Background & Aims

Pigs, recognized as an excellent model for human sensory neurons, offer an additional system to study sensory neuron responses to pruritogens and can therefore enhance our limited understanding of pruriception in the human nervous system (1–3). Our project bridges translational gaps by adapting current sensory neuron classification schemes relying on afferent fiber function in humans and gene expression patterns in mice (4,5) to Ca2+ imaging of pig primary sensory neuron cultures. Through combination with pruritogen challenge, this approach allows the simultaneous annotation of pig pruriceptive neurons in the human and mouse classification scheme, offering not only insights into the cellular logic of pig pruriception but also a promising approach for fruitful forward and reverse translation.

Methods

Thoracic and lumbar dorsal root ganglia (DRG) were extracted from male pigs (3 month) and used for snRNAseq and Ca2+ imaging.
snRNAseq: Ganglia were stored in RNAlater overnight before flash-freezing. Nuclei were isolated from 80 mg tissue, barcoded, and subjected to gene expression library construction using the 10XGenomics Chromium platform. Sequencing on an Illumina NextSeq550, read mapping with Cellranger, and further analysis in R with Seurat were performed.
Ca2+ Imaging: Single cells were dissociated from 6-8 ganglia and cultured on coated glass coverslips for 24 h. After incubation with Fluo-8 for 30 minutes, coverslips were mounted on an inverted microscope and cells were superfused with imaging buffer(6). Fluorescence image time sequences were acquired at 4 Hz, while stimulation with histamine (HIS), N-methyl Leukotriene C4 (LTC4), interleukin 31 (IL-31), capsaicin (CAP), KCl, and electrical currents (rectangular and sine wave at 4 or 50 Hz) was performed sequentially.

Results

Receptor profiling of snRNAseq data was employed to guide pharmacological activation of cultured C-fiber neurons. Most pig C-fiber types express TRPV1 making them targetable with CAP. Within TRPV1+ neurons, a single cluster selectively expressed various pruritogen receptors, suggesting that HIS, LTC4, and IL-31 can be used to functionally identify this neuron type.
In pig DRG cultures, neurons were identified based on Ca2+ responses to KCl or CAP, with CAP activating 37% of neurons. CAP+ neurons fell into two groups: those responding to HIS (23%) and those lacking HIS sensitivity (76%). Some HIS+ neurons (33%) selectively showed rapid Ca2+ transients to IL-31 and LTC4. More than 80% of CAP/HIS+ neurons had high thresholds to rectangular electrical stimulation (50-100 mA), while thresholds to 4 Hz sine wave currents were lower (20-50 mA). In comparison, CAP+/HIS- neurons had similar rectangular current thresholds but a markedly decreased responsiveness to 4 Hz sine waves.

Conclusions

In conclusion, we provide a framework for the characterization of subgroups of pig DRG neurons through the successful adaptation of pharmacological and electrical stimuli. With a particular focus on itch, we were able to use this framework to functionally identify a distinct population that likely constitutes pruriceptive neurons. Our findings pave the way for future investigations on mechanisms underlying pruritus in this functionally identified sensory neuron subgroup. Future methodological improvements are needed to extend our approach to other pig DRG neuron subgroups due to a relatively low cell viability in our cultures (only 50% of visibly identified neurons responded to KCl/CAP).

References

1. Schmelz M. How Do Neurons Signal Itch? Front Med. 2021 Mar 15;8:643006.
2. Werland F, Hirth M, Rukwied R, Ringkamp M, Turnquist B, Jorum E, et al. Maximum axonal following frequency separates classes of cutaneous unmyelinated nociceptors in the pig. J Physiol [Internet]. 2021 Mar 1 [cited 2024 Jan 29];599(5):1595–610. Available from: https://pubmed.ncbi.nlm.nih.gov/33369733/
3. Rukwied R, Thomas C, Obreja O, Werland F, Kleggetveit IP, Jorum E, et al. Slow depolarizing stimuli differentially activate mechanosensitive and silent C nociceptors in human and pig skin. Pain [Internet]. 2020 Sep 1 [cited 2024 Jan 29];161(9):2119–28. Available from: https://pubmed.ncbi.nlm.nih.gov/32379219/
4. Hockley JRF, Taylor TS, Callejo G, Wilbrey AL, Gutteridge A, Bach K, et al. Single-cell RNAseq reveals seven classes of colonic sensory neuron. Gut [Internet]. 2019 Apr 1 [cited 2024 Jan 31];68(4):633–44. Available from: https://pubmed.ncbi.nlm.nih.gov/29483303/
5. Zeisel A, Hochgerner H, Lönnerberg P, Johnsson A, Memic F, van der Zwan J, et al. Molecular Architecture of the Mouse Nervous System. Cell [Internet]. 2018 Aug 8 [cited 2024 Jan 31];174(4):999. Available from: /pmc/articles/PMC6086934/
6. 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 [Internet]. 2015 Sep 25 [cited 2024 Jan 29];10(9). Available from: https://pubmed.ncbi.nlm.nih.gov/26407014/

Presenting Author

Zeinab Abbasi

Poster Authors

Zeinab Abbasi

MSc

Heidelberg University

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Itch