The Ticking of the Trigeminal Ganglion Clock during Headache

By Neil Andrews | April 12, 2024 | Posted in

A new study identifies circadian features of trigeminal ganglion cells and animal pain responses. These features are reflected at a genetic level, too, which could set the stage for improved headache treatment.

One of the unique characteristics of cluster headache is its circadian pattern: People living with this terribly painful and debilitating condition will have headaches at the same time each day. While the circadian features of cluster headache and some other types of headache like migraine are well recognized, the underlying genetic and molecular factors responsible for them have remained unclear. A new animal study now bridges this gap by describing the trigeminal ganglion “clock.”

Using an array of techniques, a team led by Mark Burish and Seung-Hee Yoo, UTHealth Houston, US, provide evidence for circadian rhythms in trigeminal ganglion cells. The pain responses of mice in response to nitroglycerin, a model of headache, also had circadian characteristics, which were disrupted in animals genetically engineered to lack previously identified circadian clock genes.

Supporting these findings was the discovery of hundreds of genes whose expression displayed a circadian rhythm. The team also found that 10 genes expressed in a circadian manner happen to be the targets of a number of current medications for cluster headache, migraine, and trigeminal neuralgia.

“This work provides potential mechanistic explanation for the circadian nature of many headache subtypes by showing rhythmic gene expression patterns within the trigeminal ganglia, even when the neurons were removed from the animals,” wrote Greg Dussor, a migraine researcher at UT Dallas, US, in an email to Migraine Science Collaborative. “It seems likely that similar patterns would exist in other areas of the nervous system. If this is the case, signaling potential within circuits relevant for headache may change dramatically throughout the day and night, leading to rhythmic patterns in the susceptibility to attacks.”

Dussor, who was not involved with the new work, also pointed to the relevance of the results for chronotherapy – treatment based on circadian rhythms.

“The biggest strength [of the study] is the translational relevance of the circadian pattern of gene expression for common migraine drug targets. This kind of finding could lead to rapid translation to the clinic given the drugs are already in use; they just may not be given at the optimal time for efficacy” as things stand now, he said.

The research appeared in the February 2024 issue of the journal Headache.

Teaming up for a study
To get the research off the ground, Burish, who is a neurologist as well as a headache and pain researcher, went over to Yoo’s office at UTHealth Houston to discuss whether there was a way to design a study to better understand the circadian features of headache he sees in his cluster headache patients. Yoo is a circadian biologist, and she told MSC that the understanding of circadian biology has advanced significantly from its earliest days.

“At the very beginning, people focused on the brain, and specifically a very specialized area of the anterior hypothalamus called the suprachiasmatic nucleus, as the master clock,” she said. “But now we know that every individual cell and tissue is regulated by a clock. So, other than the brain, the heart, lung, kidney, liver, and muscle all have their own tissue-specific clock, and breaking those clocks has significant pathological consequences.”

But, despite clinical observations of circadian features of headache, there was a paucity of basic science research on the topic, and, in general, a dearth of studies on the circadian biology of the peripheral nervous system. So Yoo and Burish – just two of six principal investigators with their own areas of expertise who would take part in this collaborative research effort – set their sights on the trigeminal ganglion, considering it is a key structure involved in the generation of headache and facial pain.

“Both cluster headache and migraine have a daily cycle, though cluster headache much more strongly so,” Burish told MSC. “For cluster headache, headaches happen at the same time each day; there seems to be this very odd circadian pattern to them. We thought that was interesting, and so we wanted to study it at a basic level – at a genetic and molecular level.”

From cells and genes …
The team first turned their attention to trigeminal ganglion tissue they took from mice and maintained in tissue culture dishes – known as ex vivo cultures.

Before doing so, they had genetically engineered trigeminal ganglion cells in the animals to luminesce – to emit light – under the control of a circadian gene. By looking at the patterns of luminescence, which ultimately reflect patterns of gene expression, the researchers could assess whether the cells exhibited circadian rhythms.

