Optogenetics—the use of light-sensitive ion channels to regulate neuron activity—is a powerful method that has only recently been used to probe the transmission of pain signals. But thus far, most optogenetic studies have required the production of transgenic mice—a process that is costly and takes a long time. A new technique from Scott Delp and colleagues at Stanford University, US, removes this requirement and offers additional benefits: It can either activate or inhibit pain in freely moving mice. Delp’s research team described the technique and its use in a mouse model of chronic pain in a study published online on February 16 in Nature Biotechnology.
In the study, Delp and colleagues used an adenovirus to introduce light-sensitive channels into nociceptors. After injecting adeno-associated virus 6 (AAV6) engineered to express the blue light-sensitive cation channel channelrhodopsin-2 (ChR2) into one side of the sciatic nerve in wild-type mice, they found ChR2 expression in unmyelinated nociceptors that project to lamina I/IIo of the spinal cord. Shining a blue light on the mouse’s paw activated ChR2 and caused the animal to flinch and lick its paws, indicating a pain response. Lower intensity blue light did not induce pain behavior, but made the mice sensitive to normally inoffensive levels of thermal and mechanical stimulation. Additionally, the mice developed an aversion to low levels of blue light and, when given a choice, would avoid entering cage areas illuminated with blue light.
Next, the researchers asked if they could use optogenetics to make mice less sensitive to touch and heat. To test this, they used AAV6 encoding the yellow light-sensitive chloride pump halorhodopsin (NpHR)—an opsin that turns off neuronal activity. When exposed to yellow light, mice injected with this virus became less sensitive to both thermal and mechanical stimulation. According to graduate student Shrivats Iyer, one of the lead authors of the study, “to our knowledge, this is one of the first demonstrations of optogenetic inhibition of nociceptor activity that has been published.”
The group took this finding one step further and used the NpHR virus in a mouse model of chronic pain. Mice with a chronic constriction injury of the sciatic nerve (CCI) develop hypersensitivity to mechanical and thermal stimulation, but CCI mice injected with the NpHR virus did not show this hypersensitivity when a yellow laser was used to activate the opsin. According to Philippe Séguéla, McGill University, Montreal, Canada, who was not part of the study, the “effects reported are striking.”
This technique shares some similarities with previously published work—for example, a study by Séguéla’s group used optogenetics to activate pain behaviors in freely moving transgenic mice (see PRF related news story). But the new technique’s ability to decrease a mouse’s sensitivity to thermal and mechanical stimuli makes it unique—as does its viral approach. Iyer said that the virus’ quick action is a big advantage: “You can give a mouse an injection and within two weeks achieve opsin expression.” This compares well to transgenic optogenetic mice, which take several months or even years to make.
Séguéla agreed that speed is a major advantage of the technique, and also noted that “the approach is likely not restricted to mice” and could possibly “be applied to rats and non-human primates.”
According to graduate student Kate Montgomery, who was also a lead author of the study, the viral approach offers other advantages: “We wanted to develop a system that was modular so it could be changed by different neuroscientists who want to target different cell populations or want to use different opsins.” She said this is particularly important, as new opsins are constantly being developed.
Video demonstration of transdermal optogenetic activation of nociceptors. A mouse with bilateral ChR2 expression is placed in a clear cylinder on a transparent glass plate and allowed to habituate to its environment. When blue light (473 nm, 1 mW/mm2) is shone on the plantar surface of the mouse's skin, the animal immediately withdraws its paw, engages in prolonged licking behavior, and shakes its paw. However, when yellow light (593 nm, 1 mW/mm2) is shone on the skin, there is no observable change in behavior, and the mouse moves around normally. Also, when blue light (473 nm, 1 mW/mm2) is shone on the paw of a bilaterally injected YFP+ mouse, no change in behavior is seen, and the mouse moves around normally. Credit: Iyer and Montgomery. Caption provided by study authors.
Both Iyer and Montgomery see several uses for the technique. Montgomery said, “One of the first ways we see this technique being used is chronically stimulating different neuron populations and seeing how chronic stimulation (or inhibition) of these fibers changes the development of different pain models."
The new technique does have some limitations. One drawback is that gene expression of the opsins decreases over four to five weeks, likely due to properties of the AAV6 virus. Further engineering of AAV viruses might make this less of an issue in the future. Séguéla noted some other limitations: The technique is invasive because it requires surgery and nerve injection, is spatially restricted, and does not allow discrimination between the different nociceptor subtypes.
To get around that last limitation, the technique could be used in conjunction with already available transgenic mice, Iyer explained. In the current study, the opsins were expressed under the control of a pan-neuronal promoter (human synapsin-1). This meant that any neuron that was transduced by the virus—predominantly small-diameter nociceptors—expressed the opsin. Hypothetically, a researcher could use a Cre-inducible virus in a transgenic mouse that expresses Cre recombinase under a promoter with a more restricted range. This would allow for light activation of subsets of nociceptors that are both infected by the virus and express Cre recombinase.
When asked about future therapeutic use of this technique in humans, Montgomery stressed, “We see this primarily as a scientific tool, but to use optogenetics as a therapy in the future, gene therapy will be required, and there are a lot of hurdles and obstacles to overcome with that.” One hurdle is that opsins have never been introduced into humans. Another is the ability of light to activate the opsins. “If you want to control neurons that do not project to the skin or some superficial area, you would need a light-emitting implant,” said Montgomery. The group recently published a studyin which they used such implants in rats (Towne et al., 2013), but this was a challenging procedure that would be difficult to do in people.
However, Iyer noted that new opsins are being generated that are more sensitive to red light, which has a greater penetrance in tissues.
According to Séguéla, many clinical issues will have to be sorted out before viral-based optogenetics could be used in patients, but he added that it “is clearly an interesting avenue for the treatment of chronic pain.”
