Pain researchers interested in mapping nociceptive pathways and understanding opioid signaling have always grappled with a dearth of experimental tools. Now two groups report novel photoactivatable molecules to remedy the shortage. The new compounds provide on-demand and selective activation of neurons, enabling rapid and precise spatiotemporal control of pain pathways.
“With the capacity to instantaneously release neuropeptides to a localized space, one can now precisely map out how different neuropeptides modulate the physiological and biochemical mechanisms of pain sensation,” said Clifford Woolf, a pain expert at Harvard Medical School and Children’s Hospital Boston, US, who was not involved in the current research. Woolf’s recent studies elucidating the mechanism of action of the local anesthetic and lidocaine derivative, QX-314, were the basis of the photoactivatable molecule designed by the first group.
The latest findings were reported online on February 19 in Nature Methods by Richard Kramer at the University of California, Berkeley, US; Dirk Trauner at the University of Munich, Germany; and colleagues, and in the January 26 issue of Neuron by Bernardo Sabatini and Matthew Banghart at Harvard Medical School, Boston, US.
Exactly how the nervous system detects and interprets external and endogenous pain signals to regulate pain perception is poorly understood. Several obstacles have impeded research in this area, including a heterogeneity of signaling molecules and receptors involved in nociception, an inability to selectively release and quantitatively measure signaling molecules from distinct cell types, and a lack of spatiotemporal control of nociceptive activity when using signaling molecules.
In the first study, Kramer and colleagues used a photo-isomerizable compound, QAQ, which is taken up selectively by pain-sensing neurons containing the capsaicin-responsive TRPV1 subset of transient receptor potential channels. They found that QAQ, in its trans form, blocked voltage-gated Na+, K+, and Ca2+ channels, similar to how lidocaine and QX-314 function. But unlike lidocaine, which is membrane-permeable and non-selective, QAQ, like QX-314, enters neurons specifically using TRPV1 channels as its main conduit, and only when the channels are in the dilated state following treatment with the agonist, capsaicin.
Upon illumination with 500-nm light, inactive cis-form QAQ rapidly and reversibly isomerized to the active trans form. By using this unique feature of the compound, the researchers found that isolated mouse dorsal root ganglion (DRG) neurons treated with capsaicin and QAQ were photosensitized. They also found that QAQ uptake by TRPV1-containing neurons in lamina II of the spinal cord’s dorsal horn led to the photosensitization of neurons in that area. In contrast, they saw only negligible photosensitization of the lamina III-IV region that contains non-nociceptive neurons, further demonstrating that QAQ selectively enters TRPV1-containing nociceptors. Furthermore, Kramer and colleagues demonstrated that QAQ, like QX-314, functioned as a local anesthetic when they treated peripheral nerve endings in the eyes of the mice topically with a combination of QAQ and capsaicin.
This ability to photoactivate QAQ at will and to restrict its uptake to specific nociceptive neurons will be highly valuable to researchers who previously had only blunt methods at their disposal. “We want to put these tools to work and ask interesting biological questions,” Kramer told PRF. For instance, he would like to use QAQ uptake assays to compare when and where ion channels in nociceptors are active in the presence and absence of noxious stimuli.
In the second study, Banghart and Sabatini describe the rapid and exact spatiotemporal control of opioid receptors in rat brain tissue by photoactivating CYLE and CYD8, two inert “caged” analogs of endogenous opioid peptides. “The ability to photosensitize affords precision and reproducibility, enabling robust quantitative analysis of peptidergic signaling for the first time,” explained Banghart, lead author of the study.
In contrast to the selective uptake strategy used in the first investigation, Banghart and Sabatini first equilibrated rat brain tissue with a high concentration of inert neuropeptide. Then they regulated the precise area of release and local concentration of active neuropeptide by manipulating the light intensity or area of illumination. Upon spatially restricted release, the active opioid peptides bound to their target μ receptors in rat neurons of the locus coeruleus, generating outward currents through opioid receptor-mediated activation of K+ channels.
In addition to local signaling, Banghart and Sabatini also observed signaling at distant receptors (~150 mm from the site of neuropeptide release). Such neuropeptide volume transmission has long been suspected, since neuropeptide receptors are often located hundreds of microns from neuropeptide release sites in intact brain tissue. By spatially restricting neuropeptide release to a very narrow area of the brain (just ~2 mm in diameter), Banghart and Sabatini provide the first evidence for this phenomenon. Since the researchers used the smallest and most chemically stable neuropeptides, it will be interesting to see how neuropeptide signaling and volume transmission vary when using larger and less stable endogenous molecules.
While the clinical utility of photoactivatable agents remains uncertain, the ability to exert rapid and fine spatiotemporal control of nociceptors and opioid signaling using QAQ and caged neuropeptides now gives investigators an immensely appealing tool to conduct basic pain research.
CORRECTION (20 Mar 2012): In the original version of this news story, the term “opioid nociceptors” was used instead of the correct wording, “opioid receptors.” In addition, a reference to QX-314 gave the impression that the compound does not enter cells via TRPV1, which it does. The text in both cases has been corrected.
Raji Edayathumangalam is an Instructor in Neurology at Harvard Medical School (Boston, US) and a freelance science writer.