Immunohistochemical characterization of calcitonin gene-related peptide in the trigeminal system of the familial hemiplegic migraine 1 knock-in mouse

Abstract

Introduction: Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in the CACNA1A gene, with the R192Q mutation being the most common. Elevated calcitonin gene-related peptide (CGRP) levels in acute migraine and clinical trials using CGRP receptor antagonists suggest CGRP-related mechanisms are important in migraine.

Methods: Wild-type and R192Q knock-in mice were anaesthetized and perfused. Using immunohistochemical staining, the expression of CGRP in the trigeminocervical complex (TCC) and in the trigeminal and dorsal root ganglia was characterized.

Results: There was a 38% reduction in the percentage of CGRP-immunoreactive cells in the trigeminal ganglia (p < 0.001) of R192Q knock-in mice compared to wild-type animals. The size distribution profile of CGRP-immunoreactive cells within the trigeminal ganglia demonstrated no significant difference in cell diameter between the two groups (p ≥ 0.56). CGRP expression was also reduced in thoracic ganglia of R192Q knock-in mice (21% vs. 27% in wild-type group; p < 0.05), but not in other ganglia. In addition, decreased CGRP immunoreactivity was observed in the superficial laminae of the TCC in R192Q knock-in mice, when compared to the control group (p < 0.005).

Conclusion: The data demonstrates that the FHM-1 CACNA1A mutation alters CGRP expression in the trigeminal ganglion and TCC. This suggests further study of these animals is warranted to characterize better the role of these mutations in the neurobiology of migraine.

Keywords

CGRP migraine R192Q trigeminal system

Introduction

Familial hemiplegic migraine (FHM) is an autosomal dominant subtype of migraine with prominent aura symptoms. Attacks are episodic and are characterized by the presence of hemiparesis, which may be isolated or associated with other aura symptoms (1). To date three genes have been identified for migraine, which establish a molecular basis for these migraine syndromes (2). Although FHM represents a rare subtype of migraine, it is likely that genes, such as the CACNA1A calcium channel gene implicated in FHM type-1 (FHM-1), will offer insights into migraine more generally, if imperfectly.

The R192Q mutation of the CACNA1A gene is one of a number of recognized mutations implicated in FHM-1 (3–6). This mutation eliminates a conserved positive charge in the voltage sensor S4 segment of domain I on the N-terminal side of the α1A subunit of Cav2.1 channels (3). The mutation demonstrated a gain-of-function phenotype, including enhanced Ca²⁺ influx in human Cav2.1 channels, increased Cav2.1 current density in cerebellar neurons and enhanced neurotransmission at the neuromuscular junction (7,8). The R192Q mutation has so far been shown to affect cerebellar and cortical neurotransmission (8,9), therefore it is likely that the functional changes in mutant Cav2.1 calcium channels lead to secondary changes in the expression of transmitters, neuropeptides, and receptors involved in Cav2.1-related neurotransmission.

The R192Q mutation has been successfully reproduced in a knock-in mouse (8). The transgenic FHM-1 mouse model demonstrates an increased susceptibility to cortical spreading depression (CSD) (8), which is widely considered to be the animal homologue of migraine aura (10,11). In vivo experiments have shown that this knock-in mouse has a lower threshold for CSD induction, an increased velocity of propagation, and a longer duration of CSD depolarization (8). Moreover, unlike other CACNA1A transgenic mice, which show variable symptoms of ataxia and epilepsy, they exhibit no overt phenotype and are therefore considered a very promising model for further study into migraine mechanisms (12).

Cav2.1 channels in the trigeminocervical complex (TCC) contribute to the modulation of trigeminovascular nociception in vivo (13), and it is thought this may be achieved by modulating the pre-synaptic release of neurotransmitters, such as glutamate and calcitonin gene-related peptide (CGRP) (14). CGRP is a neuropeptide with a pivotal role in migraine pathophysiology. During acute migraine attacks, CGRP serum levels are elevated in the external jugular circulation of migraineurs (15) and treatment with the 5-HT_1B/1D agonist sumatriptan normalizes its levels in parallel with headache relief (16). Additionally CGRP receptor antagonists, such as olcegepant (BIBN4096BS), are effective in aborting migraine attacks (17–19), while CGRP infusion in migraineurs can induce headache (20). In animal models, activation of trigeminal sensory fibers evokes a CGRP-mediated neurogenic dural vasodilation (21), which is blocked by a number of migraine treatments, including dihydroergotamine and triptans (22), and has a calcium channel-mediated component (23). In addition, neuronal firing at the level of TCC can be blocked by CGRP receptor antagonists (24,25).

Here we studied whether the R192Q mutation in the transgenic mouse model alters CGRP expression in the TCC and in the trigeminal ganglia (TG), structures which are known to be involved in trigeminovascular activation and are implicated in migraine pathophysiology (26). CGRP expression was also characterized in the dorsal root ganglia (DRG) to ascertain whether changes seen in the R192Q knock-in mouse were specific to the trigeminovascular pathway or if they also occurred in DRG, given that triptan 5-HT_1B/1D receptors are widely distributed at all levels of the sensory ganglia (27). Elements of this study have been previously presented in a preliminary form (28).

Materials and methods

Tissue preparation

All animal procedures were carried out in accordance with a project license issued by the UK Home Office under the direction of the Animals (Scientific Procedures) Act, 1986 and in accordance with UCSF Institutional Animal Care and Use guidelines. The experiments were performed on untreated homozygous transgenic R192Q knock-in (R192Q KI; n = 18) micewith the human FHM-1 R192Q mutation knocked in and backcrossed in C57BL/6Jico mouse for at least five generations, as previously described (8). Untreated wild-type (wt) C57BL/6J littermate controls (n = 17) were used as a comparison to these knock-in mice. All mice were bred at the Leiden University Medical Center and transported to London or San Francisco. The animals were housed in a 12:12 hours light:dark cycle with food and water available ad libitum.

