(205) Cellular Mechanisms Underlying Keratinocyte Cold Responses and Subsequent Modulation of Sensory Neuron Thermal Activity

Abstract

Thermosensation is a critical process through which organisms detect temperature changes within the body and local environment. Convention proposes that thermosensation, and general somatosensation, is mediated exclusively by sensory neurons. Recent publications challenge this idea by describing the role that epidermal cells play in the detection and transmission of mechanical stimuli. Keratinocytes, which constitute 95% of the cells in the epidermis, are depolarized upon mechanical stimulation and release ATP which then modulates sensory neuron responses to mechanical stimuli via P2X4 receptors (Moehring, eLife, 2018). The ability of keratinocytes to respond to cold temperature, another omnipresent sensation, remains unknown. To answer this question, archaerhodopsin-3, an inhibitory opsin, was expressed in epidermal cells. Optogenetic inhibition of epidermal cells significantly decreased cold sensitivity in naive mice; paw withdrawal latencies to a dry ice stimulus were increased by ∼35%. To dissect the keratinocyte signaling pathways mediating the response to cold, calcium imaging was employed. Primary keratinocytes from mouse glabrous skin exhibited rapid and robust responses to a ramp of decreasing bath temperature; of 600+ cells tested, 100% were sensitive to cold stimulation. Cold responses were abolished when extracellular calcium or sodium were omitted from buffer. To specifically assess which cold-sensitive ion channels initiate and sustain this response, various pharmacological agents were included in the cold bath. Finally, the relevance of keratinocyte ATP to sensory neuron P2X4 signaling was assessed with regards to cold sensation. Local hydrolysis of ATP increased paw withdrawal latencies to cold stimuli similar to optogenetic inhibition of keratinocytes. Cell sniff assays confirmed the ability of keratinocytes to release ATP upon cold stimulation. P2X4 sensory neuron knockout mice were also significantly less sensitive to plantar cold stimulation. Collectively, these experiments illustrate the previously unknown ability of keratinocytes to respond to cold and alter downstream sensory neuron responses to cold stimuli.