CGRP Administration Into the Cerebellum Evokes Light Aversion, Tactile Hypersensitivity, and Nociceptive Squint in Mice

Abstract

The neuropeptide calcitonin gene-related peptide (CGRP) is a major player in migraine pathophysiology. Previous preclinical studies demonstrated that intracerebroventricular administration of CGRP caused migraine-like behaviors in mice, but the sites of action in the brain remain unidentified. The cerebellum has the most CGRP binding sites in the central nervous system and is increasingly recognized as both a sensory and motor integration center. The objective of this study was to test whether the cerebellum, particularly the medial cerebellar nuclei (MN), might be a site of CGRP action. In this study, CGRP was directly injected into the right MN of C57BL/6J mice via a cannula. A battery of tests was done to assess preclinical behaviors that are surrogates of migraine-like symptoms. CGRP caused light aversion measured as decreased time in the light zone even with dim light. The mice also spent more time resting in the dark zone, but not the light, along with decreased rearing and transitions between zones. These behaviors were similar for both sexes. Moreover, significant responses to CGRP were seen in the open field assay, von Frey test, and automated squint assay, indicating anxiety, tactile hypersensitivity, and spontaneous pain, respectively. Interestingly, CGRP injection caused significant anxiety and spontaneous pain responses only in female mice, and a more robust tactile hypersensitivity in female mice. No detectable effect of CGRP on gait was observed in either sex. These results suggest that CGRP injection in the MN causes light aversion accompanied by increased anxiety, tactile hypersensitivity, and spontaneous pain. A caveat is that we cannot exclude contributions from other cerebellar regions in addition to the MN due to diffusion of the injected peptide. These results reveal the cerebellum as a new site of CGRP actions that may contribute to migraine-like hypersensitivity.

Keywords: CGRP; anxiety; cerebellum; light aversion; migraine; pain.

Figure 1

Injection of CGRP into the MN induced light-aversive behavior under dim light. (A) Time in light every 5-min block during 30-min light/dark assay at 55 lux following injection of PBS (n = 11; F: n = 5; M; n = 6) or CGRP (1 μg /200 nl; n = 10; F: n = 5; M: n = 5) into the right MN of C57BL/6J mice via cannulas. Time in light for all mice (left), female mice (middle), and male mice (right). Data are from two independent experiments. All mice in A are further analyzed in B. (B) Mean time in light per 5-min block for individual mice. (C) Schematic of positions of injection cannula tips superimposed on Allen Mouse Brain Atlas coronal images. Numbers indicate the distance from bregma in the anteroposterior plane. Data are the mean ± SEM. Statistics are described in Supplementary Table 1. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 2

Injection of CGRP into the MN reduced motility. Motility data were collected at the same time as light aversion data from the same mice shown in Figure 1. Mice were given PBS (n = 11; F: n = 5; M: n = 6) or CGRP (1 μg/200 nl; n = 10; F: n = 5; M: n = 5) into the right MN of C57BL/6J mice via cannulas. Data are from two independent experiments. (A) Percentage of time spent resting in the light (upper panel) and dark (lower panel) zones every 5-min block during 30-min light/dark assay for all mice (left), female mice (middle), and male mice (right). All mice in A are further analyzed in B. (B) Mean percentage of time in light (upper panel) and dark (lower panel) zones per 5-min block for individual mice from A. (C) Number of vertical beam breaks per min in light (upper panel) and dark (lower panel) zones every 5-min block during 30-min light/dark assay for all mice (left), female mice (middle), and male mice (right). All mice in C are further analyzed in D. (D) Mean number of vertical beam breaks in light (upper panel) and dark (lower panel) zones per 5-min block for individual mice from C. (E) Number of transitions between light and dark zones every 5-min block during 30-min light/dark assay for all mice (left), female mice (middle), and male mice (right). All mice in E are further analyzed in F. (F) Mean number of transitions between light and dark zones per 5-min block for individual mice from E. Data are the mean ± SEM. Statistics are described in Supplementary Table 1. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 3

CGRP injection into the MN did not induce gait alterations. Stride length (A) and frequency (B) for all mice following injection of PBS (n = 10) or CGRP (1 μg/200 nl; n = 16) into the right MN of C57BL/6J mice via cannulas. Stride length (C) and frequency (D) for female mice (PBS: n = 6; CGRP: n = 9). Stride length (E) and frequency (F) for male mice (PBS: n = 4; CGRP: n = 7). Injection of CGRP into the MN did not change the stride length and frequency comparing before and after CGRP or PBS treatments across and within sexes. It suggests that CGRP in the MN did not change the gait. LF, left front paw; RF, right front paw; LH, left hind paw; RH, right hind paw. Data are the mean ± SEM. Statistics are described in Supplementary Table 1. Data are from one experiment.

