Transcript A New Behavioral Assay for Studying TRPA1 Channels in Fruit Flies

A New Behavioral Assay for Studying TRPA1 Channels in Fruit Flies
Madhura Manjunath, Mai Motoni, Wayne L. Silver
Department of Biology, Wake Forest University, Winston-Salem, NC 27109
Introduction
Chemesthesis is the sense of
irritation produced by chemicals.
In mammals, the trigeminal nerve
is a mediator for chemesthesis in
the head and face and is
stimulated by chemical irritants.
Transient receptor potential
(TRP) ion channels are found in
the trigeminal nerve including
TRPA1, which is known to be
activated by over 90 compounds
(Silver et al., 2008).
We are studying the ability of the
painless gene, an evolutionary
homolog of mammalian TRPA1
expressed in fly gustatory
receptor neurons, to detect
irritants. Painless flies do not
avoid the irritant allyl
isothiocyanate (AITC) a
prototypical TRPA1 agonist and
the active ingredient in mustard
oil and wasabi. We are developing
a new Drosophila feeding assay
to examine what other irritants
might stimulate Painless.
Conclusions
Two bottle preference tests are often
used in mammalian studies to
examine the ability to detect different
chemicals (e.g . Blednov et al, 2009).
In our current preliminary
experiment, we expected wild-type
flies to drink more of the sucrose
solution than the AITC solution. If
this aversion to AITC is mediated by
the painless gene, then Painless
mutants (lacking the gene) should
drink equal amounts of the two
solutions. We believe that the blue
and red dyes help the flies to
determine the irritant from the
sucrose.
This assay will allow us to quickly
screen a large number of chemical
irritants to determine whether they
activate Painless. Future experiments
will examine a bigger range of
irritants or learned flavor preferences
in flies.
Figure 1 Methods
•Wild-type and Painless flies were anesthetized with carbon dioxide. Three flies were then
placed in each vial containing two 5 µL capillary tubes.
•One capillary tube was filled with 5 μL of 1% sucrose and red dye. The other was filled
with 5 μL of 1% sucrose plus10 mM AITC and blue dye.
• The amount in μL of liquid drunk by the flies from each tube was measured and
recorded.
Mean Amount of Solutions Drank
1.8
Literature Cited
1.6
Silver WL, Roe P, Atukorale V, Li W, Xiang BS. (2008) TRP Channels
and Chemosensation. ChemoSense. 10: 1-6.
1.4
Volume (μL)
1.2
1
1% sucrose
0.8
Blednov YA, Harris RA (2009) Deletion of vanilloid receptor (TRPV1)
in mice alters behavioral effects of ethanol. Neuropharmacology. 56:
814-820.
10 mM AITC in
DMSO
0.6
0.4
0.2
0
Wild-type
Painless
Figure 2. Comparing mean volumes drank of
1% sucrose and AITC from Wild-type flies
and Painless flies. The data plotted represents
the average of four trials. Error bars are of the
+/- standard deviation.