Let the light ease your pain: Epidural optogenetics provides analgesia

Using optogenetics as a tool to evaluate pain processing

Optogenetics has emerged as a powerful tool to modulate neuronal activity in neurons expressing genetically modified light-sensitive proteins using light. Recent studies of nociception, or response to harmful stimuli, used this technique to elucidate the sensory circuits involved in pain processing, focusing on the integration of sensory signals that takes place in the spinal cord. Noxious stimuli signals are transmitted via sensory afferents from the periphery to the spinal cord for integration, and then via ascending axons to the brain stem or the thalamocoritcal system for further processing. Ultimately, these signals result in the animal (or subject) experiencing pain. Pathological conditions, such as chronic pain, often result from abnormal sensory input or processing of neuronal activity in the dorsal horn of the spinal cord.

Recently, a subcutaneous light-emitting diode (LED) system was utilized to activate channelrhodopsin-2 (ChR2)-expressing sensory afferents in the periphery and spinal cord, which allowed for the manipulation of sensory signal inputs into the spinal cord in awake mice. However, in order to stimulate neurons expressing optogenetic proteins within the sensory processing pathway of the dorsal horn, a sufficient light intensity is necessary that may not be achieved through subcutaneous transmission. To address this limitation, a group led by Dr. Yves De Koninck of Université Laval, Quebec, developed an epidural optic fiber implant that could work with LED or laser-based optogenetic systems and that is capable of delivering different light wavelengths to either activate or inhibit specific dorsal horn neurons in the spinal cord that are important in modulating pain processing.

Epidural optic fiber implantation and generation of optogenetic mouse models

The epidural optic fiber was constructed out of multimode plastic fiber and a diffusive tip allowing for multidirectional diffusion of light. The fiber implant was fed through the atlanto-occipital membrane at the base of the neck and down the spinal column, with the diffusive tip terminating at lumbar spinal segments L4-L6. Placement of the fiber was confirmed by hooking up the fiber with a light source and observing illumination in the back at the lumbar spinal level.  In general, implanted mice tolerated the implant well and showed no obvious motor impairments or pain-related behaviors.

To highlight the versatility of the epidural optic fiber, three optogenetic mouse models were created:

1. Mice expressing ChR2 in Na_v1.8⁺ nociceptive afferents (Na_v1.8⁺-ChR2) were created by crossing Na_v1.8⁺-Cre mice with B6;129-Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/EYFP)Hze}/J (Stock# 012569). In these mice, Cre expression is restricted to dorsal root ganglion (DRG).
2. Mice expressing the inhibitory optogenetic protein, archaerhodopsin (ArchT) in Na_v1.8⁺ nociceptive afferents were generated by introducing into Na_v1.8⁺-Cre pups via intraperitoneal injection an AAV8 virus carrying a Cre-activatable (lox-stop-lox) ArchT expression cassette.
3. Finally, mice expressing ArchT in spinal cord inhibitory neurons were generated by intraspinal injection of the AAV8-lox-stop-lox ArchT virus into STOCK Gad2^tm2(cre)Zjh/J (Stock# 010802) mice.

Light delivery causes hyperalgesia and results in analgesia

The first challenge was to see if the spinal delivery of blue light (488nm) would activate sensory afferents in Na_v1.8⁺-ChR2 mice and produce nocifensive responses, such as twitching, fleeing or vocalization. Not only did the acute stimulation of spinal neurons elicit nocifensive behavior, but also prolonged delivery of blue light to the hindpaw in anesthetized mice induced hyperalgesia; that is, an increased sensitivity to pain. These experiments indicate that peripheral stimulation with blue light was sufficient to activate the ChR2-expressing sensory afferents involved in pain processing, thus validating that the epidural fiber optic implants can stimulate optogentic proteins in the spinal cord and modulate pain responses. The researchers went on to show that prolonged spinal blue light delivery produced similar hyperalgesia in awake mice as in anesthetized mice, confirming that the model can be used to manipulate chronic pain responses in awake and freely moving mice.

Another goal of the novel epidural optic fiber design was to establish that enough light could be delivered to activate less sensitive ArchT proteins delivered to GABAergic inhibitory interneurons of the spinal cord in the Gad2-cre mice via AAV8-lox-stop-lox ArchT virus. Stimulation with orange light (592nm) silenced spinal cord GABAergic inhibitory interneurons resulting in hyperalgesia.  As expected, delivering orange light had no effect on Na_v1.8⁺-ChR2 mice, supporting the specificity of the mouse models and the optic fiber design.

Finally, Na_v1.8⁺-ArchT mice were utilized to investigate whether inhibiting Na_v1.8⁺–expressing sensory afferents would provide an analgesic response. Acute spinal delivery of orange light resulted in prolonged withdrawal times to painful stimuli, indicating that ArchT proteins were activated and that analgesia could be induced using optogenetics in awake and freely moving mice.

This novel epidural optic fiber design compatible with optogenetic transmission systems should serve as a powerful tool to examine sensory afferents involved in pain processing. The design provides an all-in-one system to either activate or inhibit key elements in the spinal cord that are involved in acute and chronic pain.  The epidural implant also may support the development of novel  therapeutic  treatments for treating pain in the clinic.