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

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.

Automated detection of squint as a sensitive assay of sex-dependent calcitonin gene-related peptide and amylin-induced pain in mice.

ABSTRACT: We developed an automated squint assay using both black C57BL/6J and white CD1 mice to measure the interpalpebral fissure area between the upper and lower eyelids as an objective quantification of pain. The automated software detected a squint response to the commonly used nociceptive stimulus formalin in C57BL/6J mice. After this validation, we used the automated assay to detect a dose-dependent squint response to a migraine trigger, the neuropeptide calcitonin gene-related peptide, including a response in female mice at a dose below detection by the manual grimace scale. Finally, we found that the calcitonin gene-related peptide amylin induced squinting behavior in female mice, but not males. These data demonstrate that an automated squint assay can be used as an objective, real-time, continuous-scale measure of pain that provides higher precision and real-time analysis compared with manual grimace assessments.

Investigating Migraine-Like Behavior Using Light Aversion in Mice.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Amylin Analog Pramlintide Induces Migraine-like Attacks in Patients.

OBJECTIVE: Migraine is a prevalent and disabling neurological disease. Its genesis is poorly understood, and there remains unmet clinical need. We aimed to identify mechanisms and thus novel therapeutic targets for migraine using human models of migraine and translational models in animals, with emphasis on amylin, a close relative of calcitonin gene-related peptide (CGRP). METHODS: Thirty-six migraine without aura patients were enrolled in a randomized, double-blind, 2-way, crossover, positive-controlled clinical trial study to receive infusion of an amylin analogue pramlintide or human αCGRP on 2 different experimental days. Furthermore, translational studies in cells and mouse models, and rat, mouse and human tissue samples were conducted. RESULTS: Thirty patients (88%) developed headache after pramlintide infusion, compared to 33 (97%) after CGRP (p = 0.375). Fourteen patients (41%) developed migraine-like attacks after pramlintide infusion, compared to 19 patients (56%) after CGRP (p = 0.180). The pramlintide-induced migraine-like attacks had similar clinical characteristics to those induced by CGRP. There were differences between treatments in vascular parameters. Human receptor pharmacology studies showed that an amylin receptor likely mediates these pramlintide-provoked effects, rather than the canonical CGRP receptor. Supporting this, preclinical experiments investigating symptoms associated with migraine showed that amylin treatment, like CGRP, caused cutaneous hypersensitivity and light aversion in mice. INTERPRETATION: Our findings propose amylin receptor agonism as a novel contributor to migraine pathogenesis. Greater therapeutic gains could therefore be made for migraine patients through dual amylin and CGRP receptor antagonism, rather than selectively targeting the canonical CGRP receptor. ANN NEUROL 2021;89:1157-1171.

Different forms of traumatic brain injuries cause different tactile hypersensitivity profiles.

ABSTRACT: Chronic complications of traumatic brain injury represent one of the greatest financial burdens and sources of suffering in the society today. A substantial number of these patients suffer from posttraumatic headache (PTH), which is typically associated with tactile allodynia. Unfortunately, this phenomenon has been understudied, in large part because of the lack of well-characterized laboratory animal models. We have addressed this gap in the field by characterizing the tactile sensory profile of 2 nonpenetrating models of PTH. We show that multimodal traumatic brain injury, administered by a jet-flow overpressure chamber that delivers a severe compressive impulse accompanied by a variable shock front and acceleration-deceleration insult, produces long-term tactile hypersensitivity and widespread sensitization. These are phenotypes reminiscent of PTH in patients, in both cephalic and extracephalic regions. By contrast, closed head injury induces only transient cephalic tactile hypersensitivity, with no extracephalic consequences. Both models show a more severe phenotype with repetitive daily injury for 3 days, compared with either 1 or 3 successive injuries in a single day, providing new insight into patterns of injury that may place patients at a greater risk of developing PTH. After recovery from transient cephalic tactile hypersensitivity, mice subjected to closed head injury demonstrate persistent hypersensitivity to established migraine triggers, including calcitonin gene-related peptide and sodium nitroprusside, a nitric oxide donor. Our results offer the field new tools for studying PTH and preclinical support for a pathophysiologic role of calcitonin gene-related peptide in this condition.