Figure 4

Injection of CGRP into the MN induced anxiety-like behavior in the open field assay. (A) Percentage of time spent in the center of the open field every 5-min block during 30-min testing period following injection of PBS (n = 11; F: n = 5; M: n = 6) or CGRP (1 μg/200 nl; n = 11; F: n = 5; M: n = 6) into the right MN of C57BL/6J mice via cannulas. All mice (left) separated by sex (female: middle; male: right). Data are from two independent experiments. All mice in A are further analyzed in B. (B) Mean percentage of time in the center per 5-min block for individual mice. Data are the mean ± SEM. Statistics are described in Supplementary Table 1. *p ≤ 0.05, **p ≤ 0.01.

Figure 5

Injection of CGRP into the MN induced plantar tactile hypersensitivity in the contralateral hind paw. Plantar tactile sensitivity was assessed with injection of PBS or CGRP (1 μg/200 nl) into the right MN of C57BL/6J mice via cannulas. Data are from three independent experiments. (A) The individual thresholds of left hind paws for all mice (left) (PBS: n = 17; CGRP: n = 26), female mice (middle) (PBS: n = 8; CGRP: n = 14), and male mice (right) (PBS: n = 9; CGRP: n = 12). (B) Comparison of changes in withdrawal thresholds of left hind paws between CGRP-treated female and CGRP-treated male mice. The change in thresholds was measured by subtracting respective baseline from CGRP treatment measurements. (C) The individual thresholds of right hind paws for all mice (left) (PBS: n = 17; CGRP: n = 26), female mice (middle) (PBS: n = 8; CGRP: n = 14), and male mice (right) (PBS: n = 9; CGRP: n = 12). (D) Comparison of changes in withdrawal thresholds of right hind paws between CGRP-treated female and CGRP-treated male mice. The mean ± SEM 50% thresholds are presented. Statistics are described in Supplementary Table 1. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 6

Injection of CGRP into the MN induced nociceptive squinting behavior. (A) Mean pixel area over 5-min testing period for all mice without treatment (as baseline), with injection of PBS (left) or CGRP (middle; 1 μg/200 nl) into the right MN of C57BL/6J mice via cannulas. Right panel is the mean pixel area over 5-min testing period for individual mice (PBS: n = 30; CGRP: n = 27). Data from A separated as female (B) and male (C). Data are from two independent experiments and one crossover treatment experiment. (B) Mean pixel area over 5-min testing period for female mice (left and middle) and mean pixel area over 5-min testing period for individual female mice (right; PBS: n = 17; CGRP: n = 14). (C) Mean pixel area over 5-min testing period for male mice (left and middle) and mean pixel area over 5-min testing period for individual male mice (right; PBS: n = 13; CGRP: n = 13). Data are the mean ± SEM. Statistics are described in Supplementary Table 1. *p ≤ 0.05, **p ≤ 0.01.

Figure 7

The diffusion range of CGRP. (A) Representative example of a mouse after injection of fluorescein-15-CGRP. Upper panel: In the most rostral section, dim fluorescence was detected in the inferior colliculus, the parabrachial nucleus, vermal lobules II-V, and the simple lobule. Middle panel: Fluorescein-15-CGRP at the injection site. Areas within rectangle are magnified in boxes 1 and 2. Clusters of fluorescein-15-CGRP were detected in cell bodies in the MN (box 1) and nearby cells, including the interposed and lateral cerebellar nuclei, granular, Purkinje cell, and molecular layers of vermal lobules I/III/IV/V (box 2). Dim signal was found in the simple lobule of the hemispheric regions. Lower panel: In the most caudal section, dim fluorescence was detected in lobule IX. Green: fluorescein-15-CGRP; Blue: TO-PRO-3. (B) The spread of the green fluorescence among the mice injected with fluorescein-15-CGRP. The smallest (purple shading) spread of signals covers the MN and few of nearby cells in the vermal lobules III/IV/V. The largest spread (blue shading) covers the MN and cells beyond the MN including vermal lobules I/III/IV/V/X, the simple lobule and other cerebellar deep nuclei. In summary, florescent signals were found in vermal lobules I-X, the simple lobule in the hemispheric region, and the midbrain (mainly in superior and inferior colliculus) from rostrally to caudally. (C) A representative image of a mouse with injection of Evans blue. (D) A representative image of a mouse with injection of red beads. DN, lateral cerebellar nucleus (dentate nucleus); GL, granular layer; IC, inferior colliculus; IP, interposed nucleus; ML, molecular layer; MN, medial cerebellar nucleus; PB, parabrachial nucleus; PCL, Purkinje cell layer; SIM, simple lobule. Image credit: Allen Institute. Numbers indicate the distance from bregma in the anteroposterior plane in Allen Mouse Brain Atlas coronal images.