Short Term Irritant Exposure: Field Response,

Desensitization, and Death in the Main Olfactory Epithelium

JENNIFER LU - River Hill High School G/T Intern-Mentor Program
WEIHONG LIN, TATSUYA OGURA, ZIYING FU UMBC Department of Biology

BACKGROUND
Animals are constantly exposed to
potentially harmful physical and chemical
pollutant irritants, which affect their olfactory
systems as irritants are inhaled through the
nasal cavity.
The olfactory system consists of the olfactory
bulb, main olfactory epithelium (MOE),
vomeronasal organ, and Bowmans glands.
(Figure 1) The nasal epithelium near the
posterior region of the nasal cavity, is the first
to be damaged when exposed to irritants.
The MOE is made up of brush cells,
microvillous cells, basal cells, supporting cells,
and olfactory sensory neurons (Figure 2).
Figure 1a. Mouse
olfactory system

DATA
Figure 5a. SKN Position 1
5

Unexposed
0.3461

0.4328

0.0120

0.1041

Figure 5b. SKN Position 2

3

Exposed
0.2827

0.027

0.0553

2.5

0.0154

Normally, odorant molecules bind to Gprotein channel receptors. This causes a signal
transduction cascade as the production of
cAMP opens ion channels to allow Ca and Na
ions to flow in, causing the depolarization of
the cell. Ca also activates Cl ion channels,
allowing Cl ions to flow out of the cell and
augment the depolarization. (Figure 2)

Unexposed

Exposed

0.0536

0.3935

0.2367

0.3703

0.1725

0.1487

IMBX

Ger

Cit

2-hep

DMP

Pro

0.0264

0.0008

2
3
1.5
2

0.5
0

0
IMBX

Ger

Cit

2-hep

DMP

Pro

Tri

High K+

Figure 5c. WT Position 1

4

Unexposed

3.5

0.079

0.0434

0.0848

0.1309

4.5

Exposed
0.1427

Tri

High K+

Figure 5d. WT Position 2

Unexposed

4
0.2194

0.4364 0.1085

3.5

Figure 5a-f. Average responses of individual

odorants, separated by type of mouse,
exposure, and position. P-values indicated.
Figure 6. Representative traces of individual
odorant responses, separated by type of
mouse, exposure, and position.
Figure 7a-b. Response profiles of odorants,
indicating the general trend of rise & fall of
response amplitudes among mice.
Figure 8a-c. Average, overall responses of
odorant comparisons between water/irritantexposed mice and between SKN/WT mice.
P-values indicated.

Exposed

0.0230

0.2692

0.2500

0.0275

0.2034

0.3948

0.0545

IMBX

Ger

Cit

2-hep

DMP

Pro

Tri

0.1285

2.5

2.5

1.5

Olfactory sensory neurons (OSNS) are nerve

cells whose axons project to specialized areas
in the olfactory bulb called glomeruli which
receive and interpret nerve signals. These
neurons receive and interpret environmental
chemosensory stimuli.

ANALYSIS

1.5

0.5

0.5
0

0
IMBX

Ger

Cit

2-hep

DMP

Pro

Tri

High K+

Figure 5e. Exposed Position 1

5

Wild Type
0.3782

0.2690

0.1876

0.3189

Figure 5f. Exposed Position 2

4

SKN

0.4405

0.3917

High K+

0.1458

0.0647

3.5
3

Wild Type
0.4460

0.4455

0.4816

0.4254

SKN

0.4129

0.2453

0.1070

0.1124

Figure 3. Electroolfactogram setup

2.5

2
2

1.5

WT MICE: WATER VS. IRRITANT - EXPOSED

0.5
0

0
IMBX

Ger

Cit

2-hep

DMP

Pro

Tri

High K+

IMBX

Ger

Cit

2-hep

DMP

Pro

Tri

High K+

Figure 6. REPRESENTATIVE TRACES

This electrical response can be measured

using an electroolfactogram (EOG), which
records the negative electrical receptor
potential at the surface of the MOE.

Figure 2. Main olfactory epithelium

Across all stimuli applied to water-exposed

mice in the posterior of the MOE (Position 1,
as indicated in Figure 4), the response
amplitude of the receptor potential was
significantly higher than that of the exposed
mice (p = 0.0039252). In the anterior section
of the MOE, there was no significant
difference in response (p = 0.216456). Across
most individual odorants in both positions,
there was no significant difference. However,
when looking at all odorant responses
combined, differences were found. In the
posterior region, data suggest that irritant
exposure in wild-type mice kills OSNs and thus
causes decreased cellular response.

SKN MICE: WATER VS. IRRITANT - EXPOSED

Across all stimuli on water-exposed SKN mice
applied to the posterior region, there was a
significantly larger receptor response in the
exposed mice (p = 0.000669) when using
paired t-tests. On the other hand, in the
anterior region, the average of response size
was insignificantly different (p = 0.334819).
Like wild-type mice, across some individual
odorants, there were scattered amounts of
significant differences, but there was a
general trend in response to stimuli, indicated
by graph sets 7a-c. Unlike the wild-type mice,
SKN-1a knockout mice showed a trend
towards increased cellular response. These
data suggest that in the posterior region of
the MOE, irritant exposure could have made
the olfactory sensory neurons more
susceptible to binding to odorants, thus
resulting in a higher response.