The authors detected a daily circadian pattern of luminescence not only in the ex vivo trigeminal ganglion tissue cultures, but also in cultures of single neurons freshly isolated directly from the trigeminal ganglion; these are known as primary cell cultures.

Cells from the trigeminal ganglia, including neurons and non-neuronal cells, also made core circadian clock proteins responsible for regulating circadian rhythms, according to tissue staining experiments.

… to behavior
Moving from lab culture dishes to live animals, the researchers next asked whether pain responses in mice treated with nitroglycerine (NTG) also showed a circadian pattern. In this headache model, as expected, the mice showed increased mechanical pain sensitivity in the hind paw in response to NTG.

But, compared to control animals, mice that received NTG exhibited circadian changes in their pain responses: They had more hind paw and cephalic sensitivity during the daytime compared to the nighttime, whereas control mice showed no such changes.

The researchers then repeated their NTG experiments in genetically engineered “knockout” mice lacking Per1 and Per2, two essential circadian clock genes expressed in the brain that ultimately control the clocks present in different tissues. Here, control animals with normal expression of these clock genes showed less pain sensitivity at night in response to NTG. However, the knockout animals that received nitroglycerine NTG did not show that behavior.

All in all, these findings showed that the circadian features of trigeminal ganglion cells identified in the earlier experiments were reflected at a behavioral level, too, in an animal model of headache.

The trigeminal ganglion circadian transcriptome
Next up were experiments examining the trigeminal ganglion circadian transcriptome – the full range of RNA transcripts in trigeminal ganglion tissue. This would allow the researchers to determine which circadian genes are normally expressed in trigeminal ganglion cells, and whether NTG changed the expression of those genes.

In control animals, 466 genes were expressed with a circadian rhythm (known as rhythmically expressed genes, or REGs). But in the NTG-treated animals, 71% of those genes were no longer expressed in a circadian fashion.

Further, 584 REGs were newly expressed in the NTG-treated animals, but those genes were not expressed in a circadian manner in the control animals.

“There was a huge change in how many genes have this kind of daily rhythm to them: If you give nitroglycerin, lots of the genes stop having rhythms, and lots of new ones have rhythms. It shows how powerful this headache model is in inducing a circadian pattern,” Burish said.

“The circadian transcriptomic analysis really aligned with our behavioral observations,” Yoo added.

The researchers used a different type of analysis to understand what biological processes the REGs contributed to. It turned out that REGs from both the control and NTG groups play a role in circadian gene expression and rhythmic processes, but there were some interesting differences, too.

For instance, many of the REGs from control animals have a role in glucocorticoid signaling. That seemed reasonable, considering that glucocorticoids are known to act in a circadian way at the molecular level and are recommended treatments for cluster headache and migraine. Meanwhile, REGs from the NTG group have a role in vasculature development and muscle contraction. That also made sense, considering that NTG dilates blood vessels.

Notably, the authors also identified REGs from the NTG-treated animals that are involved in the sensory perception of pain.

Additional analyses looked specifically at core clock genes – those are genes that regulate the brain’s “master” clock for the entire body. Here, the circadian rhythm of core clock genes was similar in the control and NTG-treated animals, with only a moderate effect of NTG on those genes.

But, interestingly, with regard to headache susceptibility genes, several genes for migraine and cluster headache susceptibility exhibited a circadian rhythm of expression that NTG disrupted.

“The authors demonstrate the potential for a broad reorganization of the rhythmic expression of genes in response to a migraine-relevant stimulus (in this case, repeated nitroglycerin exposure). It is possible that expression of genes at inappropriate times plays a large role in headache pathology,” according to Dussor.

But Dussor also pointed to a challenge.

“It is likely going to be difficult to show relevance of rhythmic expression of single genes, and dysregulation of this timing, in the pathology of headache. It is more likely that combinations of genes whose expression changes together are important. Figuring out those combinations will be challenging. This is where techniques like machine learning may be valuable.”

