by Simon Akerman, and Peter J. Goadsby Neuronal PAC1 receptors mediate delayed activation and sensitization of trigeminocervical neurons: Relevance to migraine by Simon Akerman, and Peter J. Goadsby Sci Transl Med Volume 7(308):308ra157-308ra157 October 7, 2015 Published by AAAS

Fig. 1. Intravital microscopy of VIP-induced dural meningeal blood vessel vasodilation. Intravital microscopy of VIP-induced dural meningeal blood vessel vasodilation. (A) Representation of the acquisition system for middle meningeal artery (MMA) vasodilation analysis. Dural blood vessel images were captured by a video microscope, and changes in vessel diameter were analyzed online via a video dimension analyzer. (B) Example of a blood vessel at baseline and after vasodilation. (C) Intravenous VIP (0.5 μg/kg, n = 8; 1 μg/kg, n = 9; 2 μg/kg, n = 8; 5 μg/kg, n = 32; and 10 μg/kg, n = 8) causes a dose-dependent increase in dural meningeal blood vessel diameter (F4,64 = 16.7, P = 0.000). Data have been normalized and represent the percentage increase from baseline, and are expressed as means ± SEM. Statistical analysis was performed with a one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test. *P < 0.05, statistically significant difference compared to responses after administration of VIP (0.5, 1, and 2 μg/kg). Simon Akerman and Peter J. Goadsby Sci Transl Med 2015;7:308ra157 Published by AAAS

Fig. 2. VPAC2 receptors predominantly mediate VIP- and PACAP-38–induced dural meningeal blood vessel vasodilation. VPAC2 receptors predominantly mediate VIP- and PACAP-38–induced dural meningeal blood vessel vasodilation. Mean changes in dural middle meningeal blood vessel diameter and duration of vasodilation in response to intravenous administration of VIP or PACAP-38 with VPAC1/2 or PAC1 receptor antagonists. (A to C) After two control responses to VIP (5 μg/kg) had been elicited, either (A) the VPAC1 receptor antagonist (PG97-269) (50 μg/kg, n = 7; diameter: F1.4,8.3 = 0.9, P = 0.41; duration: F3,18 = 2.8, P = 0.067), (B) the VPAC2 receptor antagonist (VIP6–28, 300 μg/kg) (diameter: t7 = 2.65, P = 0.033, n = 8; duration: t5 = 3.09, P = 0.027, n = 6), or (C) the PAC1 receptor antagonist (PACAP6–38) (150 μg/kg, n = 7; diameter: F3,18 = 4.8, P = 0.013; duration: F3,18 = 3.71, P = 0.031) was administered, and VIP administration was repeated every 10 min. (D to F) After two control responses to PACAP-38 (5 μg/kg), either (D) PG97-269 (50 μg/kg) (n = 6; diameter: F3,15 = 1.35, P = 0.30; duration: F3,18 = 1.1, P = 0.37), (E) VIP6–28 (300 μg/kg) (n = 6; diameter: F3,15 = 4.9, P = 0.014; duration: F3,15 = 3.21, P = 0.05), or (F) PACAP6–38 (150 μg/kg) (n = 6; diameter: F3,15 = 0.50, P = 0.69; duration: F1.3,6.6 = 6.0, P = 0.041) was administered, and PACAP-38 administration was repeated every 15 min to measure the duration of the response. The data have been normalized to represent a percentage change from baseline and are expressed as means ± SEM. Statistical analysis was performed with ANOVA for repeated measures when there were more than two time points, followed by two-way Student’s paired t test for post hoc analysis to test for the time points of significance. When there were only two groups, two-way Student’s paired t test was used. The average of the two baseline values was used for comparison. *P < 0.05, statistically significant difference compared to an average of the two baseline values (0 time point) by two-way Student’s paired t test. Simon Akerman and Peter J. Goadsby Sci Transl Med 2015;7:308ra157 Published by AAAS

