Harnessing Miniscope Imaging in Freely Moving Animals to Unveil Migraine Pathophysiology and Validate Novel Therapeutic Strategies

ABSTRACT

Migraine is a debilitating neurological disorder that affects millions worldwide. Elucidating its underlying mechanisms is crucial for developing effective therapeutic interventions. In this editorial, we discuss the potential applications of one‐photon miniscopes, which enable minimally invasive, high spatiotemporal resolution fluorescence imaging in freely moving animals. By providing real‐time visualization of vascular dynamics and neuronal activity, these cutting‐edge techniques can offer unique insights into migraine pathophysiology. We explore the significance of these applications in preclinical research with a case study demonstrating their potential to drive the development of novel therapeutic strategies for effective migraine management.

Miniscope imaging enables high spatiotemporal visualization of meningeal blood vessel vasodilation in freely moving animals. Applicable to various physiological and disease states (i.e., healthy, migraine, treatment), it allows for concurrent behavioral readouts during disease progression and therapeutic interventions.

1. Introduction

Migraine is a common neurovascular brain disorder affecting approximately 15% of the global population, posing a significant challenge to both individuals and healthcare systems worldwide (Ferrari et al. 2022; GBD 2016; ICHD‐3 2018). Its socioeconomic impacts are profound, as it is ranked the third‐highest cause of disability globally in both males and females under the age of 50 years (Steiner, Stovner, and Vos 2016). Migraine is a primary headache disorder wherein the predominant pathological symptom is pain, independent from underlying pathologies or traumatic events (GBD 2016). It is clinically diagnosed using the International Classification of Headache Disorders, 3rd edition (ICHD‐III) (ICHD‐3 2018), and is characterized by a recurring, often unilateral throbbing headache of moderate to severe intensity, lasting 4–72 h. Migraine is frequently accompanied by symptoms such as nausea, vomiting, phonophobia, photophobia, and vascular changes in intra‐ and extracerebral arteries (Ferrari et al. 2022; ICHD‐3 2018; Ashina et al. 2019).

The pathophysiology of migraines has been extensively debated. A central question in this discussion is whether the vasculature plays a primary role in the initiation and/or persistence of migraines, a topic that remains actively explored (Mason and Russo 2018). The foundational roots of the vascular theory can be traced back to Galen in the second century and were later revisited by Thomas Willis in the late 17th century (Mason and Russo 2018). However, it was not until the early 1940s that Harold Wolff established a direct link between the intensity of migraine headaches and the pulsations within the branches of the external carotid arteries. Wolff discovered that mitigating these pulsations could significantly reduce headache severity (Tfelt‐Hansen and Koehler 2008; Blau 2004). In his experiments he used ergot derivatives (previously an extensively used migraine therapy; Ferrari et al. 2022) to induce vasoconstriction in the temporal and middle meningeal arteries, which reduced throbbing and the overall intensity of the headache. Recent research recurrently observes arterial dilation in the cephalic region during migraine episodes (Olesen et al. 2009). Specifically, the frequent involvement of meningeal artery dilation, as opposed to other arteries, supports the hypothesis that migraine‐related vascular events predominantly affect meningeal arteries and the sensory fibers innervating them (Burstein et al. 1998; Khan et al. 2019; Olesen et al. 2009). The precise role of the observed vasodilation in migraine—whether it is directly linked to pain or is merely a coincidental response—remains debated, highlighting the complexity of understanding migraine pathophysiology (Goadsby et al. 2017).