Mice (5–8 months old; 20–30 g; 22 males, 13 females) were deeply anaesthetized with sodium pentobarbital (80 mg/kg i.p.) and perfused through the aorta with 0.9% saline (50 ml), followed by 4% paraformaldehyde (25 ml) in 0.1 M phosphate buffer (PB; pH 7.4). The left or right trigeminal ganglion (wt, n = 7; R192Q KI, n = 8), left or right cervical, thoracic, lumbar and sacral DRG (wt, n = 6; R192Q KI, n = 6) were dissected out. The lower brainstem and upper cervical spinal cord segments incorporating the TCC, which extends from the trigeminal nucleus caudalis to the C2 cervical spinal cord level (29), were also collected (wt, n = 7; R192Q KI, n = 8). The tissue was cryoprotected in 30% sucrose in 0.1 M PB containing 0.01% sodium azide, and stored for a minimum of 48 hours at 4°C. The ganglia and the lower brainstem incorporating the TCC were then embedded in 100% optimal cutting temperature solution (OCT; Poole, England) and cryosectioned serially. The TG were cut into 20 µm longitudinal sections and two consecutive sections every 140 µm were mounted separately on double gelatinized slides. In this way a total of 6–8 sections per animal were mounted on the same slide and used for CGRP staining, and their consecutive sections mounted on a separate slide were used for staining with cresyl violet (Sigma). As DRG (levels C5, C6, T10, T11, L4, L5, S3, S4) are smaller, they were cut into 9 µm longitudinal sections and two consecutive sections every 18 µm were mounted on separate double gelatinized slides. In this way a total of 5–7 sections per DRG ganglion, per animal, were mounted on the same slide and used for CGRP staining, whilst their consecutive sections were used for staining with cresyl violet. The TCC was dissected out and the sections were identified anatomically using a mouse atlas (30). Eight coronal sections (thickness 20 µm) per animal that spanned the trigeminal nucleus caudalis, C1 and C2 levels of the spinal cord were collected in 0.1 M PB-containing incubating wells for free floating immunohistochemical processing. Adjacent sections were stained with cresyl violet.

Immunofluorescence

The sections were rinsed in 0.1 M PB and then incubated for 1 hour in blocking solution (5% normal goat serum, 0.5% Triton, 0.01% sodium azide in 0.1M PB). The tissue was then incubated overnight at 4°C with the primary antibody, rat anti-CGRP raised in rabbit (1:8000; Sigma-Aldrich, St. Louis, MO), diluted in 2% normal goat serum in PB. The sections were then rinsed in PB and incubated for 2 hours at room temperature with secondary antibody, biotinylated anti-rabbit raised in goat (1:500 diluted in the same buffer; Vector Laboratories, Burlingame, CA, USA), followed by further washes in PB. This step was followed by a 30-minute incubation period with an avidin-biotinylated complex in accordance with the manufacturer’s protocol (Vectastain ABC kit, Vector Laboratories). The sections were then rinsed in PB and incubated for 1 hour at room temperature with avidin coupled to fluorescein isothiocyanate (1:500; Vector Laboratories) in PB. Sections were finally coverslipped for microscope visualization.

Antibody characterization

Rabbit polyclonal antibody against rat αCGRP (Sigma-Aldrich; C8198), which is commercially available as a delipidized whole antiserum has been used in this study. In principle, polyclonal antibodies that are raised against rat αCGRP should also be suitable for the detection of both αCGRP and βCGRP peptide isoforms in mice (31). Due to the high sequence homology of the murine αCGRP and βCGRP peptides, the generation of isoform selective antibodies has not yet been successful. Here we used an antibody that was raised against rat αCGRP, which is identical in sequence to mouse αCGRP and differs in only three amino acids from mouse βCGRP. Therefore this antibody should detect both CGRP isoforms in mice (31), and previous studies have demonstrated its ability to react with mouse CGRP (32–34). In dot blots there is negligible cross-immunoreactivity with calcitonin, somatostatin, and amylin, but there is 100% cross-reactivity with rat CGRP (manufacturer’s technical information). In CGRP knock-out mice, CGRP immunoreactivity assessed with this antibody is abolished (35). Additionally, staining of sections through the ganglia and the TCC produced a pattern of CGRP immunoreactivity that was identical with previous descriptions (Figures 1 and 2) (33,36).

Figure 1.

Calcitonin gene-related peptide (CGRP) immunoreactivity in wild-type (wt) and R192Q knock-in (KI) mice. A−D: Representative microphotographs of trigeminal ganglia sections from wt and R192Q KI mice immunostained for CGRP (A, C) and their corresponding adjacent sections stained with cresyl violet (B, D). The population of CGRP-immunostained cells in wt animals (A) is higher than in R192Q KI mice. E: Microphotographs of a negative control section where the primary anti-CGRP antibody was substituted with an antibody-free normal goat serum (2%). Bars, 50 µm for all images. F: Box plot showing a comparison of the percentage of CGRP-immunopositive neurons in the trigeminal and dorsal root ganglia of R192Q knock-in and wt mice.

Figure 2.

Size distribution profile of calcitonin gene-related peptide (CGRP) immunopositive cells in (A) trigeminal and (B) dorsal root ganglia of wild-type and R192Q knock-in mice. Box plots show the percentage of small (<20 µm), medium (20⊟25 µm) and large (>25 µm) diameter neurons expressing CGRP.

In the current study, the specificity of the primary antibody for its antigen was tested using a pre-adsorption control in which the primary antiserum had been neutralized overnight at 4°C with 10 µM of the antigen peptide used to raise the antibody, rat αCGRP (Sigma) before being used for immunohistochemistry as described above. This control procedure abolished the specific signal of the primary CGRP antibody. From each cryosectioned tissue (ganglia/TCC) a single section was kept for negative control studies, which were done in parallel with the CGRP-immunofluorescent reactions. In these studies the immunofluorescent procedure was performed as described above; however, the primary antibody was substituted with an antibody free normal goat serum (2%). This negative control procedure revealed no staining in any of the immunoreacted tissue.

