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Abaffy and DeFazio BMC Research Notes 2011, 4:137
http://www.biomedcentral.com/1756-0500/4/137

SHORT REPORT

Open Access

The location of olfactory receptors within
olfactory epithelium is independent of odorant
volatility and solubility
Tatjana Abaffy1* and Anthony R DeFazio2

Abstract
Background: Our objective was to study the pattern of olfactory receptor expression within the dorsal and ventral
regions of the mouse olfactory epithelium. We hypothesized that olfactory receptors were distributed based on the
chemical properties of their ligands: e.g. receptors for polar, hydrophilic and weakly volatile odorants would be
present in the dorsal region of olfactory epithelium; while receptors for non-polar, more volatile odorants would be
distributed to the ventral region. To test our hypothesis, we used micro-transplantation of cilia-enriched plasma
membranes derived from dorsal or ventral regions of the olfactory epithelium into Xenopus oocytes for
electrophysiological characterization against a panel of 100 odorants.
Findings: Odorants detected by ORs from the dorsal and ventral regions showed overlap in volatility and water
solubility. We did not find evidence for a correlation between the solubility and volatility of odorants and the
functional expression of olfactory receptors in the dorsal or ventral region of the olfactory epithelia.
Conclusions: No simple clustering or relationship between chemical properties of odorants could be associated
with the different regions of the olfactory epithelium. These results suggest that the location of ORs within the
epithelium is not organized based on the physico-chemical properties of their ligands.

Findings
The molecular events that lead to olfactory perception
can be divided into peripheral (detection by olfactory
receptors (ORs) in the nasal epithelium) and central
(olfactory bulb and cortex). The events that occur at the
peripheral level are not only represented by odorantreceptor affinity, but also include the physico-chemical
characteristics of odorants, their diffusion through the
mucus, air flow dynamics, as well as the spatial distribution of olfactory receptors within the olfactory epithelium [1-3]. The main olfactory system has a diverse
population of receptors (for review see [4]). Most of
these receptors remain orphans with no known ligand.
Thus, the functional organization of the peripheral
olfactory system remains theoretical, particularly in
mammals.

* Correspondence: tabaffy@med.miami.edu
1
Department of Molecular and Cellular Pharmacology, Miller School of
Medicine, University of Miami, 1600 NW 10thAve, Miami, 33136, Fl, USA
Full list of author information is available at the end of the article

Odorant discrimination is mediated by ORs using combinatorial coding: a single OR can be activated by multiple odorants and most odorants activate more than one
OR [5,6]. Odorants represent a vast array of different
chemical structures and each receptor samples a specific
region of “chemical space” meaning that it is activated by
one or a few combinations of chemical features [7]. A
small change in the odorant molecule can result in a fundamental change of its molecular properties (such as
functional group, length, flexibility, hydrophobicity, volatility, polarity, chemical bonds) and consequently may
change or negate detection by a given OR.
In mammals, division of olfactory epithelium into dorsal and ventral regions is based on anatomical [8], biochemical [9,10] and behavioral [11] differences. Do these
regions have different populations of receptors with distinct functional roles? Mouse olfactory receptors are
divided into Class I and Class II receptors based on phylogenetic analysis [12]. Class I genes are the only type
found in fish [13]. Both Class I and II ORs are found in
amphibians and terrestrial vertebrates [14]. Classically,

© 2011 Abaffy et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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the olfactory epithelium has been divided along a dorsoventral axis into four zones based on OR expression
[15,16]. The dorsal region (also called Zone I) expresses
about 50% of all OR genes, exclusively Class I and some
of Class II receptors. The ventral region consists of
endoturbinates II, III and IV and expresses only class II
OR genes [17-20]. The dorsal region is exposed to near
ambient concentrations of toxins and air pollutants.
Thus, it is not surprising that this region is associated
with high expression of antioxidant, chemo-protective
enzymes: NADPH quinone oxido-reductase 1 (NQO1)
[21], NADPH diaphorase [22], glutathione peroxidase,
catalase and superoxide dismutase [23]. This region is
also rich in complex glycolipids [24] and expresses an
olfactory specific medium chain acyl-CoA synthetase
(O-MACS, EC 6.2.1.2) [10]. Interestingly, mice lacked
innate responses to aversive odorants after transgenic
oblation of the dorsal zone using O-MACS driven
expression of diphtheria toxin [11].
The ventral region of the olfactory epithelium has a
complex turbinate structure, and as documented for
hamster olfactory epithelium has three times more luminal surface area than the dorsal region [25,26]. The ventral region also expresses different transcription factors
[27] and the olfactory-specific cell adhesion molecule
OCAM, also known as NCAM2 [9,28,29].
Early experiments demonstrated that different odorants activate different regions of the olfactory epithelium
[30-34]. It was hypothesized that two processes could be
responsible for this topographic code: (1) the “inherent”
patterning process, based on the idea that ORs with
similar responses are grouped in similar regions of the
epithelium and (2) the “imposed” patterning process,
based on the morphology of the nasal cavity, on the pattern of airflow during sniffing, and on the differential
adsorption of the odorants through the olfactory mucus
[35]. A “chromatographic process in olfaction” has been
proposed, in which the separation of odorants is based
on their chemical properties and flow dynamics within
the nose, combined with odorant affinity for the olfactory receptors [36,37]. Previous experiments showed
that more water soluble odorants (those with a high
sorption rate) are completely absorbed by the olfactory
mucosa and removed from the air before they get to the
ventral region of olfactory epithelium [36,38]. In addition, it is important to mention that fluid dynamic modeling showed higher air flow through the dorsal region
[3,39,40]. Higher air flow permits odorants to distribute
throughout the dorsal epithelium. Odorants with higher
sorption rates could then activate receptors in this high
air flow dorsal region, while odorants with lower sorption rates would not have time to interact with the
receptors in that region. Consequently, odorants with a
lower sorption rate will have more time to interact with

