What’s Behind the Spatial Acuity for Pain?
Compared to other areas of the body, the fingertips are especially sensitive to the threat of injury, and yet they possess fewer pain-signaling nerve fibers. Now, according to new research led by Wenqin Luo, University of Pennsylvania, Philadelphia, US, such spatial acuity may originate not in the skin but in the spinal cord.
Thanks to a technique known as sparse labeling, Luo and colleagues examine the structure of single mouse neurons expressing Mas-related G protein-coupled receptor D (Mrgprd), a marker of non-peptidergic nociceptors in the outer layers of the skin. Across areas of the body, the cells’ peripheral terminals did not vary substantially in their density or size. In the dorsal horn, however, the arbors of paw-projecting nociceptors appeared round, while those of nociceptors representing more proximal parts of the body, such as the trunk, were long and thin. Consistent with these differences, optogenetic activation of paw nociceptors evoked heightened responses in dorsal horn neurons, and they required less stimulation to trigger pain-related behaviors.
“The images and findings in this study are stunning,” says Mark Zylka, University of North Carolina, Chapel Hill, US, who was not involved in the research. “I am very happy to see that someone has done a rigorous job of determining what the peripheral and central termination fields of Mrgprd afferents look like.”
Xinzhong Dong, Johns Hopkins University, Baltimore, US, who also did not take part in the work, says it contrasts the acuity for pain with that for touch, since the latter relies on a high density of mechanoreceptors in the skin. It also serves as a reminder not to overlook the central terminals of nociceptors.
“Usually, people think these neurons are just simple cables—a single line from the periphery to the spinal cord—but the terminals are actually very complex,” according to Dong.
The findings were published online October 12 in eLife.
Complex branches
Researchers have spent decades charting the neural circuits dedicated to pain. But “this idea of systematically comparing nociceptors across body regions hasn’t received that much attention,” said William Olson, first author of the new study.
One reason why, said Dong, might be that past studies simply lacked the appropriate tools. “Now, with sparse labeling, we can label just one or two neurons per spinal cord section, and you can clearly see the differences in morphology.”
In 2013, Luo’s lab used genetically modified mice to sparsely label axons of mechanoreceptors and proprioceptors in the dorsal horn, showing that the two sets of cells are segregated and have distinct branching patterns (Niu et al., 2013). Out of this work grew the idea to also trace nociceptors. But for that, they needed a new line of mice.
So Olson and colleagues engineered animals with a small number of genetically tagged Mrgprd neurons. Confirming earlier reports, they found that the cells were non-peptidergic, expressing several genes characteristic of such neurons. And, similar to what’s seen in people, the neurons did not have a higher density of fibers in the paw compared to the trunk, nor did they change dramatically in terminal arbor size.
(Click here for a larger, PDF version of the figure below.)
Sparsely labeled Mrgprd nociceptors have region-specific central arbor morphologies. (A-F) Round and long non-peptidergic central arbors seen in top-down whole mount (A, C, D, F) and transverse section (B and E) spinal cords. Round arbors are in the medial lumbar enlargement (B and C), while long arbors are in the lateral lumbar enlargement and thoracic spinal cord (B-F). Dorsal roots are outlined and labeled in C. Red dashed lines outline the round arbor zone. Orange arrowhead marks a round arbor, purple arrowheads mark long arbors. M, medial. L, lateral. R, rostral. C, caudal. Scale bars = 50 µm (A and B, D and E), 250 µm (C and F). From Olson et al., 2017.
In contrast, upon surveying sections of the spinal cord, “we were struck by the morphological differences,” said Luo. The arbors there formed two shapes, each restricted to a separate area of the dorsal horn. Medially, the arbors were round, but laterally, they were long and thin.
But what did that mean? “We tried to decide whether this showed that there are two functional subtypes within this population of Mrgprd neurons—maybe one for itch sensation and the other for nociception—or a true somatotopic organization” of the pain system.
A human study published in 2013 by another group of researchers made a case for the latter (Mancini et al., 2013). Countering long-held assumptions, it showed that people could localize pain better on their fingertips than on the back of their hands, even though the fingertips have a lower density of nerve fibers. In other words, there appeared to be a fovea for pain, an area where the spatial resolution of pain information is enhanced.
“It provided a new angle of thinking for us,” said Luo. “It made us wonder, Could the phenomenon we see here explain pain acuity?” Indeed, the relative locations of the round and long arbors suggested that they belonged to paw- and trunk-innervating nociceptors, respectively. Perhaps, the researchers thought, the two groups of nociceptors control how pain is represented via their central terminals.
Amplified signals
To test this idea further, the authors looked to the postsynaptic potentials evoked by input from each kind of arbor. By optogenetically activating Mrgprd terminals in the spinal cord while making electrophysiological recordings from neurons in either medial or lateral dorsal horn areas, they could detect more robust responses in medial neurons. And, those cells that responded did so with less light and hence less stimulation.
“We identified a correlation between round arbors and what we see as stronger synaptic transmission,” Olson explained.
Lastly, the group evaluated pain behavior. Mice reflexively withdrew their paws when the Mrgprd fibers in these regions were optogenetically activated with a certain amount of light. But for terminals higher up on the leg, this manipulation had no effect. Only when the intensity of light was increased by at least twofold did the animals respond.
In explaining these observations, the authors propose that signals carried by nociceptors in specific locations, such as those in the paw, might be amplified in the spinal cord or brain. Hence, the nervous system would not need to depend on a higher density of nociceptive fibers in the skin to enlarge representations of pain.
As for how such amplification occurs, Luo can only speculate. “It could have something to do with how many synaptic contacts their arbors have with downstream neurons,” she said. There are other possibilities as well. “Are the central terminals of these nociceptors connected to a different type of postsynaptic neuron, or are they connected in a different way?”
A general pattern?
If the findings hold true in people, they could begin to explain the only recently discovered pain fovea. Still, the branching patterns identified by the study may be an oddity of Mrgprd neurons, rather than a general feature of nociceptors.
Beyond their neurochemical identity, Mrgprd neurons are unique in that they extend their peripheral fibers to only one tissue―the skin. Moreover, as a recent study showed, activation of this specific population in mice caused animals to withdraw and lift their paws, but it did not seem to be aversive (Beaudry et al., 2017). It’s typically assumed, however, that pain is aversive.
“To really make the connection between nociceptive neurons and pain acuity, others could do a similar set of experiments but look at a different subpopulation of neurons, such as peptidergic neurons,” said Dong.
“Peptidergic neurons are complicated in the sense that they project to the skin, but also to deeper tissues,” said Luo. She thinks those that terminate in the skin likely contribute to pain acuity as well. “But for those deeper nociceptors, do they have a somatotopic organization? I’m not sure.”
Matthew Soleiman is a science writer residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman.
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