While the pathophysiology of migraine remains elusive, it is known that the trigeminovascular system plays a critical role in peripheral and central events that ultimately lead to the experience of migraine pain. This system comprises the trigeminal ganglia (TG), where cell bodies of pseudounipolar afferent neurons are located, signaling to the meninges, and the meningeal arteries (Ashina et al. 2019; Blaeser et al. 2022; Burgos‐Vega et al. 2015). The meninges are the only pain‐sensitive tissue within the cranium, and the highest density of small‐diameter unmyelinated C‐fibers and thinly myelinated Aδ‐fiber axons of trigeminal origin are found in proximity to the blood vessels (Ashina et al. 2019; Burgos‐Vega et al. 2015; Goadsby et al. 2017; Levy and Moskowitz 2023). Exposure of perivascular fibers to mechanical, chemical, or electrical stimuli leads to sensitization of the dura mater, which is believed to contribute to the onset of painful headaches. This theory is supported by reports that electrical and mechanical stimulation of dural vasculature, but not the surface of the brain, produced referred head pain in awake patients during craniotomy (Ray and Wolff 1940; Penfield and McNaughton 1940). In addition to projecting to the meninges, the neurons of TG also project back to the spinal trigeminal nucleus (SpV). From there, second‐order neurons extend projections to various relay stations, before ultimately reaching a wide range of cortical areas, which may mediate associated symptoms of migraine, such as phonophobia and photophobia (Ashina et al. 2019; Uddman et al. 1985; Liu, Broman, and Edvinsson 2008; Burstein et al. 2010; Noseda et al. 2011). Given the intricate architecture of the meninges and their extensive innervation by trigeminovascular neurons, it becomes clear why these structures are central to understanding the underlying mechanisms of migraine pathophysiology.

1.1. Pharmacological Challenge Models

The ability of vasoactive neuropeptides and agents to provoke migraine attacks in migraineurs lends support to the hypothesis that vasodilation plays a crucial role in migraine pathophysiology (Tajti et al. 2015). Vascular tone is controlled by a sophisticated interplay of molecular mechanisms that modulate vasoconstriction and vasodilation. These mechanisms involve signaling molecules with vasoactive properties, which are crucial for the sensitization and activation of the trigeminovascular system (Tajti et al. 2015). Neuropeptides such as calcitonin gene‐related peptide (CGRP), vasoactive intestinal peptide (VIP), pituitary adenylate‐cyclase activating polypeptide (PACAP), and other agents like nitric oxide (NO), are widely expressed throughout the trigeminovascular system and are of specific interest in the context of migraine (Ashina et al. 2019; Tajti et al. 2015; Mason et al. 1984; Hannibal 2002; Martin, Baeres, and Moller 2004; Walker and Hay 2013; Lund and Hannibal 2022; Ashina et al. 2017). Furthermore, systemic administration of these neuropeptides, along with the NO donor nitroglycerin (NTG) and the ATP‐sensitive potassium channel opener levcromakalim, has been demonstrated to provoke migraine‐like attacks in migraine patients (Levy and Moskowitz 2023; Tajti et al. 2015; Al‐Karagholi et al. 2019; Ghanizada et al. 2021; Karsan et al. 2020; Sureda‐Gibert, Romero‐Reyes, and Akerman 2022; Schytz et al. 2009). These vasoactive neuropeptides induce vasodilation through the opening of potassium channels, specifically the ATP‐sensitive potassium, (KATP) channels and large (big) calcium‐sensitive potassium (BKCa) channels (Al‐Karagholi 2023). Other vasodilators, including CGRP, VIP, PACAP, amylin (AMY) and the prostaglandins E2 (PGE₂) and I2 (PGI₂), bind to their respective G‐protein coupled receptors to activate the adenylyl cyclase (AC) enzyme, causing cAMP to be converted from ATP (Shi et al. 2008; Dickson and Finlayson 2009; Russell et al. 2014). In addition to their vasoactive properties, these neuropeptides can modulate the activity of various cell types, for instance CGRP released from trigeminal neurons can promote mast cell degranulation, resulting in subsequent release of histamine (Ottosson and Edvinsson 1997).

