Identification of chemesthetic receptors responsible for

1
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 27109 liwy6_at_wfu.edu, patetd5_at_wfu.edu
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.
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. Experimental Setup
Figure 3. Comparison of water consumption from
water and
irritant-treated water bottles


C.
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.
Figure 2. Stimuli Tested
Figure 4. Comparison of water consumption from
water and irritant-treated water bottles

Literature Cited Alimohammadi, H. and Silver,
W.L. (2000) Evidence for nicotinic acetylcholine
receptors on nasal trigeminal nerve endings of
the rat. Chem. Senses 161-66. Clapham, D.E.
(2003) TRP channels as cellular sensors. Nature.
426517-524. 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. 427260-265. Julius, D. and Basbaum,
A.I. (2001) Molecular mechanisms of nociception.
Nature. 413203-210.

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. Plt0.05 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. Plt0.05
Mann-Whitney Test
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.