Novel Imaging Technique Reveals Coupled Activation of Primary Sensory Neurons After Nerve Injury
Primary sensory neurons in the dorsal root ganglion (DRG) are responsible for transmission of pain signals from the periphery to the spinal cord. Sensitization of these neurons through tissue damage or inflammation can lead to enhanced pain sensitivity and the development of chronic pain. To date, most studies examining the contributions of DRG neurons to pain signaling have looked at the activity of single neurons by using cultured or isolated ganglia, as scientists lacked the ability to examine these cells at the population level in live animals—until now.
A new imaging technique developed by senior author Xinzhong Dong and colleagues, Johns Hopkins University School of Medicine, Baltimore, US, allows for simultaneous monitoring of over 1,600 neurons per DRG in live, anesthetized mice. Using this method, the researchers show that adjacent neurons fire simultaneously, a phenomenon known as “neuronal coupling,” in two mouse models of pain. They also demonstrate coupling between neurons and satellite glia cells (SGCs), and identify gap junctions in SGCs surrounding DRG neurons as contributors to this process.
“This is an elegant study that convincingly demonstrates neuronal coupling in mouse DRG tissue under two pathological pain conditions after inflammation or nerve injury,” said Ru-Rong Ji, 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 DRG.”
“[T]ogether with the traditional electrophysiological and molecular approaches, the imaging technique demonstrated by Kim et al. will provide a big step forward in our ability to organize and understand pain coding by primary sensory neurons,” wrote Rebecca Seal, University of Pittsburgh School of Medicine, US, in an accompanying editorial.
The new study was published online August 25 in Neuron.
A new method for imaging DRG populations in vivo…
DRG neurons are very difficult to study because they lie tucked away within the vertebrae, making electrophysiological recordings in situ challenging to do, and most studies have recorded only solitary neurons. Though single-cell recording is a powerful technique, “imaging large populations of DRG neurons will allow us to unveil novel pain mechanisms that conventional techniques, like electrophysiological recordings or in vitro DRG culture imaging, cannot study,” Dong wrote in an email to PRF.
To examine these neuronal populations, co-first authors Yu Shin Kim, Michael Anderson, Kyoungsook Park, and Qin Zheng used genetically engineered mice in which a calcium indicator, called GCaMP3, is specifically expressed in DRG neurons under the control of the Pirt promotor (Pirt-GCaMP3 mice). Pirt is a membrane protein expressed in the peripheral nervous system, mainly in nociceptive neurons (Kim et al., 2008).
After surgically exposing the right lumbar 4 (L4) DRG, which innervates the right hindpaw, leg, thigh, and back in the transgenic animals, the scientists could visualize neurons expressing the calcium indicator by using a confocal microscope, in vivo in primary sensory neurons. More than 1,600 neurons per DRG—representing about 15 percent of total DRG neurons—were viewed simultaneously in live anesthetized animals, making possible the first glance at how these cells function at the population level.
The neurons were evenly distributed throughout the DRG, and the researchers further found that about 26 percent of neurons labeled with a neuronal tracer responded to mechanical force applied at the hindpaw, and about 28 percent responded to hindpaw capsaicin injection, with approximately 3 percent responding to both. The investigators further saw little response to hindpaw capsaicin or mustard oil injection in Pirt-GCaMP3 mice further engineered to lack TRPV1 or TRPA1 ion channels, demonstrating that the calcium signals they detected were ligand/channel specific.
“[The lab was] amazed at how robust the GCaMP signals in live mice were when DRG neurons were activated, and how easy the technique was,” Dong noted.
…and driving new discoveries
After demonstrating the power of their new imaging approach, the group next showed that applying mechanical force to the hindpaw of animals injected with complete Freund’s adjuvant (CFA), a model of inflammatory pain, or subjected to chronic constriction injury (CCI), a model of neuropathic pain, doubled the number of activated neurons compared to naïve mice. The investigators also observed neuronal coupling in both pain models in response to mild mechanical stress. Indeed, they saw that activated neurons were next to each other and formed clusters of two to five DRG neurons.
Coupled Activation of Adjacent DRG Neurons in In Vivo DRG Calcium Imaging. Representative images of coupled events in in vivo calcium imaging of whole L4 DRG neurons. Left panels: Adjacent DRG neurons tend to activate together when stimuli (mild mechanical press; 300 g) were applied to the hindpaws of CFA and SN-CCI animals. Right panels: Time course of the amplitude of the calcium transient that was evoked by mild press at the hindpaw from CFA and SN-CCI animals. Each trace is a response from a single DRG neuron, and number indicates the order of DRG neurons activated by press stimuli. All data for calcium transient are expressed as the percentage of baseline calcium transient (ΔF/F0). Scale bar, 50 μm. Figure and caption from Kim et al. with permission from Elsevier.