The pharmacological administration of such agents inducing migraine has been widely used to study their impact on pain mechanisms and vascular dynamics as well as to validate novel therapeutics for migraine (Rasmussen et al. 2023). Human pharmacological challenge models play a critical role in migraine research, enabling controlled induction of migraines and allowing unique studies not feasible with other diseases due to the self‐limiting nature of migraine attacks (Ashina et al. 2017). Pharmacological challenges are vital for testing antimigraine drugs and developing an experimental human headache model for screening purposes in healthy volunteers is a valuable approach that can help determine if a trial in migraine patients is warranted. Efforts have been made to establish such models (Ashina et al. 2017). Insights from human studies can then be back‐translated into in vivo preclinical models, fostering a deeper understanding of underlying mechanisms and improving treatment predictions.

Animal research is crucial for elucidating fundamental biological processes involved in migraine, including the trigeminovascular system, nociception at peripheral and central levels, and the immunohistochemical localization of peptidergic nerve fibers and their neuropeptide receptors (Ashina et al. 2017). In vivo preclinical models offer invaluable insights into the complex pathophysiology of migraine, enabling detailed exploration of mechanisms and potential therapeutic interventions. Rodents are the most widely used species in migraine research, with various animal models replicating human provocation models by utilizing the same trigger substances to achieve different translational outcomes (Harriott et al. 2019). Expected behavioral outcomes in rodent migraine models include light aversion, decreased locomotor activity (such as reduced home‐cage movement and voluntary wheel running), and distinct pain‐related facial expressions, such as grimacing and squinting (Wattiez et al. 2021). Mechanical hyperalgesia, typically assessed using von Frey filaments, is also a common finding, reflecting increased sensitivity to mechanical stimuli (Moye and Pradhan 2017). These clinically relevant phenotypes closely mirror the reduction in normal physical activity, pain hypersensitivity, and photophobia commonly observed in migraine patients (Wattiez et al. 2021; Toriyama, Horiuchi, and Hongo 2017).

1.2. Neuroimaging in Migraine

Advanced technologies enabling high spatiotemporal and minimally invasive assessment of neurovascular brain structure, function, and metabolic activity will be crucial in further elucidating migraine pathophysiology, improving diagnostic accuracy, and identifying novel therapeutic targets for more effective management of this debilitating condition. Neuroimaging plays a key role in migraine research by visualizing dynamic neurovascular interactions and capturing real‐time changes in vascular structures and neuronal activity related to nociception. These techniques are ideal for assessing potential therapeutic targets and understanding the physiological dysfunction associated with migraine in humans and animals (Harriott et al. 2019; Evans et al. 2020). Early methods for measuring regional cerebral blood flow (rCBF) using Xenon‐133 during migraine attacks paved the way for more advanced techniques, such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI). PET and fMRI offer relatively high temporal and spatial resolution in humans and rodents, making them the most frequently employed techniques for elucidating migraine mechanisms (Russo et al. 2017). Nowadays, multiple in vivo imaging technologies are employed for visualizing vascular dynamics based on their advantages and disadvantages (Kim and Lee 2022).

Recently, fluorescence‐based imaging technologies have been widely adopted as they offer high spatial and temporal resolution allowing a more detailed visualization of vascular dynamics and neuronal activity using specific biosensors (Pryazhnikov et al. 2014) in rodent models of migraine (Blaeser et al. 2022; Blaeser et al. 2024). While two‐photon microscopy ensures high‐resolution imaging, it requires animals to be head‐fixed, which restricts the ability to observe natural behaviors and movements (Blaeser et al. 2022; Blaeser et al. 2024). Consequently, the loss of the freely moving aspect in these studies compromises the ability to fully assess the effect of migraine‐related neuropeptides on vasculature and pain‐related behavior. This highlights a trade‐off between imaging resolution and behavioral authenticity in these experimental setups.

As various imaging technologies are extensively discussed elsewhere (Evans et al. 2020; Kim and Lee 2022; Schwedt and Dodick 2009), this review will focus on the capabilities of miniscope technology for screening migraine‐related treatments. This is due to its balance between parameters like spatiotemporal resolution, compatibility with free behavior, and system portability (see Table 1).

