Peripheral inflammation sets off a cascade of neuronal activity that leads to chronic pain hypersensitivity, but the exact cellular and molecular signaling processes involved are unclear. Now, new work led by Rohini Kuner and Hilmar Bading, both at the University of Heidelberg, Germany, traces a series of events in spinal neurons that mediate the transition from acute to lasting pain in mice.
Ru-Rong Ji, a pain researcher at Duke University Medical Center, Durham, North Carolina, US, who was not involved in the current work, calls the study “very impressive” in its scope and in meeting technical challenges. The work was published January 9 in Neuron.
While synaptic calcium signals are integral to neuronal activity—initiating action potentials, for example—nuclear calcium has been less well studied. Here, the authors used virally delivered recombinant protein-based indicators to show that spinal neurons that receive nociceptive afferent input in mice displayed calcium transients within the nucleus following electrical stimulation of the dorsal root. The scientists went on to deliver a protein that binds and inactivates calcium-bound calmodulin, a key nuclear molecule in the cascade linking calcium to the transcription factor CREB and its thousands of gene targets. When this gene signaling was interrupted, long-lasting pain hypersensitivity failed to develop following injection of complete Freund’s adjuvant (CFA).
Bading, Kuner, and colleagues then surveyed the genes affected by the nuclear calcium signal using microarray-based analysis and quantitative real-time polymerase chain reaction (QRT-PCR). Not surprisingly, many pain-associated genes were upregulated or downregulated by inflammation with CFA. For a subset of those genes, including cyclooxygenase-2 (COX2), a well-known inflammatory pain mediator, blocking nuclear calcium signaling blunted the inflammation-induced expression changes. But unexpectedly, also affected were genes involved in the complement system, which comprises a set of molecules normally associated with immune responses.
Kuner initially did not believe that “immune” proteins would be important in inflammatory pain signaling in neurons. “We thought it was an artifact,” she said. But bioinformatics analysis revealed that complement-related genes comprised the pathway most significantly altered in the spinal cord neurons by peripheral inflammation.
“The idea that immune proteins and genes are only for immune cells has been overtaken by recent findings,” said Michael Salter, a researcher at the Hospital for Sick Children in Toronto, Canada, who was not involved in the work. For instance, C1q, a central molecule in the complement system, has recently emerged as a player in synaptic remodeling (Stevens et al., 2007). In the current work, Bading and Kuner found that neuronal C1q was downregulated following CFA.
The team, including co-first authors Manuela Simonetti, Anna Hagenston, Daniel Vardeh, H. Eckehard Freitag, and Daniela Mauceri, then turned its attention to structural changes in the neurons’ synapses. Spinal cord dendritic spine density increased after peripheral inflammation, in line with the idea that aberrant pain signaling is rooted in synaptic plasticity (see PRF related news story and Journal Club summary of recent research implicating maladaptive dendritic spine remodeling in diabetic neuropathy). When the investigators knocked down C1q with a small interfering RNA, they saw dendritic spines multiply even in the absence of inflammation. Conversely, the CFA-driven increase in spine density was prevented by injection or overexpression of C1q. “They see complement directly affecting dendritic spine remodeling,” Ji said of the researchers.
The downregulation of C1q in neurons during persistent inflammatory pain stands in contrast to recent findings in neuropathic pain models, where nerve injury leads to upregulation of C1q in microglia (Griffin et al., 2007). “Those are quite different signaling events,” said Ji. In inflammatory pain, the authors hypothesize, neuronal C1q may act in some yet-undescribed autocrine or paracrine manner to keep synapses pruned, a process that perhaps fails following downregulation of C1q. “C1q is really a killer of synapses,” said Bading.
The work adds to an emerging picture of chronic inflammatory and neuropathic pain as distinct—even opposite—phenomena. For example, some genes—like that encoding COX2—were upregulated by CFA inflammation but downregulated in the spared nerve injury model of neuropathic pain. Other genes, including the P2X4 ATP receptor, displayed the reverse pattern, with expression levels increasing in neuropathic pain but falling with inflammation. Another striking difference lies in the type of plasticity implicated in the two forms of pain. Whereas the current study provides evidence for structural plasticity of dendritic spines, neuropathic pain has been traced to neuronal ion balance disruption driven by microglia (Beggs et al., 2012).
The differences between chronic inflammatory and neuropathic pain, said Kuner, might stem from the distinct afferent neural firing patterns that signal each type of pain. Whereas neuropathic pain might start with a barrage of activity—arising from nerve injury—that soon falls silent, inflammatory pain exhibits a steadier, more continuous firing pattern that builds as inflammatory molecules accumulate in the periphery. Exactly how those patterns are translated into the downstream signals described in the current work remains to be seen, Kuner said.
Not surprisingly, prevention of the nuclear calcium signaling had no effect on acute inflammatory pain, which does not rely on gene transcription. Furthermore, disrupting nuclear calcium only minimally attenuated established inflammatory hypersensitivity, suggesting that, once set in motion, the genetic program no longer represents a viable therapeutic target. Recombinant C1q delivered intrathecally over 24 hours following CFA did prevent normal development of hypersensitivity. In terms of therapeutics, a potentially important experiment would be to determine whether C1q might reverse established pain, perhaps by destroying extraneous synapses, Salter suggested.
In recent years, details have emerged about the regulation of long-lasting synaptic changes that underlie processes like memory consolidation and addiction. Now Bading and Kuner have shown that the alterations observed in chronic inflammatory pain bear a striking resemblance to those other long-term changes. While pain may seem very different from memory, “the rules are the same. All these adaptations”—resulting in increased neuronal connectivity—“are triggered by synaptic activity and require gene transcription,” Bading said. In each case, “a calcium signal from the synapse to the nucleus is absolutely key. It seems more and more clear that this is a signal used independent of the type of adaptation.” Other recent lines of research also support the classification of chronic pain as a form of memory (see PRF related news stories here and here).
Finally, the elucidation of the calcium signaling pathway in the new research was a direct result of collaboration among scientists in different fields, said Kuner, who works in pain per se, and Bading, who studies synaptic plasticity in various settings. Kuner encouraged other researchers to work with scientists outside of pain research. In addition to “cross-fertilizing” the pain field with other areas of neuroscience, collaborations make research “so much more fun.”
Stephani Sutherland, PhD, is a freelance neuroscience writer based in Southern California
Image: Leucine zipper: Cyclic AMP response element binding protein (CREB)-1 binding to DNA. Credit: Yikrazuul, Wikimedia Commons