More genes
The researchers also looked at another set of genes, called circadian time-dependent differentially expressed genes (TD-DEGs). These are genes that are differentially expressed at each of six circadian timepoints, between the NTG and control groups.

The team found that, at one particular timepoint in the circadian cycle, NTG produced the differential expression of many genes, including those for neuropeptides and receptors known to be involved with headache.

An example of one of those differentially expressed genes was vasoactive intestinal peptide (VIP), which is an important player in cluster headache. The delta opioid receptor, as well as a component of the calcitonin gene-related peptide (CGRP) receptor, both of which play a role in migraine, were also differentially expressed in response to NTG.

Capitalizing on the trigeminal ganglion clock to improve current treatments – and to develop new ones?
In final experiments, the authors examined whether any genes in the trigeminal ganglion transcriptome are the targets of current preventive medications for certain painful conditions.

They discovered that 10 REGs they had identified are in fact the targets of roughly two-and-a-half dozen medications (25 medications for migraine, six for cluster headache, and three for trigeminal neuralgia). A similar number of TD-DEGs were also the targets of medications for those conditions.

“Could some of the medications work better in the morning and others work better at night, and so should you actually have two different medications based on the time of day? We don’t do that much in the headache world, but our research maybe starts to ask that question,” Burish said.

Dussor said that, with further research, improving current treatment regimens appears realistic.

“The timed administration of drugs could be rapidly implemented if additional studies showed that this increases efficacy. Smartphones could remind people to take medications at specific times, which would be extremely helpful if they are on multiple medications that need to be taken at different times.”

Could a better understanding of the trigeminal ganglion clock advance the development of new drugs? Here, Dussor was more cautious.

“The difficulty in drug development is often specificity of the therapeutic to the intended mechanism. It may be challenging to selectively target clock genes with drugs without disrupting rhythmic expression of other important genes. The viability of this path for new therapeutics will ultimately depend on how clearly studies like this one can identify smaller groups of genes whose rhythmic expression and dysregulation is problematic for headache.

Questions for the future
The study does have some limitations, including “[t]he use of a single behavioral model and a single site within the nervous system,” according to Dussor. “It would be interesting to see whether similar circadian patterns exist in other migraine models and in circuits within the brain. There is only so much the authors could do in a single study, but I hope they explore this concept further.”

Meanwhile, Yoo said that understanding the communication between the trigeminal ganglion in the peripheral nervous system and the hypothalamus in the central nervous system is the next step for the group’s work. She also said that the group is collecting fibroblasts from patient samples with the goal of developing the fibroblasts into neuron-like cells to learn more about the circadian system.

In the meantime, the current study represents a milestone in understanding the circadian features of headache.

“When Mark came to my office where we started, we were stumbling a lot because nothing was really known in this area – there was no gene or mechanism [to explain the circadian aspects of headache]. We’re the first ones studying this, and now we’re in a very exciting phase where we have a lot of clues, and a lot of resources, and we are very excited that our research can really take off.”

Neil Andrews is a science journalist and executive editor of the Migraine Science Collaborative. Follow him on Twitter @NeilAndrews

Image credit: 123RF Stock Photo.

Regulation of headache response and transcriptomic network by the trigeminal ganglion clock.
Han et al.
Headache. 2024 Feb;64(2):195-210.

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Neil Andrews is a science journalist and editor based in New York City. He has over two decades of experience covering science and medicine for expert and non-expert audiences alike. He is also the executive editor of the Migraine Science Collaborative, where he manages the day to day operations of the site. Previously he was the executive editor of the Pain Research Forum.

When not thinking and writing about neuroscience, Neil spends much of his free time on his Peloton and exploring NYC. He is also on a quest to satisfy his coffee cravings by visiting every independent coffee shop in the city. Follow him on Twitter @NeilAndrews.



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