Central TRPV1 Contributes to Neuropathic Hyperalgesia
When it comes to transient receptor potential channel V1 (TRPV1), pain research has naturally focused on the peripheral tips of sensory neurons, where TRPV1 encounters its best-known activators: heat and the capsaicin found in chili peppers. But TRPV1 also shows up on the central projections of sensory neurons in the spinal cord. New research published January 21 in Neuron demonstrates that changes in TRPV1 activity at these central terminals—spurred by serotonergic inputs—underlie pain hypersensitivity in a mouse model of neuropathic pain.
Tibor Rohacs, a neuroscientist at Rutgers New Jersey Medical School, Newark, US, who was not involved in the current work, called it “a paradigm-changing paper,” because it begins to answer a question the pain field has not yet really addressed, he said. “TRPV1 was known to be on central terminals, but what it does there has not been clear,” said Rohacs. This work provides the first details of how the central channels contribute to neuropathic pain, which Rohacs says will surprise many people in the field.
The work, a collaboration between Xinzhong Dong at Johns Hopkins University School of Medicine and Feng Wei at the University of Maryland, both in Baltimore, US, involved state-of-the-art calcium imaging in live cells that allowed the researchers to visualize TRPV1-dependent neuronal activity along the entire length of afferent sensory neurons.
The findings suggest that both TRPV1 and 5HT3A-type serotonin may control the excitability of afferent sensory neurons’ spinal connections. That raises the prospect that either a TRPV1 or 5HT3A antagonist delivered to the spinal cord might stop pain signals that result in mechanical hyperalgesia, said Dong.
Special model
To look at mechanisms of central sensitization at nociceptive nerve terminals, Wei and co-first author Yuxia Chu used a chronic-constriction injury of the infraorbital nerve (CCI-ION) model of neuropathic pain. The model takes advantage of the unique anatomy of the trigeminal ganglion (TG), which receives sensory input from three nerve branches that innervate three distinct areas of the face. Cell bodies of the incoming V1, V2, and V3 nerve branches are segregated within the ganglia, and their central projections also segregate in the trigeminal caudal subnucleus (Vc, or medullary dorsal horn), the termination point for TG sensory neuron projections. In CCI-ION, the central V2 branch, which innervates the whisker pad, is constricted; the V1 and V3 branches are uninjured.
Five, 14, and 28 days after nerve injury, the mice displayed hypersensitivity to mechanical stimulation and to capsaicin injection not only in the whisker pad, but also in the adjacent area innervated by the uninjured nerves, indicating that hyperalgesia had spread beyond the injured nerve’s facial territory due to some change in the uninjured nerves.
The researchers wanted to determine what had happened to those uninjured V3 nerves to cause hypersensitivity in the face. To do that, Dong and his colleagues visualized neuronal activation in sensory neurons by using the genetically encoded calcium indicator GCaMP3 (a hybrid of green fluorescent protein [GFP], calmodulin [CaM], and part of a myosin light chain kinase; see Looger and Griesbeck, 2012). GCaMP3 glows bright green when it binds calcium and allows researchers to visualize the waves of intracellular calcium that form the crux of excitatory signals in neurons. Co-first author Yu Shin Kim and colleagues in the Dong lab developed transgenic mice that express GCaMP3 using the Pirt promoter, which drives strong and uniform sensory neuron-specific expression throughout the dorsal root ganglia (DRG) and trigeminal ganglia (TG). “The indicator was expressed by more than 96 percent of the neurons, and that level did not change with injury,” Dong said. “These features allowed us to study neuronal plasticity after injury to see how activity changed in the neurons’ skin endings, cell bodies, and central terminals,” he said.
“Just from a technological standpoint, the paper makes a significant advance with the mouse model,” Rohacs said. “The ability to have DRG and trigeminal neurons loaded with the genetically encoded calcium indicator—that in itself will be quite valuable.”
Because GCaMP was found all throughout the sensory neurons, the team was able to image calcium signaling in the nerve endings in the skin all at once, which had not been done before. “People had recorded from single nerve fibers, but had not seen population activity,” said Dong.
