Example Natural Science Poster_Biology
Investigation of Olfactory CO2 Detection in Mice. Jessica Kenemuth 1, Allison Hensler 2, and E. Lee Coates 1, 2 Neuroscience Program 1, Biology Department 2, Allegheny College, Meadville, PA
Introduction
Conclusions
Results •
Figure 4: Mammalian Ringers (Control)
Figure 1: Odorant Detection
4
8
12
25
50
Amyl Acetate
A 1 mV 10 sec
B
ATP
Cilia Membrane
6
4
2
Ca2+
R = Odorant Receptor Golf = Olfactory G Protein AC = Adenylate Cyclase
Figure 2: CO2 Detection
80
*
*
60
5
10
15
20
25
*
•
40 20 PA
50
Citralva
AA
Cyclohexanone
GC-D ClGTP
CA
cGMP PDE2A
Ca2+
GMP -
HCO3 +
Cl-
H+
Inhibition of Ca2+ activated Cl- channels with niflumic acid attenuated both the olfactory receptor responses to CO2 and odorants showing that Ca2+ activated Cl- channels may play a role in the olfactory CO2 detection.
Odorants
•
Before 0.1mM L-cis-diltiazem After 0.1mM L-cis-diltiazem
8
CO2
Inhibition of cGMP gated Ca2+ channels attenuated both the olfactory receptor responses to CO2 and odorants suggesting that L-cis-diltiazem may inhibit both cAMP and cGMP gated Ca2+ channels.
GC-D knockout mice responded to CO2 at all concentrations tested indicating that the GC-D pathway may not be the only mechanism by which CO2 stimulates CO2 -sensitive olfactory neurons (see figure below).
C 6
4
2
0
*
*
** *
0
? CA
100
0
B
CO2
GC-D
GTP
•
120
Figure 5: L-cis-diltiazem (Inhibition of cGMP gated Ca2+ Channel) A) Proposed pathway for olfactory CO2 detection showing the site of action of L-cis-diltiazem. B) Average (±SEM) EOG responses to CO2 before and after L-cisdiltiazem. C) Average (±SEM) EOG response to propyl acetate (PA), amyl acetate (AA), citralva, and cyclohexanone (cyclo) after L-cis-diltiazem. * P < 0.05. (N=6)
?
Before Mammalian Ringers After Mammalian Ringers
8
% CO2
Cl-
cAMP
D
0
A Golf
C
EOG Amplitude (% before)
2
0
R
CO2
1
5
10
15
20
*
*
25
50
EOG Amplitude (% before)
AC
Typical EOG responses to CO2 and amyl acetate before (A) and after (B) topical application of Mammalian Ringers. C) Average (±SEM) EOG responses to CO2 before and after Ringers. D). Average (±SEM) EOG response to propyl acetate (PA), amyl acetate (AA), citralva, and cyclohexanone (cyclo) after Mammalian Ringers. * P < 0.05. (N=7)
0
EOG Amplitude (mV)
Odorant molecules
% CO2
EOG Amplitude (mV)
Physiological concentrations of CO2 (less than the 4-5% CO2 in expired air) have been shown to stimulate a small subset of olfactory receptor neurons allowing mice and rats to “smell” low concentration of CO2 (3,4). While “typical” odorants are known to stimulate olfactory receptors via a cAMP mediated pathway (Fig 1) recent studies indicate that cGMP and the enzyme carbonic anhydrase (CA) are important for the detection of CO2 (Fig 2)(2,4,5). The objective of this study was to investigate the transduction pathway for CO2 detection by recording olfactory receptor responses to CO2 and odorants before and after topical application of L-cis-diltiazem, which inhibits cGMP activated Ca2+ channels or niflumic acid, which inhibits Ca2+ activated Cl- channels. Electro-olfactograms (EOG), which measure summated olfactory receptor responses, were recorded from the surface of the olfactory epithelium in areas known to contain high concentrations of CA (1,4) . EOGs were recorded in response to CO2 (0-50%) and odorants (amyl acetate, citralva, cyclohexanone, propyl acetate) before and after topical application of the inhibitors.
Application of Mammalian Ringers did not significantly affect the olfactory receptor responses to CO2 but did attenuate the responses to some odorants indicating possible non-specific effects of fluid application.
120 100
Cl-
80 60
*
*
*
* GTP
40
cGMP
CA PDE2A
20
Ca2+
GMP
0 PA
Citralva
AA
Cyclohexanone
CO2
HCO3- + H+
?
Odorants
% CO2
cGMP
GC-D
CO2
Ca2+
PDE2A GMP CA = Carbonic Anhydrase GC-D = Guanylyl Cyclase-D PDE2A = Phosphodiesterase 2A
Redrawn from Sun et al., 2009
Figure 6: Niflumic Acid (Inhibition of Ca2+ gated Cl- Channel) A
Methods
• •
GC-D
CO2
ClGTP cGMP
CA
PDE2A
Ca2+
GMP
CO2
-
HCO3 +
H+
6 4 2 0
EOGs were recorded from the surface of the olfactory epithelium using glass electrodes (tip dia. = 10-15 μm) filled with mammalian Ringers.