And the Delp research group is eager to share their new technology with other investigators who want to improve on the technology. Iyer said, "We're interested in helping to get this technique to be used in as many different labs as are interested in using it.”
Summer Allen, PhD, is a neuroscientist and freelance writer in Rhode Island, US.
Image: © Wirachapong Chanthakan | Dreamstime.com
Comments on Related Content
Mark Zylka, University of North Carolina at Chapel Hill
Just prior to setting up my
Just prior to setting up my lab in 2006, Ed Boyden and Karl Deisseroth’s seminal paper on channelrhodopsin-2 (ChR2) had been published (Boyden et al., 2005). These investigators showed how ChR2, a blue light-activated ion channel, could be used to depolarize neurons with millisecond precision. This new “optogenetic” technology was clearly amazing.
In addition, it was obvious how this new technology could be used to address fundamental questions in the pain field. For example, what types of behaviors can be driven by activating specific classes of nociceptive neurons? Could light be used to sensitize nociceptors and create an optogenetic model of chronic pain?
Being an ambitious and perhaps overeager new PI, I felt such an experiment was a no-brainer and would be easy. Thus, we knocked ChR2 into the Mrgprd class of nonpeptidergic nociceptive neurons, with the lofty goal of making a blue light-sensing mouse. We made the mouse, no problem. We could evoke ChR2 photocurrents and action potentials in dorsal root ganglia neurons (DRG) and photocurrents in spinal cord slices (see Campagnola et al., 2008; Wang and Zylka, 2009). But no matter what we tried, we couldn’t get the mouse to do anything when optically prodded with blue light.
Clearly, getting ChR2 to work in vivo, in the somatosensory system, wasn’t as easy as I had hoped.
Fortunately, several labs have now gotten ChR2 to work in vivo in the somatosensory system, and to work extremely well (see Ji et al., 2012; Daou et al., 2013; and this paper by Iyer and colleagues). The key seems to be high-level expression of ChR2.
These discoveries represent a major breakthrough for the pain field. By targeting ChR2 to nociceptors, these labs have shown that it is possible to drive nociceptive responses with high-intensity blue light, sensitize nociceptive responses, and even elicit aversion. These discoveries open up new avenues of research, such as to develop screens for chemicals that quiet light-evoked activation of nociceptors. High-throughput screens such as this could identify new analgesic drugs.
Intriguingly, Iyer and colleagues also found that a yellow light-sensitive chloride pump, called halorhodopsin, could be used to inhibit nociceptive responses. With further optimization, it is not hard to imagine how this technology could be used to turn pain off with the flick of a light.
Iyer and colleagues' paper is well written and a quick read. If you don’t have time to read the paper, at least check out Supplementary Video 1. The video is entertaining and nicely demonstrates the power of this new technology.
Patrick Stemkowski, University of Calgary
Gerald W Zamponi co-authored
Gerald W Zamponi co-authored this comment.
Illuminating pain
Optogenetic techniques have opened a new window toward understanding brain circuits in vivo. While optogenetic interrogation of central nervous system function typically involves surgical implantation of optic fibers, cutaneous peripheral neurons can be accessed directly via illumination of the skin. Hence, it is possible to stimulate or inhibit sensory afferents directly with light. A previous study from the Séguéla laboratory (Daou et al., 2013) has exploited this opportunity to activate peripheral nociceptors by breeding a mouse line that expresses channelrhodopsin-2 in Nav1.8-positive neurons.
In an exciting new paper, Iyer and colleagues have expanded on the flexibility and adaptability of optogenetics that are currently being applied to the field of pain research by moving away from a purely transgenic approach. The authors were able to show functional expression of opsins that appeared at least two weeks following an intrasciatic injection of adeno-associated virus that encoded either excitatory or inhibitory opsins in wild-type mice. This expression was reversible (lasting approximately two weeks) and was biased toward a mixed population of nociceptors. The behavioral responses to transdermal illumination are spectral specific, vary as a function of light intensity, are bi-directionally controlled, and can be assessed with reflexive as well as increasingly more sought-after operant measures. Furthermore, transdermal illumination modulates behavioral responses to more natural stimuli that are not limited to acute nociception, but can also be applied to animal models of neuropathic pain and, perhaps, inflammatory pain. Taken together, the authors’ work adds to the emerging use of optogenetics that will undoubtedly make an impact on basic pain research and perhaps as a new animal model for drug and opsin discovery purposes. It may even open the door as a clinical application in treatment of chronic pain. To move in this direction, it will be important to increase specificity of opsin expression by choice of gene promoter and ensure stability of opsin expression over longer periods of time.
Ru-Rong Ji, Duke University Medical Center
This is a very interesting
This is a very interesting paper, which has demonstrated the technical feasibility of excitation and inhibition of pain in freely moving mice via a virally mediated optogenetic approach. With further improvement, this technique may allow for rapid in vivo testing of the behavioral effects of novel opsin variants. The analgesic effect is very impressive and also surprising, given the fact that only a small population of nociceptors is activated/inhibited by this optogenetic approach. Although this is a truly novel way to activate nociceptors, it is unclear, at this moment, whether this approach will improve the understanding of pain mechanisms and the treatment of chronic pain.
Feng Tao, Texas A&M University Baylor College of Dentistry
This amazing work
This amazing work demonstrates that optogenetic manipulation is a very useful approach in pain research. By activating light-sensitive proteins expressed in sensory neurons, specific pain signaling is regulated (either excitation or inhibition). Meanwhile, pain behaviors including spontaneous pain and evoked pain can be measured during optogenetic stimulation. In the future, through combining this approach with Cre-lox technology, we would be able to specifically modulate different types of neurons in pain signaling.