PACAP Induces Light Aversion in Mice by an Inheritable Mechanism Independent of CGRP

Abstract

The neuropeptides CGRP (calcitonin gene-related peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) have emerged as mediators of migraine, yet the potential overlap of their mechanisms remains unknown. Infusion of PACAP, like CGRP, can cause migraine in people, and both peptides share similar vasodilatory and nociceptive functions. In this study, we have used light aversion in mice as a surrogate for migraine-like photophobia to compare CGRP and PACAP and ask whether CGRP or PACAP actions were dependent on each other. Similar to CGRP, PACAP induced light aversion in outbred CD-1 mice. The light aversion was accompanied by increased resting in the dark, but not anxiety in a light-independent open field assay. Unexpectedly, about one-third of the CD-1 mice did not respond to PACAP, which was not seen with CGRP. The responder and nonresponder phenotypes were stable, inheritable, and not sex linked, although there was a trend for greater responses among male mice. RNA-sequencing analysis of trigeminal ganglia yielded hierarchical clustering of responder and nonresponder mice and revealed a number of candidate genes, including greater expression of the Trpc5 and Kcnk12 ion channels and glycoprotein hormones and receptors in a subset of male responder mice. Importantly, an anti-PACAP monoclonal antibody could block PACAP-induced light aversion but not CGRP-induced light aversion. Conversely, an anti-CGRP antibody could not block PACAP-induced light aversion. Thus, we propose that CGRP and PACAP act by independent convergent pathways that cause a migraine-like symptom in mice.SIGNIFICANCE STATEMENT The relationship between the neuropeptides CGRP (calcitonin gene-related peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) in migraine is relevant given that both peptides can induce migraine in people, yet to date only drugs that target CGRP are available. Using an outbred strain of mice, we were able to show that most, but not all, mice respond to PACAP in a preclinical photophobia assay. Our finding that CGRP and PACAP monoclonal antibodies do not cross-inhibit the other peptide indicates that CGRP and PACAP actions are independent and suggests that PACAP-targeted drugs may be effective in patients who do not respond to CGRP-based therapeutics.

Keywords: CGRP; CGRP antibody; PACAP; PACAP antibody; light aversion; migraine.

Figure 1.

PACAP-38 induces light aversion in CD-1 mice. A, After two baseline pre-exposures in the light-dark box (Pre1, Pre2), mice were treated (Tx) with vehicle (Veh; n = 28), PACAP-38 (P-38; 0.3 mg/kg, n = 25), or CGRP (0.1 mg/kg, n = 18). The mean (±SEM) time spent in the light zone every 5 min over a 30 min period is shown for each pre-exposure and after treatment. B, Left, Data from individual mice are shown with the mean (±SEM) time in light per 5 min interval calculated from the entire 30 min testing period from A. Right, Data from the left panel separated as male (M) and female (F). C, Effect of treatment with vehicle (n = 21), PACAP-38 (0.3 mg/kg, n = 29), or CGRP (0.1 mg/kg, n = 13) after only one baseline pre-exposure (Tx/One Pre). Data are shown for individual mice with the mean (±SEM) time in light per 5 min interval from the entire 30 min testing period. D, Open field behavior of mice injected with vehicle (n = 20), PACAP-38 (0.3 mg/kg, n = 26), or CGRP (0.1 mg/kg, n = 10). Data are shown for individual mice with the mean time (percentage ± SEM) in the center zone. Statistics are described in Table 1. For light aversion at the later time point, see Extended Data Figure 1-1.

Figure 2.

PACAP-38 reduces motility. Motility data were collected at the same time as light aversion data from the same mice shown in Figure 1, A and B. After two baseline pre-exposures (Pre1, Pre2), mice were treated (Tx) with vehicle (Veh; n = 28), PACAP-38 (P-38; 0.3 mg/kg, n = 25), or CGRP (0.1 mg/kg, n = 18). A, Resting time in light and dark zones. Left, Percentage (±SEM) of time spent resting in each zone every 5 min over the 30 min testing period. Right, Data for individual mice from treatment day shown as the mean percentage of time (±SEM) spent resting in each zone per 5 min interval. B, Rearing in light and dark zones. Left, Mean (±SEM) number of vertical beam breaks in each zone every 5 min over the 30 min testing period. Right, Data for individual mice from treatment day shown as the mean (±SEM) number of vertical beam breaks in each zone per 5 min interval. C, Transitions between the light and dark zones. Left, Mean (±SEM) number of transitions every 5 min over the 30 min testing period. Right, Data for individual mice from treatment day shown as the mean (±SEM) number of transitions per 5 min interval. Statistics are described in Table 1. For correlation between time in light and motility, see Extended Data Figure 2-1.