TABLE 1.

Comparison of common technologies for measuring vascular dynamics. Measure: magnetic resonance imaging (MRI) detects blood flow and functional ultrasound (fUS, Iconeus) detects blood flow and vessel diameter, while miniscope imaging (Inscopix) detects fluorescence from a biosensor or dye. This allows for recordings of neuronal activity, vasodilation, and blood flow changes. Spatial and temporal resolution: miniscope imaging offers high spatiotemporal resolution compared to fUS and MRI. Animal state: The behavioral state of the animal is crucial for validating in vivo drug efficacy. Miniscope imaging can be performed in freely moving animals, whereas fUS and MRI require anesthesia in the majority of experimental studies. Invasiveness: MRI and fUS do not necessarily require surgical procedures if the skull thickness is not excessive (e.g., a craniotomy is recommended in an adult rat with high skull thickness to perform fUS). Miniscope imaging, however, typically requires a craniotomy. System portability and costs: MRI systems are generally large, restricting opportunities for portability and expansion to multiple machines. fUS is similarly constrained in portability due to the size of the equipment and probes. In contrast, miniscope technology is compact, allowing for enhanced portability and scalability such as, the ability to mount multiple systems in parallel). Costs are relative as they are dependent by the machine itself, portability, maintenance, and the number of operators required to establish and use these technologies (exact costs should be obtained from the supplier). As mentioned in the main text, a deep dive into various technologies to detect vascular changes is discussed elsewhere (Evans et al. 2020; Kim and Lee 2022; Schwedt and Dodick 2009).

Parameters	MRI	fUS	Miniscope
Measure	Blood flow	Blood vessel diameter and blood flow	Blood vessel diameter, blood flow, and biosensors
Spatial resolution	Low (whole brain or brain section: mm3)	Intermediate (Whole brain or brain section: > 50‐100um)	High (> 10 µm)
Temporal resolution	Low (min: seconds)	Intermediate (Min: > 300 ms)	High (Min: 10 Hz; Max: 100 Hz)
Animal state	Anaesthetized	Anaesthetized	Freely moving
Invasiveness	Noninvasive	Noninvasive	Minimally Invasive (typically requires craniotomy)
System portability	Low	Moderate	High
Costs	High	Relative	Relative

1.2.1. Miniscope Imaging as a Novel Tool in the Migraine Space

One‐photon miniscope technology enables in vivo, real‐time visualization of neural activity and vascular dynamics in freely moving animals. This technique relies primarily on genetically encoded calcium indicators to image specific neuronal populations and fluorescent dyes to visualize the vasculature, both with high spatiotemporal resolution. It overcomes the limitations of traditional imaging methods, offering deeper insights into brain and vascular dynamics in both health and disease (Ghosh et al. 2013). Although real‐time in vivo vessel imaging is susceptible to motion artifacts and blood volume changes that may affect vessel diameter measurements, advancements such as motion correction algorithms, fast focal plane adjustments, and standardized pipelines for extracting vessel diameter and red blood cell (RBC) velocity measurements have made this approach accessible to laboratories with limited technical and computational expertise. A significant advantage of miniscope imaging is its capacity for longitudinal studies due to the stability of chronic implants while permitting in vivo real‐time visualization of superficial and deep brain structures using cranial windows or gradient refractive index (GRIN) lenses, respectively. This allows for continuous imaging of brain and vascular structures over extended periods, which is essential for assessing changes in neural circuits, disease progression, and the efficacy and side effects of new therapeutic interventions (Ziv et al. 2013; Sheintuch et al. 2017; Parker et al. 2018). Additionally, miniscope imaging captures neurobehavioral phenotypes by allowing animals to move freely during imaging. This should enable the correlation of subsecond changes in vascular activity with spontaneous behaviors, such as responses to light stimulation (Wang et al. 2021) or repetitive stress (Avona et al. 2020), which induce migraine‐like behaviors. This integrative approach provides unprecedented insights into how various drugs modulate neural circuits and influence behavior (Yun et al. 2023).