Staining with cresyl violet

Sections taken from the ganglia, that were adjacent to CGRP-reacted sections, were stained with cresyl violet (Sigma-Aldrich) in order to provide estimates of the total number of cells in the ganglia sections that were stained for CGRP. This enabled CGRP-like immunoreactive cells to be expressed as a percentage of the total number of cells in a given area. Sections from the TCC were also stained in order to aid anatomical identification of the spinal cervical levels and the trigeminal nucleus caudalis, using the atlas of Paxinos and Franklin (30). Slides were immersed for 2 minutes in each of the following solutions: xylene, 100% alcohol, 95% alcohol and 70% alcohol. The slides were then rinsed in distilled water and incubated in 0.5% cresyl violet for 1 minute. After rinsing in water for 1 minute, the sections were dehydrated through 1 minute steps in ascending series of alcohol followed by xylene. The slides were then coverslipped using distyrene plasticizer xylene (DBX) medium (Fischer Scientific, Pittsburgh, PA, USA).

Analysis

All sections were visualized using an Axioplan Universal microscope equipped with a digital camera (MRC5; Carl Zeiss, Jena, Germany) and Axiovision software (version 4; Zeiss). For fluorescent signal visualization the same system device was used with the relevant filter block (fluorescein isothiocyanate, 450⊟490 nm).

Trigeminal and dorsal root ganglia

Before proceeding with counting cells in either CGRP or cresyl violet sections, the areas chosen for counting were visualized in both sections to ensure they were at the same level and corresponded to the area with the highest neuronal population. For TG and DRG, photomicrographs of the areas with the highest neuronal populations, were captured using a digital camera, by an investigator blinded to the respective groups. The same magnification (×100), time exposition, binning, and gain were used for each image, and no further manipulation of the photomicrographs was performed. The CGRP-immunoreactive cells in each section were counted by two independent investigators, blinded to the experimental groups. For CGRP-stained sections only strongly stained cells that displayed a fluorescent signal well above background throughout their cell body with a visible immunonegative nucleus were counted as being CGRP immunoreactive. Cresyl violet stained cells on adjacent sections were counted in the corresponding fields where CGRP-immunoreactive cells had been counted. For each section the total number of CGRP-immunoreactive neurons was then expressed as a percentage of the total number of cresyl violet stained cells in the area where the cells were counted. The diameters of these neurons were also measured in order to compare the size distribution profile of cells that express CGRP in R192Q KI and wild-type mice. The neurons were variable in shape; therefore to establish consistency in measurement, diameter was measured across the widest region for all cells. In order to characterize primary fiber types, this measurement was used to create size distribution profiles for CGRP-immunoreactive neurons in mutant and wild-type mice, with small cells being < 20 µm, medium cells 20⊟25 µm, and large cells >25 µm (37). The diameters of all the cresyl violet stained cells were also measured to determine if the size distribution profile for all cells were different in R192Q KI and wild-type mice.

Trigeminocervical complex

The TCC extends from the trigeminal nucleus caudalis to the C2 cervical spinal cord level (29,38). On average, eight sections that spanned the TCC were stained per animal. Pictures were taken on the same day to prevent the decrease of fluorescence intensity over time. Following acquisition of images at ×50 magnification, with unchanged linear properties, Image J (National Institutes of Health, Bethesda, MD) was used for densitometric analysis of CGRP fibers in each hemisection as previously described (39,40). Results are expressed as median and interquartile (IQ) ranges per hemisection for each animal group.

Statistical analysis

Cronbach’s coefficient α was used to assess intra- and inter-observer reproducibility for counts made by the two independent investigators. All observations from the two investigators had an α coefficient >0.8 (41) and thus values were pooled together. At least 700 cells were counted per animal per ganglia over 6–8 longitudinal sections and all data are expressed as median and IQ ranges per section for each animal group. As measurements were made on an ordinal scale, statistical analysis was carried out using non-parametric statistics (42). The Mann−Whitney U-test was used when making comparisons between the two animal groups. Within each animal group, the percentage of CGRP-immunoreactive neurons in the TG was compared with other DRG levels using Kruskall−Wallis analysis of variance followed by Mann−Whitney U-tests with the Bonferoni correction. Analysis was performed with SPSS version 16.0 (SPSS, Chicago, IL, USA). A two-tailed test at the p < 0.05 level was considered significant.

Results

No differences were seen between male and female animals for any of the structures considered (p ≥ 0.29) and thus the data from both sexes were pooled together. Cresyl violet-stained sections throughout the ganglia and the TCC demonstrated no gross anatomical differences between wild-type and R192Q KI mice. CGRP staining was also highly reproducible for all tissue used in this study. Cronbach’s alpha test showed that there was good correlation (α = 0.86) between the data in the ganglia obtained from the two investigators and so the counts were pooled for further analysis.

Trigeminal and dorsal root ganglia

Cell characteristics

The total number of cells stained with cresyl violet were counted in the TG and in the cervical (C5, C6), thoracic (T10, T11), lumbar (L4, L5), and sacral (S3, S4) DRG. The number of cells counted was not different between R192Q KI and wild-type mice, in both the TG (wt: n = 7, median = 137, IQ range 99−193; R192Q KI: n = 8, median = 134, IQ range 91⊟188; p = 0.31) and DRG (wt: n = 6, median = 201, IQ range 152⊟236; R192Q KI: n = 6, median = 214, IQ range 175⊟268; p = 0.09). The diameter of cells in the TG was also measured and the median diameter in both animal groups was 19 µm (IQ range for both R192Q KI and wt: 16⊟23 µm). There were no significant differences (p ≥ 0.36) in the percentage of large (wt: 19%, median diameter 27 µm; R192Q KI: 13%, median diameter 27 µm), medium (wt: 31%, median diameter 22 µm; R192Q KI: 29%, median diameter 22 µm), and small diameter cells (wt: 51%, median diameter 17 µm; R192Q KI: 58%, median diameter 17 µm).

CGRP-like immunoreactivity

In both animal groups, CGRP was intensely stained in the cell bodies and in fibers travelling in central and peripheral directions of the TG and DRG. CGRP-like immunoreactive cell bodies were distributed throughout the ganglia and the staining pattern was diffusely granular in some cell bodies and homogenous in others. No gross differences were detected in the morphology and staining pattern of CGRP-immunoreactive cells in wild-type and mutant mice.

