15: Clock genes control nociceptor excitability and pain in a sex-and time-dependent manner

Topic: Basic Science
Subtopic: Non-specific to any diagnosis (including studies in healthy animals), Neuroimmunology, Electrophysiology, Behavioral studies

Background and Aims: Nociception is critical for pain perception and survival and begins with the activation of nociceptors, specialized sensory neurons located in the dorsal root ganglia (DRGs)[1,2]. Sex plays a significant role in modulating pain perception, with important differences characterized in peripheral and central immune cells and glia[3]. However, little is known about the contribution of circadian (24-hour) rhythms to this process, governed by core clock genes within individual peripheral and central cells[4,5], We therefore sought to determine whether 1) nociception and pain are rhythmic in mice, 2) an endogenous circadian machinery exists in nociceptors, 3) these clocks might contribute to nociception and pain, and 4) there may be an interaction between circadian rhythms and sex differences. Understanding the contribution of circadian clocks at the cellular level presents a unique opportunity to decipher mechanisms regulating pain across both healthy and diseased states.

Methods: Our work used a circadian and diurnal approach to better understand the impact of time of day on pain and nociception in wildtype (C57BL/6J) mice and transgenic mice lacking the core clock gene in Nav1.8+ nociceptors. We began by deeply phenotyping the expression of core clock genes in the intact dorsal root ganglia of male and female mice across 4 times of day (ZT2, 8, 14, and 20, where ZT0 is lights-on and ZT12 is lights-off in a 12:12 facility). Ex vivo whole-cell recordings of intact DRGs were carried out at ZT2 and ZT14 to measure changes in nociceptor activity with behavioural characterization of stimulus response across the day. RNA-sequencing identified circadian expression of ion channels, which were then targeted using the novel ClC-2 antagonist AK-42[6] for in vitro and in vivo studies.

Results: We identified a rhythm to thermal sensitivity in naive male, but not female, mice. The evoked firing rate in nociceptors ex-vivo best reflects heat sensitivity in both sexes: there is no fluctuation in heat pain and nociceptor activity in females, while diurnal variation is seen for both in males. Disruption of the core clock gene Bmal1
in Nav1.8-expressing neurons not only diminished nociceptor activity but also abolished the nighttime reduction in heat sensitivity observed in males, highlighting a pivotal role for the molecular clock in regulating nociception. Transcriptomic and immunohistochemistry analyses, voltage-clamp recordings, and pharmacological experiments identified the voltage-gated chloride channel ClC-2 as a key mediator for the observed fluctuations in male nociceptor excitability and heat sensitivity.

Conclusions: Recent studies in healthy human males found that thermal nociception is rhythmic, with decreased response to stimulus during the active phase[7]; whether such rhythms exist in females is unknown[8]. We identified the same rhythmic pattern in the nocturnal mouse, but in males only. This effect was mediated by circadian expression of the voltage-gated chloride channel ClC-2. Abolishing the core clock machinery in nociceptors was able to eliminate these responses. This work opens a new avenue of study for our understanding of time-of-day effects on pain and nociception, and the potential for sexually-dimorphic responses. In particular, there is a potential for chronobiology-inspired strategies in pain management, that have until now been overlooked.

References: 1. Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell. 2009;139:267–284
2. Dubin AE, Patapoutian A. Nociceptors: the sensors of the pain pathway. J Clin Invest. 2010;120:3760–3772
3. Mogil JS. Qualitative sex differences in pain processing: emerging evidence of a biased literature. Nature Reviews Neuroscience. 2020;21:353–365.
4. Brécier A, Li VW, Smith CS, Halievski K, Ghasemlou N. Circadian rhythms and glial cells of the central nervous system. Biol Rev Camb Philos Soc. 2023;98:520–539.
5. Segal JP, Tresidder KA, Bhatt C, Gilron I, Ghasemlou N. Circadian control of pain and neuroinflammation. J Neurosci Res. 2018;96:1002–1020.
6. Koster AK, Wood CA, Thomas-Tran R, et al. Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel. Proceedings of the National Academy of Sciences. 2020;117:32711–32720.
7. Daguet I, Raverot V, Bouhassira D, Gronfier C. Circadian rhythmicity of pain sensitivity in humans. Brain. 2022;145:3225–3235.
8. Bumgarner JR, Walker WH II, Nelson RJ. Circadian rhythms and pain. Neuroscience & Biobehavioral Reviews. 2021;129:296–306.

Presenting Author: Aurelie Brecier
Submitting Author: Nader Ghasemlou

Senior Author: Nader Ghasemlou

IASP 2026 Global Year theme of Neuropathic Pain: No

• Aurelie Brécier: PhD - Queen's University
• Courtney Bannerman: PhD - Queen's University
• Yu-Feng Xie: PhD - The Hospital for Sick Children
• Christopher Dedek: BSc - The Hospital for Sick Children
• Amanda Zacharias: PhD - Queen's University
• Ciara O'Connor: BSc - Queen's University
• Aitana Rickert Llàcer: MSc - Queen's University; Krembil Brain Institute
• Steven Miller: BSc - Stanford University
• Vina Li: BSc - Queen's University
• Christina Meier: BSc - Queen's University
• Laurel Ballantyne: BSc - Queen's University
• Justin Du Bois: PhD - Stanford University
• Qingling Duan: PhD - Queen's University
• Steven Prescott: PhD - The Hospital for Sick Children; University of Calgary
• Nader Ghasemlou: PhD - Queen's University; Krembil Brain Institute