Stimulation of Posterior Thalamic Nuclei Induces Photophobic Behavior in Mice.

OBJECTIVE: A hallmark of migraine is photophobia. In mice, photophobia-like behavior is induced by calcitonin gene-related peptide (CGRP), a neuropeptide known to be a key player in migraine. In this study, we sought to identify sites within the brain from which CGRP could induce photophobia. DESIGN: We focused on the posterior thalamic region, which contains neurons responsive to both light and dural stimulation and has CGRP binding sites. We probed this area with both optogenetic stimulation and acute CGRP injections in wild-type mice. Since the light/dark assay has historically been used to investigate anxiety-like responses in animals, we measured anxiety in a light-independent open field assay and asked if stimulation of a brain region, the periaqueductal gray, that induces anxiety would yield similar results to posterior thalamic stimulation. The hippocampus was used as an anatomical control to ensure that light-aversive behaviors could not be induced by the stimulation of any brain region. RESULTS: Optogenetic activation of neuronal cell bodies in the posterior thalamic nuclei elicited light aversion in both bright and dim light without an anxiety-like response in an open field assay. Injection of CGRP into the posterior thalamic region triggered similar light-aversive behavior without anxiety. In contrast to the posterior thalamic nuclei, optogenetic stimulation of dorsal periaqueductal gray cell bodies caused both light aversion and an anxiety-like response, while CGRP injection had no effect. In the dorsal hippocampus, neither optical stimulation nor CGRP injection affected light aversion or open field behaviors. CONCLUSION: Stimulation of posterior thalamic nuclei is able to initiate light-aversive signals in mice that may be modulated by CGRP to cause photophobia in migraine.

Peripherally administered calcitonin gene-related peptide induces spontaneous pain in mice: implications for migraine.

Migraine is the third most common disease in the world (behind dental caries and tension-type headache) with an estimated global prevalence of 15%, yet its etiology remains poorly understood. Recent clinical trials have heralded the potential of therapeutic antibodies that block the actions of the neuropeptide calcitonin gene-related peptide (CGRP) or its receptor to prevent migraine. Calcitonin gene-related peptide is believed to contribute to trigeminal nerve hypersensitivity and photosensitivity in migraine, but a direct role in pain associated with migraine has not been established. In this study, we report that peripherally administered CGRP can act in a light-independent manner to produce spontaneous pain in mice that is manifested as a facial grimace. As an objective validation of the orbital tightening action unit of the grimace response, we developed a squint assay using a video-based measurement of the eyelid fissure, which confirmed a significant squint response after CGRP injection, both in complete darkness and very bright light. These indicators of discomfort were completely blocked by preadministration of a monoclonal anti-CGRP-blocking antibody. However, the nonsteroidal anti-inflammatory drug meloxicam failed to block the effect of CGRP. Interestingly, an apparent sex-specific response to treatment was observed with the antimigraine drug sumatriptan partially blocking the CGRP response in male, but not female mice. These results demonstrate that CGRP can induce spontaneous pain, even in the absence of light, and that the squint response provides an objective biomarker for CGRP-induced pain that is translatable to humans.

Anti-CGRP antibodies block CGRP-induced diarrhea in mice.

The multifunctional neuropeptide calcitonin gene-related peptide (CGRP) and its receptor are expressed throughout the gastrointestinal tract. Previous studies have shown that CGRP has roles in intestinal motility, water secretion, and inflammation. Furthermore, animal studies have demonstrated CGRP involvement in diarrhea secondary to C. difficile and food allergies. Diarrhea thus provides a convenient bioassay of CGRP activity in the GI system. In this proof of principle study, we report that prophylactic administration of an anti-CGRP antibody is able to block CGRP-induced diarrhea in mice. As a control, the CGRP-receptor antagonist olcegepant also attenuated the diarrhea response to CGRP. This preclinical study indicates that anti-CGRP antibodies may provide a new preventative therapy for gastrointestinal disorders involving CGRP.