Wild-type mice: In the TG of wild-type mice, 29% (IQ range 21⊟33%; Table 1) of cells were CGRP immunoreactive, and these were mainly cells of small and medium diameter (small: 57%, IQ range 47⊟72%; medium: 23%, IQ range 18⊟30%; large: 20%, IQ range 5⊟27%). The median percentage of CGRP-immunoreactive neurons in the DRG ranged between 20 and 27% (overall expression: 24%, IQ range 20⊟29%; Table 1). There was no difference in the percentage of CGRP-immunoreactive neurons in the trigeminal ganglion compared to cervical, thoracic, lumbar, and sacral DRG (p ≥ 0.05, Mann−Whitney after Bonferroni correction; Table 2).

Table 1.

Percentage of calcitonin gene-related peptide immunoreactive neurons in the trigeminal and dorsal root ganglia of R192Q knock-in (KI) and wild-type mice

	Wild type	R192Q KI	p-value
Trigeminal ganglia	29 (21⊟33)	18 (14⊟22)	<0.001
Cervical ganglia	24 (21⊟30)	26 (20⊟30)	0.96
Thoracic ganglia	27 (22⊟29)	21 (16⊟25)	<0.05
Lumbar ganglia	20 (17⊟27)	24 (20⊟30)	0.11
Sacral ganglia	23 (17⊟26)	21 (17⊟23)	0.46

Values are median % (interquartile range). Statistical comparisons were assessed using Mann−Whitney U-test.

Table 2.

Results of statistical comparisons (p-value) of calcitonin gene-related peptide immunoreactivity in the trigeminal ganglia compared to dorsal root ganglia in R192Q knock-in (KI) and wild-type mice

	Trigeminal ganglia
Wild type	R192Q KI
Cervical ganglia	0.45	0.001*
Thoracic ganglia	0.64	0.16
Lumbar ganglia	0.02	0.001*
Sacral ganglia	0.02	0.10

Statistical comparisons were assessed using Mann−Whitney U-test with Bonferoni correction analysis. *p < 0.0125

R192Q knock-in mice: In the TG of R192Q KI mice, 18% (IQ range 14⊟22%; Table 1) of cells were CGRP immunoreactive with a distribution profile of 56% small (IQ range 39⊟75%), 24% medium (IQ range 16⊟32%), and 20% large (IQ range 9⊟27%) diameter cells. The percentage of CGRP-immunoreactive neurons in the DRG ranged between 21 and 26% (overall expression: 23%, IQ range 17⊟28%). Across the different levels of ganglia, the overall percentage of CGRP-immunoreactive neurons was significantly different (H(4) = 18.85, p = 0.001). Post-hoc analysis (level of significance: 0.0125) revealed that R192Q KI TG had a significantly lower percentage of CGRP-immunoreactive neurons compared to cervical and lumbar (p = 0.001) but not thoracic and sacral (p ≥ 0.10) DRG (Table 2).

The percentage of CGRP-expressing neurons in the TG of R192Q KI was significantly lower compared to wild-type animals; 18% vs. 29% (p < 0.001; Figure 1). Overall CGRP expression in the DRG appeared to be unaffected by the mutation (p = 0.44; Figures 1 and 3). However when comparing the individual levels of DRG of R192Q KI and wild-type animals, although no significant difference was seen for the cervical, lumbar, and sacral ganglia (p ≥ 0.11; Table 1), significance was recorded for the thoracic ganglia (p < 0.05; Table 1). The median diameter of CGRP-expressing cells in the TG of both R192Q KI and wild-type mice was equivalent (18 µm). A size distribution profile of CGRP-immunoreactive cells within the TG demonstrated no significant difference between the two groups (p ≥ 0.56; Figure 2).

Figure 3.

Calcitonin gene-related peptide (CGRP) immunoreactivity in the dorsal root ganglia of wild-type and R192Q knock-in mice. Representative microphotographs of cervical, thoracic, lumbar, and sacral ganglia sections immunostained for CGRP (green) and their corresponding adjacent sections stained with cresyl violet. Bars, 50 µm for all images.

Trigeminocervical complex

Cronbach’s alpha test showed excellent correlation (α = 0.9) between the measurements obtained from the two investigators, allowing us to pool the data for analysis. CGRP-immunoreactive fibers were predominantly seen in layers I and II in the TCC of both mutant and wild-type mice. Fewer CGRP-immunoreactive fibers were seen in deeper laminae (IV and V). CGRP immunoreactivity in the TCC was 29% lower in R192Q KI mice compared to wild-type animals (wt: n = 8; median 1649, IQ range 1022⊟3408; R192Q KI: n = 8; median 1140, IQ range 734⊟2356; p < 0.05; Figure 4).

Figure 4.

Calcitonin gene-related peptide (CGRP) immunoreactivity in the trigeminocervical complex of wild-type (wt) and R192Q knock-in (KI) mice. A, B: Representative microphotographs of trigeminocervical hemisections (at the cervical spinal cord level 1) showing CGRP-immunopositive fibers concentrated mainly in laminae I and II. All images were acquired with a fluorescent microscope (Carl Zeiss). C: Box plot showing a comparison of the intensity of CGRP immunostaining in laminae I and II per hemisection.

Discussion

This is the first quantitative and comparative anatomical study of CGRP in the trigeminal system of the CACNA1A R192Q knock-in mouse. Our results demonstrate a decrease in the expression of CGRP in first-order trigeminal ganglion neurons of R192Q knock-in mice compared to wild-type mice. This trend was also seen in the TCC, where the intensity of staining of CGRP-immunoreactive fibers within the superficial laminae was also reduced. The mutation did not affect the size distribution profiles of CGRP-immunoreactive neurons in the ganglia. CGRP-expressing cells were mainly of small to medium diameter corresponding to unmyelinated C- and thinly myelinated Aδ-fibers, reinforcing the importance of this peptide in nociceptive processing in the R192Q KI mouse, as well as in control animals. The reduced expression was not restricted to the trigeminal system as CGRP expression was also significantly reduced in the thoracic ganglia of the R192Q knock-in mouse. The mutation did not affect the total number of cells in the TG and DRG, thus the reduced number of CGRP-immunoreactive cells reflects a decrease in the expression of CGRP and is not due to the ablation of a neuronal subpopulation. The data provide an interesting link between the FHM mutation and the neurobiology of more typical forms of migraine, in particular, with regard to CGRP mechanisms and the trigeminovascular system.

Cav2.1 channels are located at pre-synaptic terminals throughout the brain, where they have a prominent role in the control of neurotransmitter release (14,43,44). They are expressed in many structures known to have a role in migraine pathophysiology (45), including the trigeminal ganglion, where they, along with L- and N-type voltage-gated calcium channels, control the pre-synaptic release of neurotransmitters (14). One would expect the increased opening probability of mutant channels, which has been demonstrated at cerebellar and cortical excitatory synapses, would lead to enhanced exocytosis of neurotransmitters in areas where neurotransmission is predominantly Cav2.1 channel mediated. Here we report reduced expression of CGRP in the trigeminal system of the FHM-1 mouse model. One possibility is that this reflects an over-active trigeminovascular system in which there is enhanced exocytosis and a resulting depletion of intracellular CGRP stores. However, there is evidence demonstrating that CGRP release leads to a further increase in CGRP synthesis from neurons in the TG (46), but whether this is the case in this particular mouse model is uncertain. Another possibility is the result reflects a true reduction in the expression of CGRP.

The R192Q KI mouse has been shown to have a reduced threshold and an increased velocity of propagation for CSD (8), the animal homologue of migraine aura. The increased susceptibility to CSD could be a result of increased action-potential-evoked Ca²⁺ influx through pre-synaptic Cav2.1 channels and the subsequent enhancement of cortical glutamate release (9). If FHM mutations are directly responsible for the pathogenesis of migraine, it is unlikely that their effects would be confined to one neuronal process, such as CSD, relevant to migraine. This is clearly shown in our study, where CGRP expression in the trigeminal system of R192Q knock-in mice is also affected. More recent data from our laboratory shows alterations in Fos protein expression with nociceptive trigeminovascular stimulation (47) and altered Fos protein levels in central modulatory nuclei (48) in R192Q mutant mice compared to wild-type mice, again reinforcing the broader pathophysiological effects of the FHM mutation that our data imply.

CGRP plays a crucial role in migraine pathophysiology (49). It is present in key structures related to migraine pathophysiology (50–52) and its extracerebral levels are elevated in patients during migraine attacks (16). Whether CGRP plays a causative role in migraine attacks is not known but it is unlikely, as evidence now emerging demonstrates that vasodilatation is neither necessary nor sufficient to induce a migraine attack. However, when CGRP is infused in migraineurs it can trigger an attack that is indistinguishable from typical migraine, suggesting that increased CGRP levels in migraineurs may play at least a contributory role in some crucial parts of the attack (20). Clinical trials have demonstrated the efficacy of CGRP receptor antagonists in the treatment of acute migraine (18,19) and basic research has suggested that these antagonists could be acting centrally, at least at the level of the trigeminocervical complex (24,25,53), where they inhibit trigeminovascular activation. FHM shares many clinical features with common types of migraine, as the headache and visual aura symptoms are similar to those of more usual migraine with aura (54) and FHM patients can experience migraine with or without aura, devoid of hemiparesis symptoms (55). This implies the existence of common neurobiological pathways in these disorders. One might thus expect a more prominent role of CGRP in the trigeminal system of an FHM-1 animal model; interestingly our results revealed a reduced number of CGRP-immunoreactive neurons within the trigeminal system of R192Q knock-in mice. The loss of trigeminal CGRP cells and fibers seen in our study and recent data from clinical studies that showed FHM patients are insensitive to CGRP infusion (56), could reflect important differences in the neurobiology of FHM compared to other types of migraine. In support of this, other studies showed FHM-1 and FHM-2 patients demonstrate no hypersensitivity to nitric oxide (57,58), which normally causes a headache attack in migraineurs (59). Moreover, there are intriguing clinical trial data suggesting that the CSD inhibitor tonabersat (60), which is not effective in routine migraine with and without aura in terms of headache reduction (61), does reduce aura attacks (62). Both our results and these clinical studies underlie the importance of bench-to-bedside correlations that can facilitate a better understanding of migraine mechanisms and potentially predict the effect of therapeutics.

Beyond the differences in the CGRP-like staining in TG of R192Q knock-in and wild-type mice, a significant decrease of CGRP-like stained cells was also seen in the thoracic ganglia. As Cav2.1 are widely distributed in the neural system, including the DRG, it is not surprising that a mutation which affects this channel affects the expression of CGRP beyond the trigeminal system. Why such difference was seen only in the thoracic ganglia and not in other DRG, however, is unknown. The most important property of CGRP is being a vasodilator at all levels of the cardiovascular system with its major activity being local at the site of release. It remains to be investigated whether this difference in R192Q knock-in mice reflects any difference on the activity of CGRP within the cardiovascular system of FHM-1 patients. To date, no such evidence from FHM-1 patients have been presented. Investigating potential differences in the population of central CGRP-expressing cells in FHM-1 knock-in mice further represents an unexplored avenue. For example, the cerebellum represents one of the CNS areas with the highest levels of CGRP-containing neurons (63) and as the FHM-1 mutation increases Cav2.1 current density in cerebellar neurons (7), it will be interesting to see if and how it may affect the expression of CGRP in the cerebellum.

