Background & Aims

A subset of Peripheral Sensory Neurons (PSNs), including nociceptors, display pseudo-stochastic deflections of the resting membrane potential. Although these Subthreshold Membrane Potential Instabilities (SMPIs) are exacerbated in painful conditions such as neuropathies and cancer (Velasco et al., 2022), their underlying molecular mechanisms and implication in pain signaling remain largely unknown. We hypothesized that SMPIs are generated by the stochastic activation of voltage-gated ion channels and that they mediate action potential (AP) firing in response to slow and tonic stimuli. We aimed at testing these hypotheses by: (1) characterizing the relationship between SMPIs and AP firing, (2) identifying the ion channels implicated in SMPIs generation, (3) testing the role of such channels in the encoding of slow and tonic thermal noxious stimulation, and (4) developing a computational model serving to understand the underlying mechanisms and pathophysiological relevance of SMPIs.

Methods

We used whole-cell patch-clamp to record primary cultured trigeminal PSNs isolated from wild type (WT) and NaV1.9 knockout mice (NaV1.9 KO). PSNs were stimulated in current-clamp mode with square current pulses or with slow cold and heat ramps reaching noxious temperatures. SMPIs and APs produced in response to these stimuli were detected and analyzed using custom-made semiautomated software scripts. In behavioral experiments, animals were stimulated tonically or with fast or slow heat or cooling ramps and scored on their nocifensive reactions, such as paw licking and jumping. Finally, deterministic and stochastic computational models of neuronal electrical behavior were built based on the Hodgkin-Huxley formalism to assess the contribution of voltage-gated Na+ and K+ channels (NaV and KV) to the production of SPMIs and their relation to AP firing.

Results

Eighty-two % of small diameter neurons displayed SMPIs in the shape of Subthreshold Membrane Potential Transients (SMPTs). Membrane depolarization evoked SMPTs regardless of the stimulus modality (thermal, chemical or electrical), and if large and fast enough, SMPTs triggered Action Potentials (APs). SMPTs were reduced by blocking TTX-resistant NaVs, as well as in PSNs isolated from NaV1.9 KO mice. The nocifensive responses to slow and tonic thermal stimuli were deficient in NaV1.9 KO animals, whereas responses to fast stimulation were preserved. The computational models revealed that the stochasticity of channel gating is necessary and sufficient for the generation of SMPIs and for AP firing in response to slow and tonic depolarization, such as those produced by noxious thermal stimuli.

Conclusions

SMPTs are generated by the stochastic activation of NaV1.9 channels and lead to the trigger of AP firing during tonic and slow thermal stimulation. These findings unveil SMPTs as crucial regulators of peripheral sensory processing, with possible implications in pathological conditions.

References

Velasco, E., Alvarez, J. L., Meseguer, V. M., Gallar, J., & Talavera, K. (2022). Membrane potential instabilities in sensory neurons: mechanisms and pathophysiological relevance. Pain, 163(1), 64–74.

Presenting Author

Enrique Velasco

Poster Authors

Enrique Velasco

PhD

Laboratory of Ion Channel Research, VIB-KULeuven

Lead Author

Michael Mazar MSc

The Hebrew University of Jerusalem, Israel

Lead Author

Alina Milici

VIB-KU Lueven

Lead Author

Ellaline Cami MSc

Laboratory of Ion Channel Research, KU Leuven, Leuven

Lead Author

Maria José Giner García

Instituto de Neurociencias de Alicante

Lead Author

Víctor Meseguer PhD

Instituto de Neurociencias, UMH-CSIC, San Juan de Alicante, Spain

Lead Author

Juana Gallar PhD

Instituto de Neurociencias, UMH-CSIC, San Juan de Alicante, Spain

Lead Author

Alexander Binshtok PhD

The Hebrew University of Jerusalem, Israel

Lead Author

Julio Álvarez PhD

Laboratory of Ion Channel Research, KU Leuven, Leuven

Lead Author

Karel Talavera PhD

Laboratory of Ion Channel Research, KU Leuven, Leuven

Lead Author

Topics

  • Mechanisms: Biological-Molecular and Cell Biology