Background & Aims
There are clear species differences between rodents and humans with regard to the primary nociceptor classes, which hampers direct translation of experimental approaches. In the field of pain research, the pig has been established as an alternative, and it has been shown that the functional nociceptor classes in pigs are much more similar to those in humans. Functional classification is largely based on single fiber recordings from pig skin and there are still many unanswered questions regarding the expression (dynamics) of important molecules such as ion channels and receptors in the periphery, cell bodies and spinal cord. Our aim was therefore to establish a cell culture model with dorsal root ganglion (DRG) cells in order to functionally classify classes of primary afferent neurons comparing pig and mouse.
Methods
Mouse and pig thoracic DRG cell cultures were generated and used the following day(s) for non-ratiometric calcium imaging with Fluo-8-AM (1,2). Functional expression of ion channels and receptors was investigated utilizing selective agonists, antagonists and modulators. Capsaicin (CAP) was used to activate TRPV1, pregnenolone sulfate (PS) to activate TRPM3, allyl isothiocyanate (AITC) to activate TRPA1 and menthol (ME) to activate TRPM8. Electrical stimulation paradigms (square-wave [1, 2, 4 pulses], sine-wave, half-sine) were tested, optimized, established and applied in order to examine biophysical properties of the neurons.
Results
Pig neurons were mostly between twenty and seventy micrometers in diameter, with cells that respond to CAP or PS being predominantly smaller. Different proportions of all neurons reacted to CAP (38.9%), PS (22.8%) or ME (6.5%). AITC activated non-neuronal cells to an extent that precludes analysis of pig neurons. Electrical stimulation allowed separate assessment of thresholds to square-wave (23.2+/-5.6mA) and sine-wave (26.1+/-10.6mA) stimuli in pig neurons. Pig neurons that responded to both types of electrical stimulation and not to PS tended to be larger (57.8+/-8.4µm), but those that additionally responded to PS tended to be similar in size (42.7+/-13.4µm) than the average size of all neurons (44.5+/-15.3µm). While thresholds for rectangular stimulation did not vary greatly within and between pig DRG neuron subclasses, thresholds for sinusoidal stimulation were higher in cells that responded to PS (30.4+/-10.3mA) than in those that were not sensitive to PS (22.8+/-9.8mA).
Conclusions
Fluo-8-AM-based calcium imaging demonstrated that nociceptive TRP channels are expressed and functional in cultured pig DRG neurons, allowing identification and characterization of sensory neuron subclasses. Established electrical stimulation protocols, which are used for the investigation of nerve fiber classes in pigs and humans, could be adapted for DRG neurons in culture and used for refined identification and characterization of sensory neuron subclasses.
References
1. Jonas R, Klusch A, Schmelz M, Petersen M, Carr RW. Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata. PLoS One. 2015 Sep 25;10(9).
2. Behrendt M, Solinski HJ, Schmelz M, Carr RW. Bradykinin-Induced Sensitization of Transient Receptor Potential Channel Melastatin 3 Calcium Responses in Mouse Nociceptive Neurons. Front Cell Neurosci. 2022 Apr 13:16:843225.
Presenting Author
Marc Behrendt
Poster Authors
Marc Behrendt
PhD
Dept. Exp. Pain Research, MCTN, Med. Faculty Mannheim, Heidelberg University, Mannheim, Germany
Lead Author
Zeinab Abbasi
Heidelberg University
Lead Author
Richard Carr
Dept. Exp. Pain Research, MCTN, Med. Faculty Mannheim, Heidelberg University, Mannheim, Germany
Lead Author
Roman Rukwied
Dept. Exp. Pain Research, MCTN, Med. Faculty Mannheim, Heidelberg University, Mannheim, Germany
Lead Author
Martin Schmelz
Heidelberg University, Dept. Exp. Pain Research, MCTN Mannheim
Lead Author
Topics
- Mechanisms: Biological-Molecular and Cell Biology