Some methodological considerations need to be addressed in this study. CGRP immunoreactivity in ganglion neurons was quantified as a percentage of the total number of cells stained with cresyl violet. The cell bodies stained with cresyl violet are most likely to correspond to neuronal cell bodies, as glial cells only represent a small number of satellite cells in rodent TG (64) and neuroglial cells have very little detectable cytoplasm around the nucleus and thus do not stain intensely. The use of cresyl violet was chosen, as the morphology of stained ganglion structures is clearly visible and cells could be easily differentiated for the purposes of counting. Our results regarding the percentage of CGRP neurons in the TG of wild-type animals are similar to those previously reported in mice (33). This is the first study, to our knowledge, to estimate the percentage of CGRP-expressing neurons within the different levels of DRG in wild-type mice, therefore comparisons cannot be drawn with existing data. To assess the intensity of staining of fibers in laminae I and II of the TCC, the size of the area where CGRP-immunoreactive fibers were concentrated was considered and the mean intensity of staining within this area was calculated. We are not aware of an objective consistent method of quantification for fiber density, and in most previous studies an arbitrary intensity threshold had to be set (65–67). In our study all photomicrographs were taken under the same conditions and no further manipulation was applied. Furthermore, we ensured that each section had its own control by expressing the intensity of CGRP immunoreactivity as a ratio of the intensity of staining in a given area where no CGRP-fluorescent fibers were present and only background staining was seen. The same quantification methods were applied by observers blinded to the experimental groups, for both ganglia and TCC sections, thus the recorded differences in CGRP expression between knock-in and wild-type mice are valid.

The functional role of the R192Q mutation within the trigeminovascular system remains unknown. The loss of CGRP immunoreactivity seen in the TG of the R192Q knock-in mouse is also reflected in the TCC and this suggests that a less dense plexus of CGRP fibers are innervating dural structures than would normally be expected (50,68). Stimulation of dural vessels innervated by trigeminal fibers has been shown to cause a headache-like pain in humans (69). In animal models, electrical stimulation of dural structures results in CGRP-induced dural vasodilation (21) and activates second-order neurons centrally, within the TCC (29,38,70). Our results, in addition to recent clinical data imply the existence of changes in the neurobiology of trigeminal pain pathways in FHM-1 that are particular to the mutation, as compared to non-mutant animals. The use of experimental models of trigeminovascular activation in the R192Q mouse will help to evaluate further the effects of the mutation during activation of the trigeminovascular pathway. Moreover, the development of mouse models for FHM-2 and 3, or examination of a different FHM-1 mouse model, such as the S218L FHM-1 knock-in mouse (71), which also has a decreased threshold for CSD induction (72), will reveal whether the observed differences are specific to the R192Q mutation or if these changes are representative of pathophysiological mechanisms that are inherent to all FHM subtypes. Of note, the S218L KI mouse exhibits ataxia (12) and is predisposed to severe brain oedema (72), therefore it is likely that this mutation has more profound functional effects compared to those seen in R192Q knock-in mice.

It is important to put into perspective that FHM represents a rare subtype of migraine (73). Nevertheless, the increased propensity of the R192Q knock-in mouse for CSD correlates with the prominent aura symptoms that characterize FHM (1), and the decreased expression of CGRP in the trigeminal system of these mice, as recorded in the current study, may be related to the reduced sensitivity of FHM patients to CGRP infusion (56). These features imply that the R192Q knock-in mouse is a reliable model of FHM-1 in more than one dimension with further work being necessary to gain insights into how the fascinating biology of this mouse can inform our understanding of migraine more broadly.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

References

Headache Classification Committee of The International Headache Society. The International Classification of Headache Disorders (second edition). Cephalalgia 2004; 24(Suppl 1): 1–160.

van den Maagdenberg

AMJM

Haan

Terwindt

Ferrari

. Migraine: gene mutations and functional consequences. Curr Opin Neurol 2007; 20: 299–305.

Ophoff

Terwindt

Vergouwe

van Eijk

Oefner

Hoffman

SMG

. Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 1996; 87: 543–552.

Haan

Kors

van den Maagdenberg

Vanmolkot

Terwindt

Frants

. Toward a molecular genetic classification of familial hemiplegic migraine. Curr Pain Headache Rep 2004; 8: 238–243.

Alonso

Barros

Tuna

Seixas

Coutinho

Sequeiros

. A novel R1347Q mutation in the predicted voltage sensor segment of the P/Q-type calcium-channel alpha-subunit in a family with progressive cerebellar ataxia and hemiplegic migraine. Clin Genet 2004; 65: 70–72.

Kors

Vanmolkot

Haan

Frants

van den Maagdenberg

Ferrari

. Recent findings in headache genetics. Curr Opin Neurol 2004; 17: 283–288.

Tottene

Fellin

Pagnutti

Luvisetto

Striessnig

. Familial hemiplegic migraine mutations increase Ca2+ influx through single human CaV2.1 channels and decrease maximal CaV2.1 current density in neurons. Proc Nat Acad Sci USA 2002; 99: 13284–13289.

van den Maagdenberg

AMJM

Pietrobon

Pizzorusso

Kaja

Broos

LAM

Cesetti

. A Cacna1a knock-in migraine mouse model with increased susceptibility to cortical spreading depression. Neuron 2004; 41: 701–710.

Tottene

Conti

Fabbro

Vecchia

Shapovalova

Santello

. Enhanced excitatory transmission at cortical synapses as the basis for facilitated spreading depression in Ca(v)2.1 knockin migraine mice. Neuron 2009; 61: 762–773.

10.

Lauritzen

. Pathophysiology of the migraine aura. The spreading depression theory. Brain 1994; 117: 199–210.

11.

Hadjikhani

Sanchez del Rio

Schwartz

Bakker

Fischl

. Mechanisms of migraine aura revealed by functional MRI in human visual cortex. Proc Nat Acad Sci USA 2001; 98: 4687–4692.

12.

van de Ven

Kaja

Plomp

Frants

van den Maagdenberg

Ferrari

. Genetic models of migraine. Arch Neurol 2007; 64: 643–646.

13.

Shields

Storer

Akerman

Goadsby

. Calcium channels modulate nociceptive transmission in the trigeminal nucleus of the cat. Neuroscience 2005; 135: 203–212.

14.

Xiao

Richter

Hurley

. Release of glutamate and CGRP from trigeminal ganglion neurons: Role of calcium channels and 5-HT1 receptor signaling. Mol Pain 2008; 4: 12–12.

15.

Goadsby

Edvinsson

Ekman

. Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann Neurol 1990; 28: 183–187.

16.

Goadsby

Edvinsson

. The trigeminovascular system and migraine: studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats. Ann Neurol 1993; 33: 48–56.