Coupling was seldom observed in naïve mice (approximately 29 percent of total activated neurons were coupled in CFA or CCI mice compared with about 5-6 percent in naïve mice). Also, when animals were allowed to recover from CFA injection for seven days, neuronal coupling decreased from 29 percent to 13 percent of total activated DRG neurons.
To follow up on these observations, the authors performed a full dose response to mechanical pressure, with stimuli ranging from a barely noticeable 50 g to a painful 500 g of mechanical pressure. In CFA mice, the percent of coupled neurons increased as mechanical stimulation became more noxious. In contrast, neuronal coupling remained unchanged in naïve mice as mechanical pressure increased (at 500 g of pressure, more than 50 percent of cells were coupled in CFA animals compared with less than 20 percent in naïve animals). Finally, additional imaging experiments revealed that coupling occurred in nociceptors, and also in low-threshold mechanoreceptors.
Together, the data suggest a direct influence of tissue injury on neuronal coupling―an observation made possible using population-level DRG imaging. “This kind of surprising result is almost impossible to detect using conventional approaches,” said Dong.
Gap junctions may add the “how” to the “what”
What enables coupled neurons to couple? Increased coupling of satellite glia cells (SGCs) surrounding DRG neurons via gap junctions, which connect the cytoplasm of two cells, thus allowing for intercellular communication, has been demonstrated following systemic inflammation (Blum et al., 2014). Furthermore, gap junctions in the DRG appear to influence neuronal activation and subsequent visceral pain (Huang et al., 2010). Thus, gap junctions were a possible explanation for the neuronal coupling observed in the new study.
To investigate this possibility, the researchers injected rhodamine, a red dye that crosses gap junctions, into one of the coupled activated DRG neurons from CFA mice and showed dye transfer between coupled but not singly activated neurons. Patch clamp recordings of neuron-SGC pairs in dissociated DRG, performed by co-author David Spray, Albert Einstein College of Medicine, Bronx, US, revealed coupling between neurons and SGCs (patch clamp recordings were used because of the difficulty in imaging SGCs, which are very small and wrap tightly around neurons). Additional experiments suggested that coupling occurred mainly through neuron-SGC-neuron coupling rather than via neuron-neuron coupling.
Next, the group showed that the gap junction blockers heptanol and carbenoxolone (CBX), when injected into CFA mice, decreased coupling by more than 50 percent within an hour of administration. Importantly, CBX also reduced mechanical hyperalgesia in CFA and CCI mice compared with controls. Thus, these results demonstrated that coupling occurred via gap junctions, and established a link between gap junction-mediated coupling and pain behavior.
Finally, the group turned to the gap junction protein connexin 43 (Cx43), which has been shown to play a role in chronic pain. Cx43 increased significantly following administration of CFA in SGCs in the DRG but not in the spinal cord. Genetic deletion of Cx43 in the DRG decreased coupling after CFA treatment compared to controls, paralleled by a decrease in mechanical hyperalgesia, with no effect on thermal hyperalgesia or baseline pain sensitivity.
Previous studies have shown that astrocytes in the spinal cord also increase their expression of Cx43 after nerve injury (see PRF related news story). However, the relative importance of Cx43 in astrocytes versus SGCs in chronic pain has yet to be sorted out. “Further studies are needed to define whether peripheral and central Cx43 play different roles in the development and maintenance of chronic pain,” Ji told PRF.
All for transgenics and transgenics for all
The current results demonstrate the power of the group’s new method for visualizing DRG neurons at the population level in live animals and uncover a new mechanism for pain plasticity, via gap junction-mediated neuron-glia communication. The future holds the exciting possibility of learning even more about pain signaling by using the novel imaging technique with a variety of transgenic animals. “We have many visitors coming to our lab to learn the technique and collaborate,” Dong told PRF. “Since Pirt-GCaMP mice can be easily crossed to other genetically modified mouse lines, we envision that more and more researchers in the field of pain research will use Pirt-GCaMP mice to study the molecular and cellular mechanisms of pain.”
Hillary Doyle is a PhD candidate and science writer studying pain and analgesia at Georgia State University in Atlanta.
Image credit: Kim et al. with permission from Elsevier
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