Glutamate in primary afferents is required for itch transmission

Whether glutamate or itch-selective neurotransmitters are used to confer itch specificity is still under debate. We focused on an itch-selective population of primary afferents expressing MRGPRA3, which highly expresses Vglut2 and the neuropeptide neuromedin B (Nmb), to investigate this question. Optogenetic stimulation of MRGPRA3⁺ afferents triggers scratching and other itch-related avoidance behaviors. Using a combination of optogenetics, spinal cord slice recordings, Vglut2 conditional knockout mice, and behavior assays, we showed that glutamate is essential for MRGPRA3⁺ afferents to transmit itch. We further demonstrated that MRGPRA3⁺ afferents form monosynaptic connections with both NMBR⁺ and NMBR⁻ neurons and that NMB and glutamate together can enhance the activity of NMBR⁺ spinal DH neurons. Moreover, Nmb in MRGPRA3⁺ afferents and NMBR⁺ DH neurons are required for chloroquine-induced scratching. Together, our results establish a new model in which glutamate is an essential neurotransmitter in primary afferents for itch transmission, whereas NMB signaling enhances its activities.