Figure 3.

PACAP-38 responder and nonresponder subpopulations. A, After one baseline pre-exposure (Pre), mice were treated with either vehicle (Veh; n = 20) or PACAP (P-38; 0.3 mg/kg, n = 38) in the light-dark box (Tx1). After 3 d, the same mice were treated and tested again (Tx2). PACAP-treated mice are separated into responders (P-38 R; n = 18) and nonresponders (P-38 N; n = 20). Left, Mean ± SEM time spent in the light compartment every 5 min over a 30 min period is shown for each test day. Right, Data for individual mice from each treatment day shown as the mean time (±SEM) in light per 5 min interval. B, Data from A (right) separated by sex (male, M; female, F). C, Data from A (right) showing responses of individual mice to Veh or PACAP for the P-38 R and P-38 N populations during the Pre, Tx1, and Tx2. Statistics are described in Table 1. For the ratios of nonresponders, see Extended Data Figure 3-1. For the response to vehicle and PACAP in the same mice, see Extended Data Figure 3-2. For the motility, see Extended Data Figure 3-3.

Figure 4.

Light aversion in F1 responders and nonresponders. A, F1 progeny of crosses between responder and responder mice (RxR), responder and nonresponder mice (RxN), and nonresponder and nonresponder mice (NxN) were given one pre-exposure to the testing chamber (Pre), then treated (Tx1) with vehicle (Veh; RxR, n = 38; RxN, n = 40; NxN, n = 15), or PACAP-38 (P-38; 0.3 mg/kg; RxR, n = 59; RxN, n = 56; NxN n = 21). After 3 d, the same mice were treated and tested again (Tx2). The mean (±SEM) time spent in the light zone every 5 min over a 30 min period is shown for each test day. B, Data from individual mice are shown with the mean (±SEM) time in light per 5 min interval over the entire 30 min testing period from A. Statistics are described in Table 1. For the percentage of progeny responders, see Extended Data Figure 4-1. For motility, see Extended Data Figure 4-2.

Figure 5.

Differential gene expression between responder and nonresponder mice. Gene level counts were normalized to size factors calculated by DESeq2 to correct for library size. Normalized counts were compared between responders and nonresponders using the Wald test for significance. Male and female mice in each cohort are indicated by blue and red symbols, respectively. A, Heat map reflects gene expression differences between responder (R; orange) and nonresponder (N; teal) mice on the x-axis from the list of differentially expressed genes in Extended Data Figure 5-1 that are grouped on the y-axis. Mouse number and sex [male (M) and female (F)] are indicated. Mouse numbers with the same first numbers (e.g., 48) indicate mice from the same litter. B–L, Gnrhr (B), Fshb (C), Lhb (D), Cga (E), Tshb (F), Ghrhr (G), Gh (H), Nts (I), Kcnk12 (J), Trpc5 (K), and AL731706.1 (L). Statistics are described in Table 1. For further details, see Extended Data Figures 5-1, 5-2, 5-3, 5-4, 5-5, 5-6, 5-7, 5-8, 5-9, and 5-10. For qPCR results, see Extended Data Figure 5-11.

Figure 6.

Pretreatment with anti-PACAP antibody inhibits PACAP-38-induced light aversion. A, Male and female mice were divided into four groups, two of which would eventually get vehicle and two that would get PACAP-38. After a baseline pre-exposure (Pre), but before antibody treatments, mice were treated (Tx1: pre Ab) with vehicle [Veh(1), n = 9; Veh(2), n = 9] or PACAP-38 [0.3 mg/kg; P-38(1), n = 9; P-38(2), n = 9]. Three days after Tx1 mice were given an injection of control antibody (Con Ab) or anti-PACAP antibody (PACAP Ab), then 24 h later, mice were treated with vehicle or PACAP again (Tx2: 24 h post Ab). Mean (±SEM) time spent in the light compartment every 5 min over a 30 min period is shown. B, Data for individual mice from each treatment day are shown as the mean time (±SEM) in light per 5 min interval. Statistics are described in Table 1. For motility, see Extended Data Figure 6-1.