Fig. 3. PAC1 receptors mediate NDV Fig. 3. PAC1 receptors mediate NDV. Mean changes in MMA diameter caused by electrical stimulation of the trigeminal innervation of the dura mater (NDV) after intravenous administration of VPAC1/2 and PAC1 receptor antagonists. PAC1 receptors mediate NDV. Mean changes in MMA diameter caused by electrical stimulation of the trigeminal innervation of the dura mater (NDV) after intravenous administration of VPAC1/2 and PAC1 receptor antagonists. (A to C) After two control responses to NDV, either (A) the VPAC1 receptor antagonist (PG97-269) (50 μg/kg, F3,18 = 1.0, P = 0.44, n = 7), (B) the VPAC2 receptor antagonist (VIP6–28) (300 μg/kg, t5 = 0.6, P = 0.57, n = 6), or (C) the PAC1 receptor antagonist (PACAP6–38) (150 μg/kg, F6,42 = 4.22, P = 0.002, n = 8) was administered intravenously, and NDV was repeated every 10 min for up to 1 hour until the maximal response returned. The data have been normalized to represent a percentage change from baseline and are expressed as means ± SEM. Statistical analysis was performed with ANOVA for repeated measures for more than two time points, followed by two-way Student’s paired t test for post hoc analysis to test for the time points of significance. When there were only two groups, two-way Student’s paired t test was used. The average of the two baselines was used for comparison. *P < 0.05, statistically significant difference compared to an average of the two baselines (0 time point) using two-way Student’s paired t test. Simon Akerman and Peter J. Goadsby Sci Transl Med 2015;7:308ra157 Published by AAAS

Fig. 4. PACAP-38 causes delayed sensitization of central trigeminovascular neurons. PACAP-38 causes delayed sensitization of central trigeminovascular neurons. (A) Experimental setup for electrophysiological recording of neurons in the TCC while electrically stimulating the trigeminal innervation to the dural meninges and measuring the cutaneous facial receptive field (shaded area). (B) Time course of ongoing spontaneous trigeminal neuronal firing [action potentials per second (Hz)] in response to a 20-min infusion (shaded area) of 0.9% NaCl (saline, 60 min: F8,64 = 1.1, P = 0.35, n = 9; 3 hours: F6,48 = 0.3, P = 0.93, n = 9), VIP (60 min: F4,31.9 = 2.0, P = 0.12, n = 9; 3 hours: F6,48 = 0.9, P = 0.48, n = 9), and PACAP-38 (60 min: F2.1,20.9 = 0.74, P = 0.5, n = 11; 3 hours: F1.9,15.0 = 4.9, P = 0.025, n = 9). The data have been normalized to represent a percentage change from baseline and are expressed as means ± SEM. Statistical analysis was performed with ANOVA for repeated measures for more than two time points. *P < 0.05, statistically significant difference compared to an average of the three baselines (0 time point) using two-way Student’s paired t test. (C and D) Representative peristimulus time histograms from one animal demonstrating ongoing spontaneous trigeminal neuronal firing before VIP (C) and PACAP-38 (D) infusion and at 1, 2, and 3 hours after infusion. The numbers in parentheses indicate the mean firing (Hz) over the displayed time period, green neuronal firing indicates baseline and no change in responses, blue neuronal firing indicates an increase that is not significant, and red indicates a significant increase in neuronal firing. The color-coding is extrapolated from the data averaged across all animals in a group (n = 9 to 11), as detailed above. TG, trigeminal ganglion; SuS, superior salivatory nucleus. Simon Akerman and Peter J. Goadsby Sci Transl Med 2015;7:308ra157 Published by AAAS

Fig. 5. PACAP-38 causes delayed neuronal hypersensitivity to cranial-evoked stimulation. PACAP-38 causes delayed neuronal hypersensitivity to cranial-evoked stimulation. (A) Time course of changes in the average response of intracranial dural-evoked Aδ-fiber trigeminal neurons in response to 20 min infusion (shaded area) of 0.9% NaCl (saline, 60 min: F4.2,33.2 = 0.4, P = 0.82, n = 9; 3 hours: F6,48 = 1.6, P = 0.18, n = 9), VIP (60 min: F1.6,13.1 = 2.5, P = 0.13, n = 9; 3 hours: F1.6,12.9 = 0.56, P = 0.55, n = 9), and PACAP-38 (60 min: F2.4,24.3 = 1.13, P = 0.35, n = 11; 3 hours: F2.1,16.5 = 5.95, P = 0.011, n = 9). (B and C) Original tracings from dural-evoked Aδ-fiber neuronal responses before VIP (B) and PACAP-38 (C) infusions, and 3 hours after infusion. (D and E) Histograms of the time course in the average response of innocuous (D) and noxious (E) cutaneous facial-evoked central trigeminal neuronal firing after 20 min infusion of saline (innocuous: F4,28 = 0.41, P = 0.8, n = 8; noxious: F4,28 = 0.32, P = 0.86, n = 8), VIP (innocuous: F4,28 = 0.01, P = 1.0, n = 8; noxious: F1.5,10.3 = 0.18, P = 0.77, n = 8), and PACAP-38 (innocuous: F1.9,15.4 = 6.5, P = 0.009, n= 9; noxious: F2.2,19.7 = 3.7, P = 0.041, n = 10) infusion. The data in (A), (C), and (D) have been normalized to represent a percentage change from baseline, and all data are expressed as means ± SEM. Statistical analysis was performed with ANOVA for repeated measures for more than two time points. *P < 0.05, statistically significant difference compared to an average of the baselines (0 time point) using two-way Student’s paired t test. (F and G) Representative peristimulus time histograms from one animal depicting trigeminal neuronal firing in response to innocuous brush (F) and noxious pinch (G) of the cutaneous facial receptor field before VIP (top row) and PACAP-38 (bottom row) and at 90 min and 3 hours after infusion. The numbers in parentheses indicate the mean firing (Hz) in response to stimulation (bordered area), green neuronal firing indicates baseline and no change in responses, blue neuronal firing indicates an increase that is not significant, and red indicates a significant increase in neuronal firing. The color-coding is extrapolated from the data averaged across all animals in a group (n = 8 to 10), as detailed above. Simon Akerman and Peter J. Goadsby Sci Transl Med 2015;7:308ra157 Published by AAAS

