on IOS responses. (C and D) Acquisition of cortical slice images before (Pre) and after KCl application at different time points illustrating CSD propagation after a first induction (first episode) (C), as well as after a second and then a third KCl application (second and third episodes) (D). Note that multiple KCl applications in the same slice can induce reproducible CSD episodes. (E) Time course of three consecutive KCl applications showing reproducible IOS changes measured in one of three different ROIs (ROI 2) in the same slice presented in C. (F) Histogram revealing no differences across the parameters chosen to measure IOS—early peak, delayed phase, and slice area—after three consecutive KCl applications. (G) Time course of reproducible effects on cortical field potential (FP) amplitude during three episodes of CSD induced by repetitive KCl applications. (Upper) Representative traces showing a cortical field potential evoked under control conditions (a), during a CSD episode (b), and immediately after the return of field potential amplitude to baseline level (c).

Fig. 2.

Effects of NMDA and AMPA glutamate receptor antagonists, TTX, and AEDs on CSD. (A) Schematic representation of two CSD episodes propagating on a cortical slice before (first episode; blue region) and after drug administration (second episode; red region). (B) (Upper) Images of a cortical slice showing CSD propagation before KCl application (Left), in a predrug condition (Center), and after incubation with 50 μM of the NMDA receptor antagonist l-APV (Right). (Lower) Time course graphs of IOS changes in three different ROIs during CSD, in the predrug condition, and after incubation with l-APV. (C) Time course graphs of IOS changes in three different ROIs during CSD, in the predrug condition, and after incubation with 10 μM of the AMPA receptor antagonist CNQX. (D) Histogram illustrating the effects of l-APV and CNQX on IOS early peak, delayed phase, and slice area measured during KCl-induced CSD. (E–G) Time course graphs of IOS changes in three different ROIs during KCl-induced CSD propagation in the predrug condition and after incubation of the slice with 1 μM TTX (E), 30 μM CBZ (F), and 100 μM TPM (G). (H) Histogram illustrating the effects of TTX, CBZ, and TPM on IOS early peak, delayed phase, and slice area measured during KCl-induced CSD. *P < 0.05; ***P < 0.001, Student t test.

Fig. 3.

CGRP-R antagonism reduces CSD. (A) (Upper) Images of a cortical slice before CSD induction (Left), in the predrug condition (Center), and after incubation with 10 μM CGRP-R antagonist MK-8825 (Right). (Lower) Time course graphs of KCl-induced changes in IOS in the predrug condition and after incubation with MK-8825. (B) Time course graphs of KCl-induced changes in IOS in the predrug condition and after incubation with 0.1 μM BIBN4096BS. (C) Dose–response curves of the effects of MK-8825 (Left) and BIBN4096BS (Right) on CSD area. (D) Histogram comparing IOS changes in the presence of 10 μM MK-8825 or 0.1 μM BIBN4096BS. **P < 0.01; ***P < 0.001, Student t test.

Fig. 4.

Characterization of the roles of endogenous and exogenous CGRP in CSD. (A) Histogram showing IOS area during a CSD episode in control slices (white bar) and in slices incubated in the presence of 3 μM and 10 μM CGRP-R antagonist CGRP 8-37 (gray bars). (B) The increase in CGRP-LI outflow from rat cortical slices evoked by 26 mM KCl under control conditions (gray bar) was absent in experiments performed in a calcium-free medium (light-gray bar). (C) Time course of IOS measurements in control slices (black traces) and in slices incubated with 1 μM CGRP (gray traces) in the presence of increasing extracellular K⁺ concentrations (13–26 mM KCl). Note that CSD is induced in the presence of 26 mM KCl, but not at lower KCl concentrations both in control conditions and in the presence of 1 μM CGRP. (D) Histogram showing the area of slices covered by IOS changes during CSD episodes in the presence of 1 μM CGRP (light-gray bar), 10 μM MK-8825 (white bar), and CGRP plus MK-8825 (gray bar). *P < 0.05; **P < 0.01, Student t test.