Figure 7.

Immediate injection of PACAP-27 induces light aversion that can be blocked by anti-PACAP antibody. A, After a baseline pre-exposure (Pre), male and female mice were treated (Tx1: P-38) with vehicle (Veh; n = 20) or PACAP-38 (P-38; 0.3 mg/kg, n = 25) and assessed for light aversion 30 min postinjection. Three days later, the same mice were treated (Tx2: P-27) with vehicle or PACAP-27 (P-27; 0.2 mg/kg) and assessed for light aversion 30 min postinjection. Left, Mean (±SEM) time spent in the light every 5 min over a 30 min period is shown for each test day. Right, Data for individual mice from treatment days shown as the mean (±SEM) in light per 5 min interval. B, After Pre, mice were treated and tested immediately (Tx: immed) after injection of vehicle (n = 12), P-38 (0.3 mg/kg, n = 14), or P-27 (0.2 mg/kg, n = 13). Left, Mean (±SEM) time spent in the light compartment every 5 min over the 30 min test period. Right, Data for individual mice from the first 15 min of treatment shown as the mean (±SEM) in light per 5 min interval. C, Male and female mice were divided into four groups, two of which would eventually get vehicle and two that would get PACAP-27. After a baseline Pre, but before antibody treatments, mice were treated (Tx1: pre Ab) with vehicle [Veh(1), n = 10; Veh(2), n = 10] or PACAP-27 [0.2 mg/kg; P-27(1), n = 13; P-27(2), n = 13]. Three days after Tx1 mice were given an injection of control antibody (Con Ab) or anti-PACAP antibody (PACAP Ab), then 24 h later, mice were treated with vehicle or PACAP again (Tx2: post Ab). Mean (±SEM) time spent in the light compartment every 5 min over a 30 min period is shown. D, Data for individual mice from the first 15 min of treatment before antibody [Tx1: pre Ab (0–15 min)] and after antibody [Tx2: post Ab (0–15 min)] shown as the mean ± SEM in light per 5 min interval. Statistics are described in Table 1. For motility, see Extended Data Figures 7–1 and 7–2.

Figure 8.

Anti-PACAP antibody does not inhibit CGRP-induced light aversion. A, Male and female mice were divided into four groups, two of which would eventually get vehicle and two that would get CGRP. After a baseline pre-exposure (Pre), but before antibody treatments, mice were treated (Tx1: pre Ab) with vehicle [Veh(1), n = 25; Veh(2), n = 28] or CGRP [0.1 mg/kg; CGRP(1), n = 25; CGRP(2), n = 24]. Three days after Tx1, mice were given an injection of control antibody (Con Ab) or anti-PACAP antibody (PACAP Ab), then 24 h later, mice were treated with vehicle or CGRP again (Tx2: post Ab). The mean (±SEM) time spent in the light compartment every 5 min over a 30 min period is shown. B, Data for individual mice from each treatment day are shown as the mean time (±SEM) in light per 5 min interval. Statistics are described in Table 1. For motility, see Extended Data Figure 8-1.

Figure 9.

Anti-CGRP antibody does not inhibit PACAP-induced light aversion. A, Male and female mice were divided into four groups, two that would eventually get vehicle and two that would get PACAP. After a baseline pre-exposure (Pre), but before antibody treatments, mice were treated (Tx1: pre Ab) with vehicle [Veh(1), n = 26; Veh(2), n = 25] or PACAP-38 [0.3 mg/kg; P-38(1), n = 29; P-38(2), n = 30]. Three days after Tx1 mice were given an injection of control antibody (Con Ab) or anti-CGRP antibody (CGRP Ab), then 24 h later, mice were treated with vehicle or PACAP again (Tx2: post Ab). Mean (±SEM) time spent in the light compartment every 5 min over a 30 min period is shown. B, Data for individual mice from each treatment day are shown as the mean time (±SEM) in light per 5 min interval. Statistics are described in Table 1. For motility, see Extended Data Figure 9-1.