1.2.2. Meningeal Miniscope Imaging—A Case Study

As a proof of concept for the application of miniscope imaging technology to the investigation of migraine pathophysiology and therapy, we present here a case study in which miniscope technology was used to image meningeal blood vessels in nonanesthetized freely moving mice (Figure 1). To our knowledge, it is the first time this type of technology has been employed for this specific purpose. This case study is presented here as a qualitative example of the promise and general capabilities of this technique, and we acknowledge that reproducible results must yet be shown to firmly establish conclusions. The experiments described below were conducted under an experimental protocol approved by an Institutional Animal Care and Use Committee administrated by Lifesource Biomedical Services (Mountain View, California).

FIGURE 1.

Miniscope imaging enables monitoring of vascular dynamic in a freely moving animal model of migraine. (A) A standardized workflow for vascular imaging in a migraine model using miniscopes. Created in BioRender. Ye, T. (2024) BioRender.com/p37×969. (B) A newly developed algorithm enables accurate diameter estimation of a broad range of vessel sizes. First, users manually draw a line that bisects a vessel to be analyzed (left, middle). To estimate the vessel diameter, the algorithm uses the pixel intensity profile along each label, calculating the local minimum on each side of the curve, and comparing to the global maximum. On each side of the curve, the pixel with the value just above the half point between the global maximum and the corresponding minimum is determined to be the edge of the vessel. The distance between the two pixels is calculated as the vessel diameter (right). (C) Comparison of algorithm diameter estimates versus manual estimates of the same vessels. Correlation between the two methods is high (Pearson R = 0.992). (D) Example images before (top left) and 30 min after levcromakalim administration (top right). Orange lines overlaying the images denote the original width of the underlying vessels. Teal lines show the change in vessel width after levcromakalim administration. Traces (below) show the algorithmic diameter estimation of the corresponding vessels in the images above, normalized to the predrug mean. Dashed gray lines indicate the timing of levcromakalim administration. (E) Synchronized behavior videos can be collected alongside miniscope acquisition. The nVision deep learning algorithm can automatically detect a mouse in the camera's field of view (top). Position data can be preserved frame by frame. Mouse position data from the first 5 min of a session is plotted below.

In this case study, 12‐week old male C57BL6 mice (Jackson Laboratory, Sacramento) housed in a 12‐h reverse light dark cycle (lights on at 6:00pm) were used. Chronic cranial window implants (Figure 1A) with an affixed baseplate for docking the miniscope (Inscopix, Inc.) allowed for repeatable imaging of the same field of view for several months. Here, we present data from implants involving a craniotomy, but thin‐skull cranial windows are also suitable with this workflow. After 3 weeks of recovery from surgery, mice were habituated to the weight of the miniscope by wearing the miniscope for 60 min in an open field (42×42 cm) in two sessions across three days. The mice were also habituated to the restraining tube used for tail vein injections for 5 min before each habituation session, and intraperitoneal injections of saline (1% volume by mass) were administered during each session. In the week following the final habituation session, imaging of the meningeal vasculature was performed by mounting the miniscope in the baseplate affixed above the cranial window. Fluorescein isothyocyanate (FITC) dextran (Millipore Sigma, 2000 kDa, 9 mg/mL, 1% volume by mass) was administered via a tail vein injection prior to imaging to enable high‐contrast visualization of blood vessels using the miniscope (Wang et al. 2022). Vascular images were acquired over the course of 60 min using the nVista miniscope system (Inscopix, Inc.). The resulting videos were subsequently processed, motion‐corrected, and analyzed in the Inscopix Data Processing Software (IDPS, Inscopix, Inc.). A newly developed algorithm was used to estimate the diameter of vessels frame‐by‐frame (Figure 1B). This algorithm accurately estimates the diameter of vessels of various sizes, ranging from a few microns to around 200 µm (Figure 1C). In order to test the potential of the system, we performed a pilot experiment to image the dynamics of meningeal vasculature before and after the administration of levcromakalim, a potent vasodilator known to induce migraine in humans (Clement et al. 2022). Importantly, the focal plane was stable and vessel diameters changed very little over the course of 90 min in a control, vehicle‐injected animal (Figure 2). On the other hand, administration of levcromakalim (1 mg/kg) induced a robust and prolonged dilation of nearly all visible vessels (Figure 1D, full data set not shown).

