As human pain proves more and more complex, researchers are finding ample reasons to seek out simpler models. The nematode C. elegans and the fruit fly Drosophila melanogaster have proven themselves valuable tools for probing processes from embryonic development to neurodegeneration. But can these tiny animals really illuminate the principles of pain?
There is no question that C. elegans and Drosophila react to noxious stimuli. Poke a worm with a sharp wire, or set a fly on a hot plate, for instance, and they move away from the danger. In recent years, researchers have found that these avoidance responses involve neurons that look and behave strikingly like mammalian nociceptors. Today, a number of labs are following up on those suggestive similarities by using worms and flies to piece together the molecular machinery of nociception. Surprisingly, the researchers are finding that these simple creatures can offer insights into higher order handling of pain signals as well.
For many fly and worm biologists, the attraction of their models lies in the simplicity of the sensory systems, and the prospect of defining the exact functions of the channels and pathways that operate there. “It’s been difficult to figure out exactly the roles of these molecules, because in mammals, there are lots of different types of sensory neurons, and their functions overlap,” says William Schafer, who studies C. elegans at the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK. “That’s what happens when you have an animal with billions of neurons.”
By contrast, one look under a confocal microscope shows that the roster of sensory neurons in C. elegans and Drosophila is quite short. C. elegans makes the point especially dramatically: Two neurons called PVDs are the only nociceptors in the body of the worm (there are two more nociceptors in the head, and two in the nose).
The list in Drosophila is only slightly longer: Six nociceptors have been identified in each of the 14 body segments of the larva (although it’s possible that additional neurons also contribute to nociception). In fly larvae and in worms, the number of sensory neurons, and their locations, are precisely known. W. Daniel Tracey of Duke University in Durham, North Carolina predicts that because of this simplicity, “We will be able to understand, in exquisite detail, how the nociception pathways of Drosophila work in the next 10 to 15 years.” (See Getting to the bottom of some strange behavior for the story of how Tracey in 2003 identified nociceptive behavior in Drosophila and discovered the first fly nociception gene, painless.)
In C. elegans, touch sensation was one of the first behaviors to be genetically dissected, starting in the 1970s. Early work suggested that specific classes of neurons, the PVDs, mediate the animals’ response to harsh touch (Way and Chalfie, 1989). Recently, imaging studies from Millet Treinin at Hebrew University in Jerusalem and David Miller at Vanderbilt University in Nashville, Tennessee showed that PVD neurons have highly branched dendritic arbors that look like mammalian nociceptors. Further, profiling of PVD-enriched transcripts turned up many of the same genes found in mammalian nociceptors (Smith et al., 2010). Additional studies from these collaborators showed that the PVDs are involved in a complex set of escape behaviors elicited by handling the worms with a wire pick (Albeg et al., 2011).
With these results, the C. elegans PVD neurons join the class IV multidendritic neurons of Drosophila (Hwang et al., 2007) as model nociceptors. For both sets of neurons, their complex dendritic branching, the genes they express, and their behaviors all line up with those of mammalian nociceptors.
In the UK, Schafer and his team have started to watch the worm PVD neurons in action, using in vivo calcium imaging. In a study published in 2010, they found that either poking insults or cold temperatures cause a rise in intracellular calcium in PVDs, indicating that these neurons, like many mammalian nociceptors, are polymodal (Chatzigeorgiou et al., 2010). Using genetic analysis, they discovered that the PVDs detect noxious cold and touch using members of the two major cation channel superfamilies that have been implicated in nociceptive sensation: the transient receptor potential (TRP) and DEG/ENaC channels. Schafer and his collaborators found that the PVDs use different channels to respond to each type of stimulus: The cold reaction required TRPA1, while the harsh touch response required MEC-10 and DEGT-1, two DEG/ENaC proteins.
Schafer’s results followed closely on a report from Tracey and colleagues that Drosophila larvae use a DEG/ENaC protein, called Pickpocket, to respond to noxious touch (Zhong et al., 2010). With these data, both fly larvae and worms have wriggled their way into one of the trickiest questions in the nociception field: What channels sense mechanical pain? Interestingly, the fly TRPA1 homolog painless, which Tracey originally identified as a heat sensor, is also needed for registering harsh touch. Tracey and his group speculate that in flies the DEG/ENaC channel may be a direct mechanosensor, with the TRPA1 boosting the signal. Despite this progress in invertebrates, a receptor for mechanical pain in humans has yet to be definitively identified.
Worms also weigh in on another controversy: Is TRPA1 a cold sensor in humans? In mammals, TRPA1 is known to sense irritating electrophilic compounds including those in hot mustard and cinnamon. Common TRPA1 variants have been associated with both cold and heat pain sensitivity in humans. Whether we use TRPA1 to register cold pain is still uncertain, however (for a recent review of the conflicting evidence, see Dubin and Patapoutian, 2010).
In fact, how the function of any of the worm or fly channels might translate to mammals is not yet understood. One problem is that invertebrates and mammals frequently have different numbers of molecules in a given channel family, and it is often not clear which are the real homologs. Nonetheless, there is an expanding body of evidence that many aspects of pain sensation carry over from worms and flies to humans. Recently, for example, Paul Garrity’s group at Brandeis University in Waltham, Massachusetts found that the Drosophila TRPA1 channel detects electrophilic irritants using a structure that is highly conserved among vertebrates and invertebrates (Kang et al., 2010).