17.

Ferrari

Dodick

Galet

Kost

Fan

. Efficacy and tolerability of MK-0974 (telcagepant), a new oral antagonist of calcitonin gene-related peptide receptor, compared with zolmitriptan for acute migraine: a randomised, placebo-controlled, parallel-treatment trial. Lancet 2008; 372: 2115–2123.

18.

Mannix

Fan

Assaid

Furtek

Jones

. Randomized controlled trial of an oral CGRP antagonist, MK-0974, in acute treatment of migraine. Neurology 2008; 70: 1004–1012.

19.

Olesen

Diener

Husstedt

Goadsby

Hall

Meier

. Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N Engl J Med 2004; 350: 1104–1110.

20.

Lassen

Haderslev

Jacobsen

Iversen

Sperling

Olesen

. CGRP may play a causative role in migraine. Cephalalgia 2002; 22: 54–61.

21.

Williamson

Hargreaves

Hill

Shepheard

. Intravital microscope studies on the effects of neurokinin agonists and calcitonin gene-related peptide on dural blood vessel diameter in the anaesthetized rat. Cephalalgia 1997; 17: 518–524.

22.

Williamson

Shepheard

Hill

Hargreaves

. The novel anti-migraine agent rizatriptan inhibits neurogenic dural vasodilation and extravasation. Eur J Pharmacol 1997; 328: 61–64.

23.

Akerman

Williamson

Goadsby

. Voltage-dependent calcium channels are involved in neurogenic dural vasodilation via a pre-synaptic transmitter release mechanism. Br J Pharmacol 2003; 140: 558–566.

24.

Storer

Akerman

Goadsby

. Calcitonin gene-related peptide (CGRP) modulates nociceptive trigeminovascular transmission in the cat. Br J Pharmacol 2004; 142: 1171–1181.

25.

Fischer

Koulchitsky

Messlinger

. The nonpeptide calcitonin gene-related peptide receptor antagonist BIBN4096BS lowers the activity of neurons with meningeal input in the rat spinal trigeminal nucleus. J Neurosci 2005; 25: 5877–5883.

26.

Goadsby

Lipton

Ferrari

. Migraine- current understanding and treatment. N Eng J Med 2002; 346: 257–270.

27.

Classey

Bartsch

Goadsby

. Immunohistochemical examination of 5HT1B, 5HT1D and 5HT1F receptor expression in rat trigeminal ganglion (TRG) and dorsal root ganglia (DRG) neurons. Cephalalgia 2002; 22: 595–596.

28.

Mathew

Chami

Bergerot

van den Maagdenberg

AMJM

Ferrari

Goadsby

. Reduced expression of calcitonin gene related peptide (CGRP) in mice with the familial hemiplegic migraine (FHM) 1 mutation. Cephalalgia 2007; 27: 1190–1190.

29.

Classey

Knight

Goadsby

. The NMDA receptor antagonist MK-801 reduces Fos-like immunoreactivity within the trigeminocervical complex following superior sagittal sinus stimulation in the cat. Brain Res 2001; 907: 117–124.

30.

Paxinos

Franklin

KBJ

. The mouse brain in stereotaxic coordinates 20032nd ednLondon: Academic Press.

31.

Schutz

Mauer

Salmon

Changeux

Zimmer

. Analysis of the cellular expression pattern of beta-CGRP in alpha-CGRP-deficient mice. J Comp Neurol 2004; 476: 32–43.

32.

Salmon

Damaj

Sekine

Picciotto

Marubio

Changeux

. Modulation of morphine analgesia in alphaCGRP mutant mice. Neuroreport 1999; 10: 849–854.

33.

Price

Flores

. Critical evaluation of the colocalization between calcitonin gene-related peptide, substance P, transient receptor potential vanilloid subfamily type 1 immunoreactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse. J Pain 2007; 8: 263–272.

34.

Deumens

Lubbers

Jaken

Meijs

Thurlings

Honig

. Mice lacking L1 have reduced CGRP fibre in-growth into spinal transection lesions. Neurosci Lett 2007; 420(3): 277–281.

35.

Zhang

Hoff

Wimalawansa

Cote

Gagel

Westlund

. Arthritic calcitonin/alpha calcitonin gene-related peptide knock-out mice have reduced nociceptive hypersensitivity. Pain 2001; 89: 265–273.

36.

Sugimoto

Fujiyoshi

Xiao

Ichikawa

. Central projection of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive trigeminal primary neurons in the rat. J Comp Neurol 1997; 378: 425–442.

37.

Simonetti

Fabbro

D'Arco

Zweyer

Nistri

Giniatullin

. Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin. Mol Pain 2006; 2: 11–11.

38.

Hoskin

Goadsby

. Exposure and isolation of the superior sagittal sinus elicits Fos in the trigeminal nucleus caudalis and dorsal horn of the cervical spinal cord: how long should you wait?. Brain Res 1999; 824: 133–135.

39.

Rohn

Ralvenius

Paul

Borter

Hernandez

Witschi

. A virus-like particle-based anti-nerve growth factor vaccine reduces inflammatory hyperalgesia: potential long-term therapy for chronic pain. J Immunol 2010; 186: 1769–1780.

40.

Aita

Maeda

Seo

. The effect of neonatal capsaicin treatment on the CGRP-immunoreaction in the trigeminal subnucleus caudalis of mice. Biomed Res 2008; 29: 33–42.

41.

Kline

. The handbook of physiological testing 19992nd ednLondon: Routledge.

42.

Siegel

. Non-parametric statistics for the behavioural sciences 1956Kogakusha, Tokyo: McGraw-Hill.

43.

Llinas

Sugimori

Hillman

Cherksey

. Distribution and functional significance of the P-type, voltage-dependent Ca2+ channels in the mammalian central nervous system. Trends Neurosci 1992; 15: 351–355.

44.

Dunlap

Luebke

Turner

. Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci 1995; 18: 89–98.

45.

Westenbroek

Sakurai

Elliott

Hell

Starr

TVB

Snutch

. Immunochemical identification and subcellular distribution of the alpha(1a) subunits of brain calcium channels. J Neurosci 1995; 15: 6403–6418.

