Not Just for Pain, Opioid Receptors Found to Modulate Touch Neurons, Too
Opioid receptors are among the most intensely studied molecules in pain and the linchpin of opiate drug analgesia. Now, researchers reveal a striking new role for the δ (delta)-type opioid receptor (DOR) in mechanosensing neurons that normally signal touch, not pain. Critically, these DOR-expressing cutaneous mechanosensors are thought to give rise to the mechanical hypersensitivity and allodynia that accompany neuropathic pain, and the results suggest the DOR could provide a switch to quiet the neurons’ activity.
The work from Grégory Scherrer at Stanford University, Palo Alto, US; Allan Basbaum at the University of California, San Francisco, US; and first author Rita Bardoni at the University of Modena and Reggio Emilia, Italy, was published February 27 in Neuron.
Frank Porreca, University of Arizona, Tucson, US, who was not involved in the work, called it “a tremendous scientific study,” and said the authors had done “an incredible job” of characterizing the DOR-expressing sensory neurons.
To detect cells that make DOR, the researchers used a green fluorescent protein (GFP) reporter mouse, in which they had inserted the GFP sequence into the DOR gene to produce a DOR-GFP fusion protein in place of DOR (Scherrer et al., 2006). “That allowed expression of DOR-GFP just in the cells that normally express the receptor,” Scherrer explained.
To confirm that the fluorescent signal accurately reported the DOR expression pattern, the researchers used an ultra-sensitive technique to detect DOR messenger RNA in wild-type mice, which revealed a similar pattern to the DOR-GFP expression. They also compared the DOR-GFP expression pattern in the central nervous system to binding with two radio-labeled DOR-specific ligands, which also matched.
Next, the team carefully characterized the DOR-expressing sensory neurons by using a variety of markers. Two-thirds of dorsal root ganglia (DRG) neurons that expressed DOR also expressed neurofilament 200 (NF200), a marker of myelinated axons. About one-third of myelinated DOR neurons expressed the nerve growth factor receptor TrkA and the neuropeptide calcitonin gene-related peptide (CGRP), but not the heat-sensitive transient receptor potential vanilloid type 1 (TRPV1) channel, all hallmarks of mechanically sensitive myelinated nociceptors.
The rest of the myelinated DOR neurons expressed the neurotrophic factor receptors TrkC and/or Ret, identifying these neurons as cutaneous low-threshold mechanoreceptors (LTMRs). To confirm this identity, the researchers examined DOR-GFP-labeled nerve endings in the skin and found them associated with cutaneous touch-sensing structures including hair follicles, Merkel cells, and Meissner corpuscles.
Porreca said that while the finding of DORs in these peripheral end-organs is absolutely credible, mysteries remain about their physiological role there. “What is the ligand that reacts with the receptor, and when is that agonist normally released?”
Some unmyelinated neurons also expressed DOR; one-third of DOR+ neurons were nonpeptidergic C-fiber nociceptors that bound the isolectin B4 (IB4) and expressed the G protein-coupled receptor MrgprD, indicating they were mechanically sensitive cutaneous nociceptors.
MOR discovery
The team next examined expression of the μ (mu) opioid receptor (MOR) and found that MOR and DOR expression was mostly segregated in separate populations of sensory neurons, with only about 5 percent of DRG neurons containing both receptors. Most of the neurons co-expressing MOR and DOR were myelinated A-fibers; small C-fiber nociceptors with both receptors were extremely rare.
These results build on previous work from the same researchers (Scherrer et al., 2009) and contrast with years of studies indicating that many neurons in the DRG—including small-diameter nociceptors—express both MOR and DOR (see Wang and Wessendorf, 2001, and Wang et al., 2010). That work used a DOR antibody that, in retrospect, Scherrer believes is not specific to the receptor.
The controversy stems from a disagreement over technique more than science, Scherrer said. “It is really about what type of reagent will more faithfully represent the receptor,” he said. Whether the new paper will settle the question remains to be seen, but Scherrer says he is “confident that what we have found is correct, given all the approaches we’ve used, including in wild-type mice.”
The importance of which cells do—or do not—co-express MOR and DOR lies in the possibility that the two receptor subtypes could dimerize, forming hybrid receptors. These potential dimers might in turn be targeted with drugs specific to the hybrid dimers, avoiding potential side effects at MORs (see Fujita et al., 2014). Scherrer pointed out that the new finding is not necessarily bad news for the prospects of dimer targets. “With so many sensory cell subtypes, that 5 percent [co-expressing MOR and DOR] could play very important roles. In fact, the low number could provide an advantage” to dimer-specific drugs, like increased specificity, he said.
Activation of MORs on the terminals of pain-sensing neurons in the spinal cord dampens glutamatergic neurotransmission by inhibiting voltage-gated calcium channels (VGCCs) on the presynaptic side. To ask if DOR activation would work the same way to mute neurotransmission from mechanosensors to spinal cord neurons, the researchers made spinal cord slices and measured excitatory currents in spinal neurons in response to electrical stimulation of the attached DRG mechanosensing projections. They found that a DOR-specific agonist reduced glutamate release from the afferents to the spinal neurons. In addition, they showed that voltage-evoked currents through VGCCs in isolated DRG neurons were 38 percent smaller in the presence of the DOR agonist.
Opening a DOR to new therapeutics?
The finding of DOR in myelinated mechanoreceptors—the neurons that likely mediate allodynia—provides a potential window to affect aberrant signaling in neuropathic pain. However, Porreca cautioned, silencing these neurons carries the risk of blocking the normal sense of touch, which could result in injury or other problems. One way to alleviate allodynia without losing touch sensation—or causing other adverse events in the central nervous system—might be to deliver a DOR agonist locally to the skin.
The presence of DORs on peripheral nerve endings holds out that exciting possibility. Could a DOR-targeted agent affect neuropathic signaling with topical application? “Yes, it absolutely might,” said Scherrer. “That would be the beauty of this finding,” if a patch or ointment could quell mechanical allodynia.
Porreca agreed, with a caveat that any treatment would need to maintain touch sensation where it is most needed, such as in the hands and feet. “One possibility to imagine might be [restricted] local application to areas of the body not essential for touch, like on the back, using, for example, a patch.”
Scherrer raised another caveat to consider before targeting DOR in the skin. “We need to know what the receptor is doing in the periphery first,” he said, which may or may not resemble what they observed in the spinal cord experiments. That question is currently under investigation, Scherrer added.
Another pressing question is whether the anatomical findings in this strain of mice translate to humans.
The idea of targeting DORs for neuropathic pain is not a new concept, Scherrer said, but the new findings could go a long way toward providing a rationale for receptor-specific drug use. Scherrer stressed the importance of appreciating the heterogeneity of sensory neurons and how different drugs might affect different painful ailments. Now, it seems that tactile allodynia from the skin, at least, could one day be targeted with DOR-specific drugs.
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California, US.
Image: Distribution of DOR-GFP-expressing axons around a guard hair follicle in mouse skin. Reprinted from Neuron, Bardoni et al., © 2014, with permission from Elsevier.
Article Contributors:
