New Optogenetic System Shines a Light on Pain-Sensing Neurons

To study pain in animals, researchers can injure a nerve or present a noxious stimulus to a rat or a mouse, and see how the creature responds. But animal models of pain are rather blunt tools that not only activate nociceptors but other cells, too, making it difficult to pinpoint the consequences of activating pain-sensing neurons per se. To overcome that dilemma, Philippe Séguéla and colleagues at McGill University in Montreal, Canada, have now devised an optogenetic system in mice in which primary afferent nociceptors are activated in freely moving animals by blue light. The light produces accompanying pain behaviors, but without affecting neighboring cells. The technique will enable scientists to probe the pain system noninvasively and with a degree of precision not possible with current methods.

The new work is “an elegant proof of principle,” according to Robert Gereau, a neuroscientist at Washington University, St. Louis, US, who was not involved with the new research. The investigators present experiments, he said, that provide “a very nice example of how this [optogenetic system] can be applied to studying pain in many ways, from cellular physiology to behavioral outcomes.”

Séguéla and collaborators presented the work in October at the 2012 annual meeting of the Society for Neuroscience in New Orleans, US.

Virtually all in-vivo investigations of pain in animals, regardless of the stimulus used to provoke nociceptive responses, cause tissue damage and produce “a soup of mediators,” Séguéla said. “You don’t know which cells are activated,” he added. For instance, glia, keratinocytes or immune cells can respond along with sensory neurons. Another drawback of invasive animal models is that injected drugs or other stimuli may take effect over minutes or hours. Optogenetics address both issues, allowing specific and instantaneous activation of cells.

In the new work, Séguéla and colleagues crossed two previously generated transgenic mouse lines to create mice in which primary nociceptors express the light-sensitive ion channel channelrhodopsin-2 (ChR2). Specifically, the investigators crossed mice expressing Cre recombinase under the control of the promoter for Nav1.8, a voltage-gated sodium channel (Stirling et al., 2005), with another transgenic line carrying a Cre-dependent conditional allele of ChR2 fused to yellow fluorescent protein (YFP) (Madisen et al., 2012). Once expression of Nav1.8 switched on during development—as it does in all primary nociceptors—ChR2 became irreversibly turned on as well in the newly created mice.

External light can elicit currents and activate nociceptors only when there are sufficient levels of ChR2 at the periphery; ChR2 must be trafficked from cell bodies in dorsal root ganglia (DRG) down long axonal processes. Considering the channel’s tiny, subpicosiemen conductance, “You need a lot of protein at the membrane of peripheral sensory terminals,” said Séguéla, to get a physiological response. The researchers found that, indeed, the mice expressed ChR2 in the right place: Fluorescent nerve fibers glowed brightly in the dermis and epidermis of the glabrous skin, as well as in the central projections coming from the same cells to the dorsal horn of the spinal cord. Meanwhile, electrophysiological experiments showed that blue light of around 470 nm, a wavelength at which ChR2 opens and passes excitatory ion currents, evoked “large photocurrents” in cultured neurons, according to Ihab Daou, a graduate student who presented the work. Currents were sufficient to drive action potential firing at a frequency up to 10 Hz.

Light not only activated nociceptors—it elicited characteristic pain behaviors in the mice, too. Although normal room light includes the wavelengths that activate ChR2, the investigators found that strong flashes of blue or white light were required to provoke the behaviors, which included paw withdrawal, licking, jumping, and vocalization. The behaviors displayed a dose dependence with light intensity, as did the light-evoked currents, Gereau noted. Also, while the researchers had worried initially that mice might have some intrinsic aversion to the flashes of light, Séguéla said that control mice were “utterly unperturbed” by such stimulation.

The researchers next exposed mice to levels of blue light that did not evoke pain behaviors, and found that later, those same mice developed thermal and mechanical hyperalgesia. The results suggested that prolonged, low-level nociceptor activation was sufficient to incite these phenomena. Since hyperalgesia is thought to result primarily from central sensitization subsequent to nociceptor activation, the finding makes sense.

The mice also exhibited pain-related emotional learning. In a place aversion test, mice were first habituated to an “orange zone” of a Plexiglas box, where they were bathed in harmless orange light. Next, the team removed a barrier that had confined the mice to the orange zone, allowing the animals access to a “blue zone,” where they faced pain-evoking light. In later trials, even when the blue light remained off, mice chose to stay confined to the safe orange zone.

Further experiments indicated that centrally acting analgesic drugs blunted the mice’s behavioral responses to light. Pregabalin, which regulates voltage-gated calcium channels, or morphine reduced some of the acute pain behaviors provoked by light; thermal and mechanical hyperalgesia were also sensitive to morphine. Allan Basbaum, a pain physiologist at the University of California, San Francisco, US, noted that the dose of morphine required to blunt the effects was very high, making it a bit difficult to relate the effect of morphine on natural versus light-evoked pain behaviors. Séguéla suggested this low drug sensitivity might in fact be indicative of some “new modality of pain” evoked by blue light’s simultaneous activation of several nociceptive subtypes.

Basbaum, who was not involved with the current study, called the work a “lovely idea” that was well executed. He said he would like to see future genetic tweaks to the system that would enable investigation of individual nociceptor subtypes. “That specificity is really what we’re after,” he said.

The current work isn’t the first time photosensitive cells have been employed in the service of sensory physiology. For instance, one group engineered a population of polymodal sensory nociceptors to make ChR2 based on their expression of the Mas-related G protein-coupled receptor D (Mrgprd) and then surveyed the neurons’ connectivity in the mouse spinal cord (Wang and Zylka, 2009). Another study used the promoter for Thy-1.2, an antigenic marker, to drive ChR2 expression in a subset of rat DRG neurons, which the investigators subsequently identified as pressure-sensing cells (Ji et al., 2012). Recent work has also used optogenetics to investigate nociceptive networks in the nematode C. elegans (Husson et al., 2012) and in larval Drosophila (Honjo et al., 2012). And a non-genetic line of experiments utilized a photo-switchable ion channel inhibitor that allows optical silencing of nociceptors, leading to light-induced analgesia in rats (see PRF news story on Mourot et al., 2012). Other potential directions for optogenetic models include exploration of nociception during development and imaging studies.

It is likely that the new system won’t be the last optogenetic model aimed at pain; Gereau and others are already developing their own systems based on similar strategies.

Stephani Sutherland, PhD, is a freelance neuroscience writer based in Southern California.