FIGURE 2.

Miniscope‐derived vessel images are stable over 90 min. After FITC dextran was administered in a single mouse, three short (3–5 min) videos were acquired, one each shortly after the dye injection (baseline), then 4 and 90 min later. (A) Mean projection images from the baseline (top) and 90‐min (bottom) videos. White and colored lines overlaying the images denote where bisecting vessel labels were drawn for algorithmic diameter estimation. (B). Diameter traces normalized to the 3 min preinjection mean (top) correspond to their respective labels from the images in A, showing examples of individual vessel traces from the three videos collected. Below, mean normalized diameter estimates were calculated for each vessel across each of the three timepoints collected, and the mean of all vessels analyzed were plotted, showing little variation in diameter over time. Error bars denote the mean standard deviation of individual vessel normalized diameter means.

During the session, video of the mouse's behavior was simultaneously recorded using the nVision system (Inscopix, Inc.), a behavioral camera and data acquisition hardware unit that pairs with the nVista miniscope system via a network connection. Each sample from both systems is timestamped from a common clock, allowing for precise synchronization of behavioral and vascular events. In this example, the mouse was recorded from a bottom‐up perspective under white light in a 42 × 42 cm open field arena (Figure 1E, top). From the behavioral video, animal position was estimated frame‐by‐frame using the nVision deep learning animal tracking model on the Inscopix Data Exploration, Analysis, and Sharing (IDEAS) cloud‐based platform, which was trained on previously collected behavioral data (Figure 1E, bottom).

The use of miniscope technology proved successful in monitoring the effects of this pharmacological challenge, known to induce migraines in patients, with high spatiotemporal resolution in freely moving mice during the experiment.

1.2.3. Potential Applications of Miniscope Imaging in Migraine Research

A major challenge in discovering new migraine treatments has been the absence of reliable preclinical models of chronic migraine (Mungoven, Henderson, and Meylakh 2021). Although several promising models exist for episodic migraine attacks induced by acute injection of vasoactive substances such as levcromakalim (Al‐Karagholi et al. 2019), studying the transition from acute to chronic migraine has been challenging. The miniscope imaging approach provides sensitive and quantitative time courses of vasodilation and vasoconstriction in response to acute treatment. Therefore, this approach could be used for repeated, in‐vivo measurements to longitudinally characterize the adaptive response to chronic pharmacology, such as the induction of a chronic migraine state by NTG (Pradhan et al. 2014). Repeated electrical stimulation of the TG upregulates the expression of neuropeptides and increases their release into the blood (Harriott et al. 2019; Guo et al. 2021). Combining meningeal vessel imaging with the optogenetic capability of the miniscope technology, stimulation of the TG using the excitatory, red‐shifted opsin, ChrimsonR (Stamatakis et al. 2018), could replace pharmacological challenges and represent a new method for inducing endogenous release of neuropeptides while monitoring vascular and pain responses (Figure 3A). This approach would have the potential to mimic a chronic migraine state, potentially increasing translatability and providing better predictions of treatment principles.

FIGURE 3.

Potential applications of miniscope imaging in migraine research. (A) Optogenetic stimulation of Trigeminal Ganglia (TG) afferents expressing the excitatory red‐shifted opsin, ChrimsonR, while imaging meningeal vasculature. (B) Neurovascular coupling—simultaneous dual color imaging of vascular dynamics and neurons (e.g., during Cortical Spreading Depression, CSD). (C). Simultaneous dual color imaging of vascular dynamics and genetically encoded vasoactive peptide sensors. Created in BioRender. Ye, T. (2024) BioRender.com/l07l553.