0
** *
**
**
**
**
*
120 100
1. Coates, E.L. (2001) Olfactory CO2 chemoreceptors. Respir. Physiol., 129(1-2):219-229.
80 60
* *
40 20
**
0
5
10
15
20
25
PA
50
Citralva
AA
Cyclohexanone
Odorants
% CO2
CO2 (0-50%) and odorants were delivered through a multi-barrel olfactometer with a background humidified airflow of 500 ml/min. EOG responses to odorants and CO2 were recorded before and after topical application of Mammalian Ringers, 0.1mM L-cis-diltiazem, or 0.1mM niflumic acid (with
Introduction
Conclusions
Results •
Figure 4: Mammalian Ringers (Control)
Figure 1: Odorant Detection
4
8
12
25
50
Amyl Acetate
A 1 mV 10 sec
B
ATP
Cilia Membrane
6
4
2
Ca2+
R = Odorant Receptor Golf = Olfactory G Protein AC = Adenylate Cyclase
Figure 2: CO2 Detection
80
*
*
60
5
10
15
20
25
*
•
40 20 PA
50
Citralva
AA
Cyclohexanone
GC-D ClGTP
CA
cGMP PDE2A
Ca2+
GMP -
HCO3 +
Cl-
H+
Inhibition of Ca2+ activated Cl- channels with niflumic acid attenuated both the olfactory receptor responses to CO2 and odorants showing that Ca2+ activated Cl- channels may play a role in the olfactory CO2 detection.
Odorants
•
Before 0.1mM L-cis-diltiazem After 0.1mM L-cis-diltiazem
8
CO2
Inhibition of cGMP gated Ca2+ channels attenuated both the olfactory receptor responses to CO2 and odorants suggesting that L-cis-diltiazem may inhibit both cAMP and cGMP gated Ca2+ channels.
GC-D knockout mice responded to CO2 at all concentrations tested indicating that the GC-D pathway may not be the only mechanism by which CO2 stimulates CO2 -sensitive olfactory neurons (see figure below).
C 6
4
2
0
*
*
** *
0
? CA
100
0
B
CO2
GC-D
GTP
•
120
Figure 5: L-cis-diltiazem (Inhibition of cGMP gated Ca2+ Channel) A) Proposed pathway for olfactory CO2 detection showing the site of action of L-cis-diltiazem. B) Average (±SEM) EOG responses to CO2 before and after L-cisdiltiazem. C) Average (±SEM) EOG response to propyl acetate (PA), amyl acetate (AA), citralva, and cyclohexanone (cyclo) after L-cis-diltiazem. * P < 0.05. (N=6)
?
Before Mammalian Ringers After Mammalian Ringers
8
% CO2
Cl-
cAMP
D
0
A Golf
C
EOG Amplitude (% before)
2
0
R
CO2
1
5
10
15
20
*
*
25
50
EOG Amplitude (% before)
AC
Typical EOG responses to CO2 and amyl acetate before (A) and after (B) topical application of Mammalian Ringers. C) Average (±SEM) EOG responses to CO2 before and after Ringers. D). Average (±SEM) EOG response to propyl acetate (PA), amyl acetate (AA), citralva, and cyclohexanone (cyclo) after Mammalian Ringers. * P < 0.05. (N=7)
0
EOG Amplitude (mV)
Odorant molecules
% CO2
EOG Amplitude (mV)
Physiological concentrations of CO2 (less than the 4-5% CO2 in expired air) have been shown to stimulate a small subset of olfactory receptor neurons allowing mice and rats to “smell” low concentration of CO2 (3,4). While “typical” odorants are known to stimulate olfactory receptors via a cAMP mediated pathway (Fig 1) recent studies indicate that cGMP and the enzyme carbonic anhydrase (CA) are important for the detection of CO2 (Fig 2)(2,4,5). The objective of this study was to investigate the transduction pathway for CO2 detection by recording olfactory receptor responses to CO2 and odorants before and after topical application of L-cis-diltiazem, which inhibits cGMP activated Ca2+ channels or niflumic acid, which inhibits Ca2+ activated Cl- channels. Electro-olfactograms (EOG), which measure summated olfactory receptor responses, were recorded from the surface of the olfactory epithelium in areas known to contain high concentrations of CA (1,4) . EOGs were recorded in response to CO2 (0-50%) and odorants (amyl acetate, citralva, cyclohexanone, propyl acetate) before and after topical application of the inhibitors.
Application of Mammalian Ringers did not significantly affect the olfactory receptor responses to CO2 but did attenuate the responses to some odorants indicating possible non-specific effects of fluid application.
120 100
Cl-
80 60
*
*
*
* GTP
40
cGMP
CA PDE2A
20
Ca2+
GMP
0 PA
Citralva
AA
Cyclohexanone
CO2
HCO3- + H+
?
Odorants
% CO2
cGMP
GC-D
CO2
Ca2+
PDE2A GMP CA = Carbonic Anhydrase GC-D = Guanylyl Cyclase-D PDE2A = Phosphodiesterase 2A
Redrawn from Sun et al., 2009
Figure 6: Niflumic Acid (Inhibition of Ca2+ gated Cl- Channel) A
Methods
• •
GC-D
CO2
ClGTP cGMP
CA
PDE2A
Ca2+
GMP
CO2
-
HCO3 +
H+
6 4 2 0
EOGs were recorded from the surface of the olfactory epithelium using glass electrodes (tip dia. = 10-15 μm) filled with mammalian Ringers.
0
** *
**
**
**
**
*
120 100
1. Coates, E.L. (2001) Olfactory CO2 chemoreceptors. Respir. Physiol., 129(1-2):219-229.
80 60
* *
40 20
**
0
5
10
15
20
25
PA
50
Citralva
AA
Cyclohexanone
Odorants
% CO2
CO2 (0-50%) and odorants were delivered through a multi-barrel olfactometer with a background humidified airflow of 500 ml/min. EOG responses to odorants and CO2 were recorded before and after topical application of Mammalian Ringers, 0.1mM L-cis-diltiazem, or 0.1mM niflumic acid (with