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the receptors that are located in the lower air flow ventral region, and higher sorption rate odorants might not
even make it to the ventral zone due to complete
absorption in the dorsal zone.
Based on these results it has been predicted that the
strongly absorbed odorants (i.e. more water soluble)
interact with dorsal region of the olfactory epithelium
(zone I) and consequently activate the dorsal region of
the olfactory bulb [3]. In turn, odorants with lower solubility have more time to reach the ventral regions of the
olfactory epithelium, and subsequently lead to the activation of the ventral regions of the olfactory bulb
[38,39,41]. The relationship between differences in odorant solubility and the topography of the projections
from the epithelium to the olfactory bulb has been
extensively studied [42-46]. Recently, Johnson et al. analyzed results of glomerular mapping from over 350
odorants and found that highly water-soluble odorants
activated posterior regions of OB (halfway between dorsal and ventral extremes) [47]. These results are in contrast with a generalized notion that highly water soluble
odorants are recognized by Class I ORs located in the
dorsal region of the olfactory epithelium, which projects
to the dorsal aspect of the bulb.
In order to study the relationship between odorant
solubility and volatility properties with the topographical
location of their cognate ORs within the olfactory
epithelium, we implemented the novel method of membrane microtransplantation developed by Miledi [48].
Plasma membranes rich in ORs from either the dorsal
or ventral regions of the olfactory epithelia were directly
injected into X. laevis oocytes and tested against a panel
of odorants with a broad range of solubility and volatility. Olfactory receptor activation was tested via electrophysiology, using the cystic fibrosis transmembrane
regulator (CFTR) as a reporter [49].

Methods
Mouse olfactory epithelium was obtained under a tissue
sharing protocol approved by the University of Miami
Internal Animal Care and Use Committee.
O-MACS immunolabeling and two photon microscopy

Mice, strain C57BL, age 10-12 months, were sacrificed
by CO2 asphyxiation for 2 min and by cervical dislocation. The skin was removed, and the head was split
along the sagittal plane into left and right hemispheres.
Each hemisphere was first fixed in 50 mL 4% paraformaldehyde (about 10× volume of the sample) in the 0.1
M phosphate buffer, pH 7.4 for 5 hours at room temperature. After fixation, each hemisphere was washed 3×
for 15 min in TBS-T (50 mM Tris, 140 mM NaCl and
0.2% Triton X-100). Blocking was done in 10% NGS
(Normal goat serum, Rockville) in TBS-T for 2 hours.

Abaffy and DeFazio BMC Research Notes 2011, 4:137
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Samples were washed 3× in TBS-T for 30 min. Primary
anti-OMACS antibody (a kind gift from Dr. Hitoshi
Sakano) was applied at 1:1000 dilution in blocking buffer and incubated for 16 hours at room temperature.
Samples were again washed in TBS-T 3× for 30 min.
Labeling was visualized using a fluorescent secondary
antibody (goat anti-rabbit antibody conjugated to the
HiLyte Fluor 488 fluorophore, AnaSpec #61056-H488).
The secondary antibody was applied at 1 μg/mL in
blocking buffer for 16 hours. We used Hoechst 33258
for nuclear staining at 10 μM. The images were
obtained by two-photon microscopy (Zeiss/BioRad Radiance 2100 MP coupled with a Coherent Chameleon
Ultra) of the intact olfactory epithelium at 955 nm excitation and using standard blue and green emission filter
sets. Images are maximum Z-projections of 10-20
images at 5 micron steps. Each image is a Kalman average (n = 4) acquired at 16-bit resolution. Post-processing was accomplished with NIH ImageJ.
Isolation of dorsal and ventral regions of olfactory
epithelium