Beyond naming the key players in nociception, fly and worm biologists are also investigating the mechanisms by which those molecules and neurons operate. Schafer is keen to use worms to explore how nociceptors pick up on multiple types of stimuli. “People have generally thought that the way polymodality works is that there are TRP channels that can be activated by lots of different stimuli,” he says. However, his recent work suggests the opposite: In PVD neurons, entirely different channels respond to cold versus mechanical insults. Furthermore, different regions of the neurons may be stimuli-specific, too. TRPA1, required for cold sensation, is diffusely distributed near the cell body of PVD neurons, Schafer showed, whereas the DEG/ENaC proteins required for sensing harsh touch are located in clusters scattered all over the dendritic branches.
On the other hand, TRPA1 channels are not always cold specific, and their modality can vary from neuron to neuron. It was previously shown that some C. elegans sensory neurons use TRPA1 to respond to harsh touch (Kindt et al., 2007), and those neurons—even though they express TRPA1—are unable to react to cold.
All of that leaves Schafer wondering what determines the channels’ functions—whether structural features of the neurons, trafficking factors that shuttle channels to designated spots in a cell, or cofactors that activate or repress the sensation of different stimuli in different cells. C. elegans, with its very limited repertoire of nociceptor types, may be well positioned to help distinguish between these different possibilities.
More than skin deep
Nociception is just the beginning of pain. Actually feeling discomfort requires a signaling chain that involves modulation at the level of the spinal cord and processing in higher cortical areas. As a result, what flies and worms perceive with their simple nervous systems is likely a far cry from human pain. Surely, higher animal models—mice and rats, for example—are necessary to model what we humans experience. Right?
Not always, says Clifford Woolf, a researcher at Children’s Hospital, Boston, Massachusetts who has spent many years studying pain in rodent models. Most mouse or rat behavioral assays, Woolf says, simply check to see whether or not an animal reflexively responds to a painful stimulus, something that flies do just fine. On that score, he says, “Pain in Drosophila is as valid as many of the pain models we use in rodents.”
Woolf recently collaborated with Drosophila researcher Josef Penninger of the Austrian Academy of Sciences in Vienna to carry out a global screen for genes involved in heat nociception in flies (Neely et al., 2010). In the study, Penninger used the Vienna Drosophila RNAi library to individually knock down, in all neural tissues, most of the genes in the fly genome. The group then tested each of the ~16,000 resulting fly strains and identified 580 genes whose knockdown impaired flies’ ability to avoid noxious heat (46oC). Among those candidate thermal nociception genes, 80 had no previously known function. One of them, straightjacket, looked especially interesting because of its homology to the mammalian gene α2δ3, a subunit of voltage-gated calcium channels and a close homolog of another subunit (α2δ1) that is the target of the analgesics gabapentin and pregabalin.
Woolf and Penninger carried the result back to mice and found that, like the flies, α2δ3 knockout mice had a delayed response to applied heat. They also discovered that the gene plays a role in pain in humans. A common variant in the human homolog, CACNA2D3, associated with reduced sensitivity to heat in healthy adults. In people who had recent back surgery, the variant tracked with less chronic back pain.
Intriguingly, functional magnetic resonance imaging (fMRI) of the α2δ3-knockout mice indicated that this protein acts at a higher level than the nociceptors, in transmitting pain signals from the thalamus to other pain centers in the brain. Thus, Woolf, Penninger, and their colleagues uncovered a gene that acts beyond the initial events of sensation, and that is relevant to human pain—and they did it in flies.
New layers of complexity
Other data suggest that lower organisms may model additional complex features of human pain, including modulation of nociceptive responses. Sensitization by tissue injury or inflammation is useful just after an injury, but when the nervous system maintains a hypersensitive state longer than needed, chronic pain can ensue. Therefore, models of pain modulation could be especially helpful in understanding the most troubling aspects of human pain.
Michael Galko and his colleagues at the University of Texas MD Anderson Cancer Center in Houston have shown that tissue damage from UV light sensitizes nociception pathways in Drosophila larvae, and that this process uses a tumor necrosis factor (TNF)-like cytokine, similar to vertebrates (Babcock et al., 2009).
With modulatory systems like this, says Tracey, “Maybe our system is not as simplified as we think it is.”
In C. elegans, a long line of studies have dissected how the presence of food can create lasting changes in worms’ avoidance responses by enhancing the activity of a pair of nociceptors in the nose, called ASH neurons. These changes depend on neuromodulators including serotonin, dopamine, and a variety of neuropeptides. (See, for example, Ezcurra et al., 2011 and Esposito et al., 2010.)
There is even a model in worms for the role of common genetic variations in pain sensitivity. A group led by Miriam Goodman at Stanford University in California recently reported that natural isolates of C. elegans differ in their threshold for heat avoidance (Glauser et al., 2011). The researchers linked the difference to a polymorphism in the neuropeptide receptor gene npr-1, which appears to operate past the nociceptors, in the interneurons. The list of common human genetic variants that can tune our individual responses to painful stimuli is growing (see Pain Gene Resource for the latest); now, it appears, the same may be true in worms.