Fig. 6. VPAC1 and PAC1 receptors mediate intracranial nociceptive activation of central trigeminovascular neurons. VPAC1 and PAC1 receptors mediate intracranial nociceptive activation of central trigeminovascular neurons. (A) Time course of the average response of intracranial dural-evoked Aδ-fiber trigeminal neurons to intravenous administration of 0.9% NaCl (saline, 0.3 ml: F7,49 = 0.4, P = 0.9, n = 8), VPAC1 (PG97-269, 50 μg/kg: F7,49 = 1.2, P = 0.33, n = 8), VPAC2 (VIP6–28, 300 μg/kg: F2.4,16.7 = 0.8, P = 0.51, n = 8), and PAC1 (PACAP6–38, 150 μg/kg: F2.6,18 = 1.9, P = 0.091, n = 8) receptor antagonists. (B) Time course of the average response of dural-evoked Aδ-fiber firing of trigeminocervical neurons to intracerebroventricular administration of saline (3 μl, F3.0,18.1 = 0.9, P = 0.52, n = 7), PG97-269 (3 μg in 3 μl, F7,49 = 4.4, P = 0.001, n = 8), VIP6–28 (3 μg in 3 μl, F7,49 = 0.72, P =0.66, n = 8), and PACAP6–38 (3 μg in 3 μl, F7,49 = 2.7, P = 0.02, n = 8). The data have been normalized to represent percentage change in responses from baseline and are expressed as means ± SEM. Statistical analysis was performed with ANOVA for repeated measures for more than two time points. *P < 0.05, statistically significant difference compared to an average of the three baselines (0 time point) using two-way Student’s paired t test. Simon Akerman and Peter J. Goadsby Sci Transl Med 2015;7:308ra157 Published by AAAS

Fig. 7. Model of the role of VPAC1/2 and PAC1 receptors in the trigeminovascular system and in triggering migraine. mRNA and protein for VPAC1/2 and PAC1 receptors are present in human (32) and rat (21) middle meningeal arteries, protein expression in human trigeminal ganglia (TG) (36), mRNA expression in Model of the role of VPAC1/2 and PAC1 receptors in the trigeminovascular system and in triggering migraine. mRNA and protein for VPAC1/2 and PAC1 receptors are present in human (32) and rat (21) middle meningeal arteries, protein expression in human trigeminal ganglia (TG) (36), mRNA expression in rat trigeminal ganglia and trigeminal nucleus caudalis (TNC) (37), and protein expression in the sphenopalatine ganglia (SPG) in both human and rat (38). PACAP-38 is expressed in the TNC in rat (30). Exogenous VIP and PACAP-38 cause dural blood vessel dilation via actions on VPAC2 receptors directly on dural blood vessels. PAC1 receptors on presynaptic nerve terminals of the trigeminal innervation to the dural vasculature block the release of the vasoactive neuropeptides that cause NDV. Central PAC1 receptors are involved in both exogenously and endogenously mediated central trigeminovascular activation and sensitization in migraine. This may occur on trigeminocervical neurons (TCC), although actions in brainstem, such as the periaqueductal gray (PAG), locus coeruleus (LC) or nucleus raphe magnus (NRM), and hypothalamic or thalamic nuclei, cannot be discounted. Indeed, PACAP6–38 in the paraventricular hypothalamic nucleus modulates trigeminovascular nociceptive transmission (39). Ach, acetylcholine; NPY, neuropeptide Y; NO, nitric oxide; CGRP, calcitonin gene–related peptide; SP, substance P; NKA, neurokinin A. Simon Akerman and Peter J. Goadsby Sci Transl Med 2015;7:308ra157 Published by AAAS