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Quantifying Changes in Pain Sensitivity Using Reproducible Transcutaneous Optogenetic Stimulation in Behaving Mice
Yu-Feng Xie1, Christopher Dedek1,2,3, Steven A Prescott4,2,5,3,6
1Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
Abstract:
Optogenetics provides an unprecedented opportunity to delineate how different somatosensory afferents contribute to sensation, including pain. Afferents expressing channelrhodopsin-2 (ChR2) can be selectively activated by transcutaneous photostimuli applied to behaving mice. Despite targeting expression of ChR2 to specific cell types, imprecise photostimulation has hindered quantitative optogenetic-based behavioral testing. Here, we used a robot to reproducibly apply transcutaneous optogenetic stimuli to the hindpaw of mice while measuring nocifensive withdrawal. Different photostimulus waveforms (pulses and ramps) and response metrics (threshold and latency) were compared in mice of either sex expressing ChR2 in all afferents (Advillin-ChR2) or preferentially in nociceptors (NaV1.8-ChR2). Inflammation induced by complete Freund's adjuvant (CFA) caused withdrawal from ramped photostimuli to become faster relative to baseline and to vehicle-injected controls whereas pulsed photostimuli revealed a modest increase in threshold. Analgesia caused by NaV1.7 and 1.8 channel blockade was evident in both testing protocols. Overall, ramp-based testing was more effective and more efficient (i.e., required less time and total stimulation) than pulse-based testing. Electrophysiological measurements confirmed that CFA increases nociceptor excitability without affecting phototransduction, suggesting that withdrawal latency depends on the number of nociceptors activated rather than how strongly each nociceptor is activated. Consistent with changes described in nociceptor somata, the behavioral consequences of peripherally blocking different voltage-gated sodium (NaV) channels showed that nociceptor axons normally rely on NaV1.8 but upregulate NaV1.7 after CFA, with important clinical implications for drug efficacy. Collectively, these results demonstrate the utility of optogenetic pain testing when reproducibly delivered and strategically designed photostimuli are used.
Insights
This study introduces a robot-assisted optogenetic method for precise pain testing in mice. Ramp-based photostimulation is more effective than pulse-based methods for measuring inflammation and analgesia.
Area of Science:
- Neuroscience
- Pain Research
- Optogenetics
Background:
- Optogenetics enables selective activation of somatosensory afferents to study pain.
- Previous optogenetic pain studies were limited by imprecise photostimulation.
- Quantitative behavioral testing requires reproducible and precise stimulation methods.
Purpose of the Study:
- To develop and validate a robot-assisted transcutaneous optogenetic stimulation system for quantitative pain behavior testing in mice.
- To compare the efficacy of different photostimulus waveforms (pulses vs. ramps) and response metrics (threshold vs. latency) in assessing pain states.
- To investigate the role of specific sodium channels (NaV1.7, NaV1.8) in nociception and analgesia.
Main Methods:
- Utilized a robotic system for reproducible transcutaneous optogenetic stimulation of mouse hind paws.
- Employed channelrhodopsin-2 (ChR2) expressed in Advillin-ChR2 and NaV1.8-ChR2 mice.
- Compared withdrawal responses to pulsed and ramped photostimuli under baseline, inflammatory (CFA), and analgesic conditions.
- Performed electrophysiological recordings to correlate afferent excitability with behavioral outcomes.
Main Results:
- Ramped photostimuli effectively detected inflammation-induced hypersensitivity (faster withdrawal latency) and analgesia.
- Pulsed photostimuli showed a modest increase in withdrawal threshold during inflammation.
- Electrophysiology indicated that inflammation increases nociceptor excitability, affecting withdrawal latency.
- Nav1.8 channel blockade reduced withdrawal, while NaV1.7 upregulation was observed after CFA, impacting analgesia.
Conclusions:
- Robot-assisted optogenetic stimulation with strategically designed ramped photostimuli provides a quantitative and efficient method for pain testing.
- Ramp-based latency measurements are superior to pulse-based threshold measurements for detecting inflammatory pain and analgesia.
- Findings on sodium channel involvement in nociception have significant clinical implications for developing targeted analgesics.
