All animal experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 under Project Licenses granted to E.St.J.S. (P7EBFC1B1 and PP5814995) by the Home Office and approved by the University of Cambridge Animal Welfare Ethical Review Body. Experiments were performed using a mixture of male and female C57BL6/J mice (10–15 weeks old) and a mixture of male and female, non-breeder/subordinate, NMRs (23–145 weeks old). Age matching mice and NMRs is not straightforward, but considering the long-lifespan of NMRs vs. mice, all animals used in this study can be considered young adults. Mice were purchased from Envigo and housed conventionally with nesting material and a red plastic shelter in a temperature-controlled room at 21 °C, with a 12-hour light/dark cycle and access to food and water ad libitum. NMRs were bred in-house and maintained in an interconnected network of cages in a humidified (~ 55%) temperature-controlled room at 28–32 °C with red lighting (08:00 − 16:00). NMRs had access to food ad libitum. NMRs used in this study came from five different colonies. Animals were humanely killed by CO2 exposure followed by cervical dislocation (mice) or decapitation (NMRs). To limit the total number of animals used, DRG were taken from each animal for both immunohistochemistry (IHC) and electrophysiological analysis.

DRG neuron isolation and culture were performed as previously described (Ai et al. 2023; Pattison et al. 2024). In brief, before collecting DRG, poly-D-lysine glass-bottomed dishes (MatTek, USA) were coated with laminin (1 mg/ml, Invitrogen UK, 23017-015) and incubated at 37 °C for 1 h before removing excess laminin, washing with H2O and drying. Lumbar DRG (L2-L5, i.e. those that predominantly innervate the knee joint (da Silva Serra et al. 2016; Chakrabarti et al. 2020) were collected post-mortem and placed into cold dissociation medium (L-15 Medium (Gibco™ UK, 31415-086) with 1.5% NaHCO3 (Gibco™ UK, 25080-094). DRG were enzymatically digested in prewarmed collagenase solution (dissociation medium with 1 mg/ml collagenase (Sigma-Aldrich UK, C9891) and 6 mg/ml bovine serum albumin (BSA, Sigma-Aldrich UK, A2153) for 15 min followed by digestion in trypsin solution (dissociation medium with 1 mg/ml trypsin (Sigma-Aldrich UK, T9935), 6 mg/ml BSA, Sigma, UK) for 30 min at 37 °C before mechanical trituration with a P1000 tip 20–25 times in culture medium (dissociation medium with 1% penicillin-streptomycin (Gibco UK, 15140-122) + 10% fetal bovine serum (Sigma-Aldrich UK, F7524). Following trituration, brief centrifugation (1000 g, 30 s) was used to separate remaining DRG tissue from dissociated neurons. The resulting supernatant, containing the dissociated neurons, was collected in a separate tube, and then 2 ml of culture medium was added to pelleted DRG tissues for further trituration. Trituration and brief centrifugation were repeated 5 times until 10 ml of supernatant was collected. Collected supernatant was centrifuged at 1000 g for 5 min and the pellet was resuspended in culture medium and 100 µl plated into the center of each glass-bottomed dish. Neurons were incubated at 37 °C with 5% CO₂ for 2 h before then adding 1.9 mL of culture medium; neurons were maintained in the incubator for 18–24 h before electrophysiological recordings.

Immunohistochemistry was performed as previously described (Chakrabarti et al. 2020). Briefly, DRG were collected post-mortem and post-fixed for 1 h in 4% paraformaldehyde (PFA), followed by overnight incubation in 30% (w/v) sucrose at 4 °C for cryoprotection. DRG were next embedded in Shandon M-1 Embedding Matrix (Thermo Fisher Scientific, 10056778), snap frozen in 2-methylbutane (Honeywell International, M32631) on dry ice and stored at −80 °C. Embedded DRG were sectioned (12 μm) using a Leica Cryostat (CM3000; Nussloch, Germany) on to Superfrost Plus microscope slides (Thermo Fisher Scientific, MIC30220) and stored at −20 °C until staining.

