Identification of chemesthetic receptors responsible for the detection of irritants in mice Winston Li and Tulsi Patel Department of Biology, Wake Forest University, Winston-Salem, NC Methods C57Bl6 wild type mice are taken from their group-housed cages and placed individually in plastic cages. Food is available ad lib. The mice are offered water from a choice of two drinking tubes made from 25- ml plastic pipettes. The level of water in the tubes is measured to the nearest 0.1 mL. The spouts of the two tubes are fitted with a 3/8” felt washer. One of the washers is soaked in the irritant and the other in water. The apparatus is shown in figure 1. After 24 hours the amount of water drunk from each of the 2 tubes is recorded and the position of the tubes is switched to prevent side preferences. After 48 hours the amount of water drunk from each bottle is again recorded and the mice are returned to their original home cages. Figures 3 and 4 shows results in C57Bl6 wild type mice. We will compare the effect of the irritants on the amount of water drunk by C57Bl6 wild type and TRPV1-knockout mice. Knockout mice are transgenic animals that are bred to lack a specific protein. TRPV1-knockouts e,g,, lack the receptor that responds to capsaicin, the active ingredient of chili peppers. Introduction The trigeminal nerve provides sensory information from the eyes, nose, and mouth. It is a multisensory nerve, responding to a variety of chemical irritants in the environment. Little is known, however, about the mechanism of stimulation for many of these chemicals. We know there are a wide variety of receptor proteins associated with trigeminal nerves, including TRPV1, TRPA1, TRPM8, P2X3, nAChR and ASIC3, (Alimohammadi and Silver, 2001; Julius and Basbaum, 2001; Clapham 2003; Jordt et al., 2004) which bind specific chemicals leading to the sensation of irritation. The goal of the present study is to develop a behavioral assay to help identify receptors which certain irritants stimulate to produce the sensation of irritation. Literature Cited Alimohammadi, H. and Silver, W.L. (2000) Evidence for nicotinic acetylcholine receptors on nasal trigeminal nerve endings of the rat. Chem. Senses 1: Clapham, D.E. (2003) TRP channels as cellular sensors. Nature. 426: Jordt, S.E., Bautista, D.M., Chuang, H.H., McKemy, D.D., Zygmunt, P.M., Hogestatt, E.D., Meng, I.D. and Julius, D. (2004) Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature. 427: Julius, D. and Basbaum, A.I. (2001) Molecular mechanisms of nociception. Nature. 413: Conclusions C57Bl6 wild type mice easily discriminated drinking tubes treated with a chemical irritant from drinking tubes treated with water. In each case, when one of the two felt washers placed around the sipper tube (Figure 1A) contained a saturated chemical irritant, the mice drank significantly more water from the control water bottles (Figures 3 and 4). There were no significant differences in drinking between water bottles treated with water. We will now use this behavioral assay to help identify receptors which irritants stimulate to produce the sensation of irritation. We will do this by comparing drinking in TRPV1-knockout mice and normal controls. The normal controls should drink only from the water bottle without the irritant. If the irritant works by stimulating TRPV1 receptors, the TRPV1- knockout mice should drink equally from both bottles. If the irritant works by stimulating non-TRPV1 receptors, the knockout mice should continue to drink more from the control water bottle. Figure 1. Apparatus. A. Drinking tube made from 25ml pipette. B. Top view of the lid on the testing cage. A plastic screen was placed between the felt washer and the cage top to prevent the mice from contacting the washers. C. Picture of entire apparatus Figure 2. Stimuli tested. Figure 1. Experimental Setup Figure 2. Stimuli Tested Figure 3. Comparison of water consumption from water and irritant-treated water bottles Figure 4. Comparison of water consumption from water and irritant-treated water bottles Figure 3. Graph showing that mice were significantly able to discriminate between water and benzaldehyde as well as water and toluene. It is not known which receptors these two compounds activate in the nasal trigeminal nerve. Mice drank equally when each water bottle was treated with water-soaked washers. * P<0.05 Mann-Whitney Test Figure 4. Graph showing that mice were significantly able to discriminate between water and acetic acid as well as water and cyclohexanone. These two compounds activate TRPV1 receptors in the nasal trigeminal nerve. * P<0.05 Mann-Whitney Test C. A. B. * * * *