46.

Zhang

Winborn

Marquez de Prado

Russo

. Sensitization of calcitonin gene-related peptide receptors by receptor activity-modifying protein-1 in the trigeminal ganglion. J Neurosci 2007; 27: 2693–2703.

47.

Park

Moon

Akerman

Holland

Lasalandra

Andreou

. Blunted trigeminovascular nociceptive responses in mice with the CACNA1A mutation. Headache 2010; 50(Suppl 1): 61–61.

48.

Moon

Park

Akerman

Holland

Andreou

Lasalandra

. Altered responses in the descending modulatory system of transgenic mice with a CACNA1A mutation. Headache 2010; 50(Suppl 1): 6–7.

49.

Goadsby

. Calcitonin gene-related peptide antagonists as treatments of migraine and other primary headaches. Drugs 2005; 65: 2557–2567.

50.

Uddman

Edvinsson

Ekman

Kingman

McCulloch

. Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: trigeminal origin and co-existence with substance P. Neurosci Lett 1985; 62: 131–136.

51.

Smith

Hill

Edvinsson

Longmore

. An immunocytochemical investigation of human trigeminal nucleus caudalis: CGRP, substance P and 5-HT1D-receptor immunoreactivities are expressed by trigeminal sensory fibres. Cephalalgia 2002; 22: 424–431.

52.

Villalon

Olesen

. The role of CGRP in the pathophysiology of migraine and efficacy of CGRP receptor antagonists as acute antimigraine drugs. Pharmacol Ther 2009; 124: 309–323.

53.

Sixt

Messlinger

Fischer

. Calcitonin gene-related peptide receptor antagonist olcegepant acts in the spinal trigeminal nucleus. Brain 20093134–3141.

54.

Haan

Terwindt

Bos

Ophoff

Frants

Ferrari

. Familial hemiplegic migraine in The Netherlands. Clin Neurol Neurosurg 1994; 96: 244–249.

55.

Thomsen

Ostergaard

Romer

Andersen

Eriksen

Olesen

. Sporadic hemiplegic migraine is an aetiologically heterogeneous disorder. Cephalalgia 2003; 23: 921–928.

56.

Hansen

Thomsen

Olesen

Ashina

. Calcitonin gene-related peptide does not cause the familial hemiplegic migraine phenotype. Neurology 2008; 71: 841–847.

57.

Hansen

Thomsen

Marconi

Casari

Olesen

Ashina

. Familial hemiplegic migraine type 2 does not share hypersensitivity to nitric oxide with common types of migraine. Cephalalgia 2008; 28: 367–375.

58.

Hansen

Thomsen

Olesen

Ashina

. Familial hemiplegic migraine type 1 shows no hypersensitivity to nitric oxide. Cephalalgia 2008; 28: 496–505.

59.

Olesen

Iversen

Thomsen

. Nitric oxide supersensitivity: a possible molecular mechanism of migraine pain. Neuroreport 1993; 4: 1027–1030.

60.

Read

Hirst

Upton

Parsons

. Cortical spreading depression produces increased cGMP levels in cortex and brain stem that is inhibited by tonabersat (SB-220453) but not sumatriptan. Brain Res 2001; 891: 69–77.

61.

Goadsby

Ferrari

Csanyi

Olesen

Mills

. Randomized double blind, placebo-controlled proof-of-concept study of the cortical spreading depression inhibiting agent tonabersat in migraine prophylaxis. Cephalalgia 2009; 29: 742–750.

62.

Hauge

Asghar

Schytz

Christensen

Olesen

. Effects of tonabersat on migraine with aura: a randomised, double-blind, placebo-controlled crossover study. Lancet Neurol 2009; 8: 718–723.

63.

Edvinsson

Eftekhari

Salvatore

Warfvinge

. Cerebellar distribution of calcitonin gene-related peptide (CGRP) and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) in rat. Mol Cell Neurosci 2011; 46: 333–339.

64.

Ajima

Kawano

Takagi

Aita

Gomi

Byers

. The exact expression of glial fibrillary acidic protein (GFAP) in trigeminal ganglion and dental pulp. Arch Histol Cytol 2001; 64: 503–511.

65.

Schafers

Geis

Svensson

Luo

Sommer

. Selective increase of tumour necrosis factor-alpha in injured and spared myelinated primary afferents after chronic constrictive injury of rat sciatic nerve. Eur J Neurosci 2003; 17: 791–804.

66.

Taylor

Rosenzweig

McDonald

3rd Sakiyama-Elbert

. Delivery of neurotrophin-3 from fibrin enhances neuronal fiber sprouting after spinal cord injury. J Control Release 2006; 113: 226–235.

67.

Vamos

Pardutz

Fejes

Tajti

Toldi

Vecsei

. Modulatory effects of probenecid on the nitroglycerin-induced changes in the rat caudal trigeminal nucleus. Eur J Pharmacol 2009; 621: 33–37.

68.

Andres

During Mv, Muszynski

Schmidt

. Nerve fibres and their terminals in the dura mater encephali of the rat. Anat Embryol 1987; 175: 289–301.

69.

Wolff

. Headache and other head pain 19481st ednNew York: Oxford University Press.

70.

Strassman

Mason

Moskowitz

Maciewicz

. Response of brainstem trigeminal neurons to electrical stimulation of the dura. Brain Res 1986; 379: 242–250.

71.

Tottene

Pivotto

Fellin

Cesetti

van den Maagdenberg

Pietrobon

. Specific kinetic alterations of human CaV2.1 calcium channels produced by mutation S218L causing familial hemiplegic migraine and delayed cerebral edema and coma after minor head trauma. J Biol Chem 2005; 280: 17678–17686.

72.

Eikermann-Haerter

Dilekoz

Kudo

Savitz

Waeber

Baum

. Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1. J Clin Invest 2009; 119: 99–109.

73.

Thomsen

Eriksen

Roemer

Andersen

Olesen

Russell

. A population-based study of familial hemiplegic migraine suggests revised diagnostic criteria. Brain 2002; 125: 1379–1391.