Slides were defrosted, washed with phosphate-buffered saline (PBS)-tween and blocked in antibody diluent solution: 0.2% (v/v) Triton X-100, 5% (v/v) donkey serum and 1% (v/v) bovine serum albumin in PBS for 1-h at room temperature before overnight incubation at 4 °C with anti-TRPV1 (1:500, guinea pig polyclonal, Alomone Labs, ACC-030-GP) and anti-GFRɑ3 (1:300, goat polyclonal R&D systems, AF2645) primary antibodies in antibody diluent. Slides were washed three times with PBS-Tween and incubated with anti-guinea pig AF594 (Invitrogen, A11076) and anti-goat Alexa-568 (Invitrogen, A11057) secondary antibodies for 2 h at room temperature. Slides were washed in PBS-Tween, mounted and imaged with an Olympus BX51 microscope (Tokyo, Japan) and QImaging camera (Surrey, Canada). Exposure levels were kept constant for each slide, and contrast enhancements were made the same for all slides. Negative controls without the primary antibody showed no staining.

For analysis, sections were blinded to experimental groups, and ImageJ software (version 1.53k) was used. An automatic “minimum error” threshold algorithm was applied to 8-bit images to distinguish objects from the background. Neurons within each DRG section were manually selected, and their mean grey values were measured and normalized to the range defined by the lowest and highest intensity neurons in that section. A neuron was classified as positive for the stain if its normalized grey value exceeded the global minimum across all sections plus 2 standard deviations (SD).

Recordings from DRG neurons took place after 15 min incubation with 0.5 µg/ml isolectin B4 (IB4)-Alexa 488 (Invitrogen I21411) or IB4-Alexa Fluor 568 (Invitrogen I21412). Glass patch pipettes (3–6 MΩ, Hilgenberg) were pulled by a P-97 Flaming/Brown puller (Sutter Instruments, USA) from borosilicate glass capillaries and loaded with intracellular solution (ICS), which (in mM) contained KCl (110), NaCl (10), MgCl2 (1), EGTA (1), and HEPES (10), adjusted to pH 7.4 with KOH and osmolality adjusted to 300–310 mOsm using sucrose. DRG neurons were bathed in extracellular solution (ECS) (in mM): NaCl (140), KCl (4), CaCl2 (2), MgCl2 (1), glucose (4), HEPES (10), adjusted to pH 7.4 with NaOH, and osmolality was adjusted to 280–295 mOsm using sucrose. Cells were observed using an Olympus IX70 Fluorescence Microscope. Solutions were applied with a gravity-driven multi-barrel perfusion system (Automate Scientific). Data were acquired with a Multiclamp 700 A amplifier (Molecular Devices, USA) and digitized via Digidata 1440 A digitizer (Molecular Devices, USA).

For current-clamp recordings, bridge-balance compensation was used to reduce steady-state voltage errors. Data were sampled at 20 kHz and filtered at 5 kHz. Step current (−100 pA to 2000 pA) for 80 ms through 21 steps or no current was injected to generate action potentials (APs) under current-clamp mode. For voltage-clamp recordings, solutions were applied using a gravity-driven multi-barrel perfusion system. All recordings were made using an Multiclamp 700 A amplifier in combination with Clampex (Molecular Devices, USA) software. Pipette and membrane capacitance were compensated, with series resistance being compensated by ~ 70% to minimize voltage errors. IB4-positive neurons were visualized by observation of Alexa-488 or Alexa-568 fluorescence (CellCam Kikker 100MT).

Escherichia coli derived mouse artemin (Ala112-Gly224, R&D Systems, 1085-AR) was used to assess artemin’s ability to sensitize DRG neurons. Baseline intrinsic excitability was first measured using current-clamp recordings, in which action potential parameters, including resting membrane potential (RMP), rheobase, peak amplitude, afterhyperpolarization (AHP) duration, and AHP amplitude, were analyzed with Clampex (Molecular Devices, USA). In the same recordings, TRPV1 function was assessed in voltage clamp configuration by applying 1 µM capsaicin (Sigma-Aldrich, M2028) for 5 s. To evaluate the sensitizing effects of artemin, neurons were exposed to a 7-minute perfusion of 100 ng/ml artemin, before repeating current-clamp, voltage-clamp and capsaicin sensitivity recordings; in control experiments, neurons were exposed to ECS for 7-minutes.

Data were assessed for normality using the Shapiro-Wilk test, and parametric or non-parametric statistics were used as appropriate. IHC results are reported as mean ± standard error of the mean (SEM) Patch clamp data were analyzed using paired or unpaired t-tests as appropriate. Results are reported as mean ± SEM. Statistical analysis and graph generation was carried out in GraphPad Prism 8.0 software (USA), Biorender®, R studio® and Python.