VPAC1 and VPAC2 receptors mediate tactile hindpaw hypersensitivity and carotid artery dilatation induced by PACAP38 in a migraine relevant mouse model

Abstract

Background: Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide pivotal in migraine pathophysiology and is considered a promising new migraine drug target. Although intravenous PACAP triggers migraine attacks and a recent phase II trial with a PACAP-inhibiting antibody showed efficacy in migraine prevention, targeting the PACAP receptor PAC1 alone has been unsuccessful. The present study investigated the role of three PACAP receptors (PAC1, VPAC1 and VPAC2) in inducing migraine-relevant hypersensitivity in mice.

Methods: Hindpaw hypersensitivity was induced by repeated PACAP38 injections. Tactile sensitivity responses were quantified using von Frey filaments in three knockout (KO) mouse strains, each lacking one of the PACAP-receptors (N_total = 160). Additionally, ex vivo wire myography was used to assess vasoactivity of the carotid artery, and gene expression of PACAP receptors was examined by qPCR.

Results: PACAP38 induced hypersensitivity in WT controls (p < 0.01) that was diminished in VPAC1 and VPAC2 KO mice (p < 0.05). In contrast, PAC1 KO mice showed similar responses to WT controls (p > 0.05). Myograph experiments supported these findings showing diminished vasoactivity in VPAC1 and VPAC2 KO mice. We found no upregulation of the non-modified PACAP receptors in KO mice.

Conclusions: This study assessed all three PACAP receptors in a migraine mouse model and suggests a significant role of VPAC receptors in migraine pathophysiology. The lack of hypersensitivity reduction in PAC1 KO mice suggests the involvement of other PACAP receptors or compensatory mechanisms. The results indicate that targeting only individual PACAP receptors may not be an effective migraine treatment.

Keywords: Knockout mice; Migraine; PAC1; PACAP; VPAC1; VPAC2; von Frey.

Fig. 1

Design and experimental timeline of test paradigm for subcutaneous PACAP38 or saline injection in KO or WT mice. Following 1 day of habituation (day 0), five tests were done every other day over the course of 9 days. On every test day, the basal threshold of cutaneous sensitivity was measured using von Frey filaments prior to injections and the acute response was measured 1 h after PACAP38 injection. On day 9, tissues were sampled for qPCR. Carotid artery from saline treated mice were sampled between 1–4 weeks after day 9 for wire myography. Reproduced with modifications from reference [28]. Created with BioRender

Fig. 2

PACAP38 caused less hypersensitivity in VPAC1 KO mice (red line) than in WT (blue line). Concurrently, PACAP38 induced migraine-relevant hypersensitivity in both WT and VPAC1 KO mice when compared to saline controls (p < 0.01). (A) Responses one hour after subcutaneous administration of PACAP38 (2 g/kg) or saline to VPAC1 KO mice on five test days. (B) Descriptive representation of individual absolute data points for baseline and day 9 (acute response) with mean bars and SEMs

Fig. 3

PACAP38 induced less hypersensitivity in VPAC2 KO mice (red line) than in WT (blue line). Concurrently, PACAP38 induced migraine-relevant hypersensitivity in both WT and VPAC2 KO mice when compared to saline controls (P < 0.0001). (A) Responses one hour after subcutaneous administration of PACAP38 (2 g/kg) or saline to VPAC2 KO on five test days. (B) Descriptive representation of individual absolute data points for baseline and day 9 (acute response) with mean bars and SEMs

Fig. 4

PACAP38 induced significant hypersensitivity in both WT and PAC1 KO mice (p < 0.0001), but we found no significant difference in PACAP38 response between WT and PAC1 KO mice. (A) Responses one hour after subcutaneous administration of PACAP38 (2 g/kg) or saline to PAC1 KO mice on five test days. (B) Descriptive representation of individual absolute data points for baseline and day 9 (acute response) with mean bars and SEMs

Fig. 5

Myograph studies comparing the response to PACAP38 of the common carotid artery of 10 VPAC1 KO, 6 VPAC2 KO and 6 PAC1 KO mice and their WT control mice. After precontraction (0.03 µM U46619), arteries were exposed to 1 µM PACAP38. Data are presented as mean ± SD. Student’s unpaired t-test was used, each dot representing artery segment from individual mice

Fig. 6

mRNA levels of PACAP38 receptors in TNC for VPAC1, VPAC2 and PAC1 KO mice strains vs. WT. We found no differences between KO mice and WT mice regarding mRNA expression of the non-modified PACAP receptors in any of the three strains. Results are presented as individual points of mRNA expression fold change with geometric mean ± SD. WT is set to 1 and results are normalized to the levels of β-actin. Statistics are calculated from individual ΔCt (delta cycle threshold)