Fig. 2.

Conjunctival MrgprA3⁺ sensory neurons mediate acute ocular itch. (A) Scratching response induced by conjunctival application of leukotriene D₄ (LTD₄, 10 pmole in 2.5 μl), interleukin 31 (IL-31, 25 pmole in 2.5 μl), or nape intradermal injection of IL-31 (100 pmole in 50 μl) in WT mice (n=5/group). (B) Ocular scratching responses evoked by saline vehicle and beta-alanine (500 nmole in 2.5 μl) in WT mice (n=5/group). (C) Ocular scratching responses induced by histamine (250 nmole in 2.5 μl), chloroquine (24 nmole in 2.5 μl), serotonin (940 pmole in 2.5 μl) in MrgprA3⁺ neuron-ablated (n=11/group for histamine and chloroquine assays, and n=9 for serotonin assay)and WT mice (n=8/group). Statistical analysis by two tailed Student’s t-test (histamine, ***P=0.0001; chloroquine, ***P=0.000001; serotonin, ***P=0.000007). (D) Representative images showing MrgprA3⁺ fibers (labeled by tdTomato, red) in the conjunctiva from Pirt^GCaMP3/+; Mrgpra3^tdTomato mice (n=3). (E-G) Representative images showing the fluorescence changes of GCaMP3 in the conjunctival sensory fibers upon stimulation with serotonin (100 μM) and chloroquine (2 mM). Scale bars: 50 μm. (H) shows Ca²⁺ transients of representative MrgprA3⁺ sensory fibers (highlighted by colored arrows in F-G). (I) Percentages of MrgprA3-tdTomato sensory fibers that were activated by different pruritogens (5-HT: 40.7±2.5%, CQ: 89.4±2.8%, His: 45.3±2.9%). Each dot represents a conjunctiva explant from Pirt^GCaMP3/+; Mrgpra3^tdTomato mouse (n=5 conjunctivae from three mice per group). (J) Diagram showing the actions of both histamine and non-histamine pruritogens converge onto MrgprA3⁺ sensory fibers to induce ocular itch. All data are expressed as mean±s.e.m.