Other applications of miniscope technology in migraine research range from imaging migraine‐related neurotransmitters to examining neuronal circuits and chronic conditions. The dual‐color imaging capabilities of the miniscope technology represent a significant advancement in neuroscience research, allowing for the simultaneous visualization of different aspects of brain and vascular physiology. This innovation opens novel avenues to explore intricate interactions between neuronal activity and vascular dynamics. The introduction of such a system facilitates simultaneous imaging of both neural circuits and key measures used in migraine research, such as red blood cell (RBC) velocity (blood flow) and vessel diameter. By providing valuable insights into vascular dynamics under various conditions, including pain‐related behaviors such as allodynia monitored in the Von Frey test (Hua et al. 2020), this approach offers access to neurovascular coupling (Figure 3B). This is particularly relevant for understanding migraine pathophysiology, where impaired neurovascular coupling is thought to play a role in disease development (Fabjan, Zaletel, and Žvan 2015). Already, this application has demonstrated its capability in investigating how changes in blood flow and vessel diameter influence neuronal activity (North et al. 2023) and represents a powerful preclinical tool for identifying new targets for migraine studies.

Miniscope dual‐color imaging also allows researchers to concurrently visualize neurons expressing different neurotransmitter or peptide receptors related to migraine. For instance, one color channel can be employed to label VIP receptors (Wang et al. 2023), histamine (HA) receptors (Dong et al. 2023), or meningeal release of glutamate (Guerrero‐Toro et al. 2022), while the other monitors vascular dynamics (Figure 3C). This concurrent imaging capability would facilitate a deeper understanding of how vascular dynamics and neuropeptide release interact and contribute to the pathophysiology of migraine.

Finally, dual color miniscope imaging could be particularly advantageous for studying the phenomenon of cortical spreading depression (CSD). CSD is a wave of depolarization followed by a suppression of neural activity, which is believed to be a critical trigger for migraine auras. Fluorescence‐based imaging has already demonstrated the capabilities for the simultaneous monitoring of CSD waves associated with brain injury (Zhao et al. 2021). Moreover, dual color imaging can be used to capture cortical calcium waves indicative of CSD while monitoring vascular dynamics (Figure 3B). This dual‐channel approach would provide a more comprehensive view of how CSD propagates through different neural circuits and affects brain function while enhancing our knowledge of migraine pathophysiology.

2. Conclusions

The development and application of neuroimaging technologies that allow for minimally invasive imaging without the need for anesthetics in freely moving animals are crucial in advancing preclinical research, especially in understanding migraine pathophysiology. These innovative tools will improve diagnostic accuracy and lead to the identification of novel therapeutic targets, ultimately enhancing the management of this debilitating condition. Advanced neuroimaging techniques enable researchers to visualize and quantify dynamic neurovascular interactions, neuronal activity, and vascular responses in real time, providing valuable insights into the mechanisms of migraine in both humans and animals.

Although techniques such as PET and fMRI have been instrumental in migraine research, the development of fluorescence‐based imaging technologies like one‐photon miniscopes offers a significant advantage by providing high spatiotemporal resolution while maintaining the animals' ability to move freely. By using one‐photon miniscope technology, researchers can monitor neural activity and vascular dynamics in freely moving animals, allowing for more accurate assessments of the effects of migraine‐related neuropeptides on neurovascular coupling and pain‐related behavior. This innovative technique overcomes the limitations of traditional imaging methods, paving the way for a deeper understanding of neuronal and vascular dynamics in both health and disease.

The importance of utilizing minimally invasive neuroimaging technologies that enable high‐resolution imaging in freely moving animals cannot be overstated. These tools will be essential in further elucidating the underlying mechanisms of migraine, improving diagnostic capabilities, and identifying new therapeutic targets for effective migraine management.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.