To look at nerve hypersensitivity, the researchers applied capsaicin to an explant of ear skin innervated by the uninjured V3 branch of the TG nerve or to another area served by a DRG nerve unaffected by CCI-ION. Fluorescence imaging of the intact skin-nerves showed greater calcium responses to capsaicin—and therefore more peripheral TRPV1 activity—in the V3 nerve endings compared to the adjacent DRG nerve endings or contralateral V3 endings. “Once we observed this robust hyperactivity in uninjured neurons, we wanted to know the underlying mechanisms,” Dong said.
The team next shifted their focus from peripheral terminals to neurons’ cell bodies by imaging trigeminal ganglia explants. Neurons belonging to both the injured and uninjured nerve branches displayed greater calcium signaling in response to capsaicin than did contralateral neurons, indicating that cell bodies, too, had increased TRPV1 signaling in response to injury. Interestingly, more of the capsaicin-responsive neurons in the injured compared to contralateral V2 area of the ganglia were large-diameter neurons—a sign that non-nociceptive, mechanosensing neurons began to express TRPV1 after injury.
The researchers next applied capsaicin to slices taken from the Vc, containing the sensory neurons’ central terminals. V3 nerve endings throughout the nucleus showed increased TRPV1 signaling, including those from pain-sensing and mechanosensing neurons.
Descending influence
What could trigger this TRPV1 sensitization in the central nervous system? Wei’s group had previously shown that serotonin projections from the rostral ventral medulla (RVM) to the Vc contribute to pain sensitization after nerve injury (Okubo et al., 2013). When the team looked at these projections two weeks after CCI-ION, serotonergic inputs to the Vc had dramatically increased. When the scientists blocked synthesis of serotonin in the RVM, the injury-associated increase in TRPV1 activity seen in Vc sensory neuron terminals was prevented, as was behavioral pain hypersensitivity in both nerve-injured and non-injured territories.
Calcium imaging of sensory neuron central terminals in the Vc in response to a specific agonist of 5HT3A-type serotonin receptors showed that more terminals expressed the receptor in nerve-injured than in sham-operated mice. Furthermore, blocking 5HT3A receptors by microinjection of an antagonist into the Vc prevented mechanical hyperalgesia.
To look at the effect of neuronal hypersensitivity on the signaling from primary afferents into the Vc, the researchers recorded electrophysiological responses from the Vc neurons while stimulating their trigeminal inputs. Frequency and amplitude of excitatory responses (miniature excitatory post-synaptic currents, or mEPSCs) were increased in nerve-injured compared to naïve mice, suggesting that both the afferent sensory neurons and their targets in the Vc had become sensitized. Inhibition of 5HT3A receptors or TRPV1 reduced only the frequency—not amplitude—of mEPSCs, indicating the receptors’ pivotal role is on the sensory neuron terminals. Blocking those TRPV1 channels prevented an increase in spontaneous EPSCs recorded from dorsal horn neurons and mechanical hyperalgesia behaviors in CCI-ION mice.
All together, the results indicate that TRPV1 plasticity at the peripheral, cell body, and central levels contributes to neuropathic pain sensitivity. “TRPV1 is not a mechanosensitive channel,” Dong said, “but it could be targeted for mechanical hyperalgesia.”
Rohacs agreed, saying that although heat is TRPV1’s most well known activator, the channel clearly serves as a signal integrator. The new evidence cements the idea that TRPV1 might participate in nerve sensitization at sites other than the periphery. But its heat-sensing properties should not be counted out, even in the spinal cord. In their sensitized state, Rohacs speculated, TRPV1 might make neurons spontaneously active at body temperature.
The new work leaves many open questions. For example, how exactly does activation of the 5HT3A receptor—an ion channel—lead to TRPV1 sensitization in the spinal cord? And what are the endogenous ligands for TRPV1 in the central nervous system? “These are open questions,” said Rohacs, “that obviously need further study.”
The new report makes a therapeutic recommendation as well: Intrathecal injection of a TRPV1 antagonist—or perhaps even better, a 5HT3A antagonist—might prove to be effective against neuropathic pain. Whether that will be useful in humans remains another open—but pressing—question.
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California, US.
Image: Nerves in mouse skin that are actively responding to the capsaicin have been genetically engineered to glow green. Hairs appear in yellow. Credit: David Rini
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