Abstract Introduction Rodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations. Methods This study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single‐cell ratiometric calcium imaging, with the activation of neurons to the following pruritogenic and algogenic substances: histamine, chloroquine, canine protease‐activated receptor 2 (PAR2) activating peptide (SLIGKT), compound 48/80, 5‐hydroxytryptamine receptor agonist (5‐HT), bovine adrenal medulla peptide (BAM8‐22), substance P, allyl isothiocyanate (AITC), and capsaicin. Results This study demonstrates a simple dissection and rapid processing of DRG collected from canine cadavers used to create viable primary sensory neuron cultures to measure responses to pruritogens and algogens. Conclusion Ratiometric calcium imaging demonstrated that small‐diameter canine sensory neurons can be activated by multiple stimuli, and a single neuron can react to both a pruritogenic stimulation and an algogenic stimulation.
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