From One at a Time to Many at Once

A new imaging technique visualizes neurons working together to contribute to chronic pain. Image credit: rolffimages/123RF Stock Photo.

A dorsal root ganglion (DRG) is a group of nerve cells that relay pain signals coming from the body into the spinal cord. Ultimately, these signals are sent up to the brain, for further processing. Nerve injury or inflammation can cause neurons in the DRG to become too electrically excitable, leading to exaggerated sensations of pain and the development of chronic pain.

To date, scientists have only been able to examine DRG neurons one at a time and almost exclusively in a petri dish. Now, a new imaging technique developed by Xinzhong Dong and colleagues at Johns Hopkins University School of Medicine, Baltimore, US, allows for simultaneous monitoring of over 1,600 neurons per DRG in live, anesthetized mice.

By examining many cells at a time, the investigators observed that neurons near each other “fire” together during pain—that is, they show synchronized electrical activity— a phenomenon referred to as “neuronal coupling.” They further show that DRG neurons also couple to satellite glial cells (SGCs). SGCs are cells that tightly surround neurons and contribute to many functions performed by them.

“This is an elegant study that convincingly demonstrates neuronal coupling,” in the DRG neurons during chronic pain, says Ru-Rong Ji, a pain researcher at Duke University School of Medicine, Durham, US, who was not involved in the study. “This study not only reveals a novel mechanism of chronic pain, but also provides a very useful imaging tool to study neuronal activities in DRGs.”

The study was published online August 25 in the journal Neuron.

When teamwork is not always a good thing
DRG neurons are located deep inside vertebrae, making them very difficult to study. Before now, DRG neurons had only been examined individually. This has prevented an understanding of how populations of neurons might function together to contribute to pain.

To tackle this issue, Dong and his team created genetically engineered mice in which the activity of populations of DRG neurons can be visualized using a microscope. Using this technique, they could see the activity of more than 1,600 neurons per DRG simultaneously in live, anesthetized animals, making possible the first glance at how these cells function at the population level.

The group then used this powerful technique to examine how DRG neurons respond under painful conditions. To do so, they applied mechanical force (in this case, a pinch) to the hindpaw of mice that were previously subjected to a nerve injury or to inflammation (these animal models of pain are commonly used to study the pain system in rodents).

Results showed that the number of neurons activated by the pinch doubled in the injured or inflamed mice, compared to mice without nerve injury or inflammation. This result showed a link between pain and activation of the neurons, and the ability of the new imaging technique to make the discovery.

The investigators also observed coupling. That is, they saw that a mild pinch activated clusters of 2-5 DRG neurons; these neurons were activated simultaneously, in both pain models. However, coupling was seldom observed in mice without injury or inflammation. In mice with inflammation, more coupling was seen as the pinch became stronger, but no such changes were seen in mice without inflammation. Finally, after the animals had a week to recover from the inflammation, less coupling was observed. Together, these results indicated a link between coupling and pain.

“This kind of surprising result is almost impossible to detect using conventional approaches,” wrote Dong in an email to RELIEF.

Mind the gap
What accounts for coupling? Previous studies had shown that inflammation causes coupling between SGCs (the cells that surround neurons) via gap junctions. Gap junctions are formed by proteins and they connect one cell to another, allowing the cells to communicate with each other.

As a result, the investigators thought that gap junctions might also explain the increased coupling they saw in their study. Indeed, this was case. A closer look revealed that neurons coupled not only to other neurons, but also to SGCs.

The group also saw that when they used chemicals to block gap junctions, the amount of coupling went down by more than 50%. This demonstrated that gap junctions contributed to the coupling phenomenon.

Importantly, blocking the gap junctions also reduced the amount of pain the inflamed or nerve-injured mice experienced. This result indicated that gap junctions, through their ability to control coupling, played an important role in pain.

Dong expects numerous additional discoveries about pain as more investigators use the new imaging technique with many different types of genetically engineered animals. “We have many visitors coming to our lab to learn the technique and collaborate. We envision that more and more researchers in the field of pain research will use [the technique] to study the molecular and cellular mechanisms of pain,” he said. –Hillary Doyle

To read about the research in more detail, see the related Pain Research Forum news story here.

Hillary Doyle is a PhD candidate and science writer studying pain and analgesia at Georgia State University in Atlanta.