The function of microvillous cells is less

understood. However, it is know that there
are different types of microvillous cells. This
includes TRPM-5 microvillous cells, which are
known to release acetylcholine, promoting
an inhibitory effect on OSNs in the MOE and
lowering the membrane potential. As a
result, OSNs have a smaller electrical
response to odorant stimuli.
Overstimulation with potent odorants, or
irritants, is believed to cause cell death in
the MOE, lowering potential binding areas
and thus yielding lower EOG responses.
TRPM-5 microvillous cells are believed to
help prevent this overstimulation, and thus
help prevent cell death after irritant
exposure. SKN-1 knockout (SKN) mice, which
lack the gene that codes for TRPM-5
microvillous cells, are used in the experiment.

EOG PROTOCOL
Immediately following exposure, a mouse
was euthanized using CO2 and the head was
disarticulated to prepare for the EOG
recording of field response to odorants. The
head was then bisected sagittally. The septal
cartilage and the septum was removed from
the left side. The MOE was mounted in
agarose, with a stream of Ringers solution.
Volatile odorants dissolved into Ringers
solution were used to stimulate the MOE with
three non-consecutive 1000 millisecond
pulses, where receptor potential of the
epithelial cells was recorded with Axograph.
Odorants included 100 M solutions of 3isobutyl-1-methylxanthine (IBMX), 2heptanone, geraniol, citral, 2,5dimethylpyrazine, propionic acid, and
trimethylamine, and a 40 M K+ solution.
EOG recording responses were measured in
two sections of the MOE, indicated in Figure
4. Comparisons of the largest amplitudes
were made between water and irritantexposed wild-type mice, water and irritantexposed SKN mice, and irritant-exposed SKN
and WT mice.

IRRITANT-EXPOSED MICE: WT VS. SKN

Figure 7b. Position 2

Figure 7a. Position 1

WT Water Exposed

SKN Water Exposed

WT Water Exposed

SKN Water Exposed

WT Irritant Exposed

SKN Irritant Exposed

WT Irritant Exposed

SKN Irritant Exposed

3.5

3.5

Overall paired odorant responses compared

in WT vs. SKN mice in both Position 1 and
Position 2 did not show any indication that
the odorant responses were any different
between the two types of mice. Similarly, in
the unpaired tests, no p values were found to
be under 0.05, but they were under 0.1, as
compared to wild-type mice. Similarly to the
other trials, there is scattered significance in
individual odorant comparisons. Despite the
data not showing anything too statistically
significant, the differences are indicating that
SKN mice could have higher responses after
being exposed to irritants.

2.5

2.5
2
2
1.5
1.5
1

0.5

0.5
0

0
IMBX

Ger

Cit

2-hep

DMP

Pro

Tri

Figure 8a. SKN Water

Exposed vs. Irritant Exposed
Water Exposed
1.6
1.4

High K+

IMBX

Ger

Water exposed
2.5

P = 0.0613

0.8
0.4
0.2
0

0
1

SKN

0.6

0.5

High K+

P = 0.0958

1.5

P = 0.0352

0.2

Wild Type

1.4

0.4

Tri

1.2

0.8
0.6

Pro

Figure 8c WT Irritant Exposed

vs. SKN Irritant Exposed
1.6

P = 0.0049
P = 0.1388

DMP

Irritant Exposed

1.2

2-hep

Figure 8b. WT Water Exposed

vs. Irritant Exposed

Irritant Exposed

P = 0.00621

Cit

Figure 4. Recording positions of the MOE

left (posterior) is Position 1, right (anterior) is
Position 2.

CONCLUSION AND FUTURE DIRECTION

It was hypothesized that SKN-1a knockout mice, which are deficient of TRPM-5 microvillous cells, would experience more cell death as a result of irritant exposure. This was based on the hypothesis that these
microvillous cells suppress OSN response: these cells release acetylcholine, which is an inhibitory neurotransmitter that lowers the membrane potential of OSNs and thus causes a smaller response to volatile
odorants. Without this diminished membrane potential, it was proposed that cells overexcite themselves in response to strong odorants and die as a result. This was also derived from past results from same lab,
where long-term (2-4 week) exposure resulted in OSN death and decreased field response in the irritant-exposed SKN-deficient mice compared to the irritant-exposed wild-type mice.
Shown by the results, there is a significant difference in wild-type water and irritant-exposed mice, indicating that in mice with TRPM-5 microvillous cells experience cell death after irritant exposure. Between SKN1a water and irritant exposed knockout mice, there was a significant difference in responses, where irritant-exposed mice had a higher response than water-exposed mice. However, this was only in the posterior
region this could suggest that most of the irritant damage occurred in the anterior region, which was closer to the nasal cavity, and that the posterior region was actually unaffected. This suggests that
acetylcholine released by the microvillous cells in the TRPM-5 mice were a factor. In wild-type mice, the microvillous cells would inhibit the field response, whereas in SKN-1a knockout mice, the lack of TRPM5
microvillous cells would lead to no acetylcholine release, thus producing a larger response. This is further supported by the data presented by the SKN vs. WT irritant-exposed comparisons: in the posterior region
of the MOE, there was a slightly significant difference, where SKN mice had a higher response. Overall, or the anterior region, there were not many significant responses, so conclusions cannot be drawn from
that. From that, the data suggests that TRPM-5 microvillous cells do serve as a sort of buffer for irritant exposure, inhibiting field response to prevent OSNs from overexciting themselves.