8 mice (C57BL/6J) 7.5 months old were killed by CO2
asphyxiation for 2 minutes and cervical dislocation.
Mouse heads were separated in two halves by splitting
along sagittal plane. Hemi-sections were put into icecold DMEM for 2 minutes and later immersed in ice
cold PBS with protease inhibitors (100 μL PI/10 mL buffer, Sigma P2714). Both the dorsal and the ventral
epithelia, and in some cases the respiratory epithelium,
were carefully removed. Each part was gently blotted on
the filter paper and immediately frozen in liquid N 2 .
Results from previous experiments [16,50,51] have
demonstrated bilaterally symmetric spatial pattern of
ORs expression, thus allowing us to combine left and
right hemi-sections. Tissue samples from 8 animals
were pooled for the dorsal and ventral regions (totaling
approximately 170 mg, 320 mg, respectively).
Isolation of cilia

To detach cilia from dorsal and ventral pooled epithelium samples, we used a modified “calcium shock”
method [52-54]. Pooled frozen samples from dorsal and
ventral regions (isolated as discussed above) were placed
into an ice-cold small Petri dish containing 1000 μL
Buffer A, at pH 8.0 ("Buffer A": 30 mM Tris-HCl, 100
mM NaCl, 2 mM EDTA, 1 mM PMSF). We slowly
added 10 μL of 1 M CaCl2 in 2 μL increments to give a
final concentration of 10 mM CaCl2 while the solution
was continuously stirred for 18 min at 4°C. Next, the
solution containing the tissue was centrifuged for 10
min at 1500 g and at 4°C. The supernatant (cilia) was
carefully removed and centrifuged at 12000 g for 10
min. The pellet containing cilia was re-suspended in the

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glycine buffer, pH 9.0 and stored in aliquots, for not
more than a month at -70°C and used for the plasma
membrane preparation.
Plasma membrane preparation and microtransplantation

Isolated cilia from dorsal or ventral regions were resuspended in glycine buffer, pH 9.0, (200 mM glycine, 150
mM NaCl, 50 mM EDTA, 300 mM sucrose), gently
homogenized (manually) and centrifuged at 9,500 g for
15 min at 4°C [48,55]. The supernatant was ultracentrifuged for 2 h at 100,000 g and the pellet was resuspended in 5 mM glycine. Protein concentration was
measured using Bradford Coomasie blue assay (Pierce)
and adjusted to 1 mg/mL. 50 nL was injected into X.
oocytes. We injected (microtransplanted) dorsal preparations into 600 oocytes and ventral preparations into 800
oocytes.
Preparation of oocytes and cRNA injection

We first injected cRNA for CFTR (a cAMP-activated Clchannel) and Ga olf as previously described [49]. The
next day, when both CFTR and Gaolf proteins were
expressed in the oocytes we microtransplanted ciliary
plasma membranes from either dorsal or ventral regions
into oocytes. The construct containing human M1 muscarinic G-protein coupled receptor was purchased from
the UMR cDNA Resource Center (Missouri University
of Science and Technology, Rolla, MO 65409).
Electrophysiology

Our electrophysiological assay for olfactory receptor
activation was accomplished as previously described
[49]. 24 hours after microtransplantation and 48 hours
after Ga olf and CFTR injection, we recorded
responses. Odorants were selected with a broad range
in volatility and solubility. All odorants and compounds (like GABA, isoproterenol and IBMX) were
either from Sigma Aldrich (St. Louis, MO) or Fluka
(Fluka Chemie AG, Switzerland). Odorants were first
dissolved in DMSO to 1 M or 100 mM solution, and
then diluted in regular buffer ND96 as described in
[56] and applied for 15 sec. Odorants with lower solubility were heated at 37°C water bath for few minutes.
Activation of ORs results in an inward current
recorded in two-electrode voltage clamp mode using
the OpusXpress 6000A (Molecular Devices). Each
oocyte was also challenged with 1 mM IBMX to confirm successful expression of CFTR. Our control
oocytes were injected with M1 receptor, CFTR and
Ga olf and challenged with 10 μM Ach for 5 sec. All
odorants used in our study were tested with these
control oocytes to guard against false positives resulting from direct activation of the CFTR reporter or
non-specific activation of the M1 receptor.

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For preliminary studies and method optimization,
mouse olfactory and respiratory epithelium and brain
were dissected, the tissue was homogenized and the
plasma membrane preparations where isolated as
described above, while skipping both ciliary membrane
preparation step and the separation of dorsal and ventral
parts (results shown in Figure 1). Oocytes expressing
membranes from mouse brain were challenged with
GABA to demonstrate successful microtransplantation
of brain plasma membranes [57]. In addition, oocytes
with brain preparations were challenged with 1 mM glutamate. Initially very small currents were observed;

Figure 1 Functional expression of receptors in X. oocytes
microinjected with plasma membranes. The current responses
were recorded 24 hours after microtransplantation. A. GABA and
glutamate responses in X. oocytes microinjected with mouse brain
tissue. B. Responses from mouse respiratory epithelium to the
odorant heptanal, the CFTR activator IBMX and the b2 adrenergic
agonist, isoproterenol. C. Responses from mouse olfactory epithelial
preparations to heptanal and IBMX. The response to 100 μM
heptanal indicates specific responses from olfactory receptors, since
it is absent from respiratory epithelium (B). Similar results were
obtained from 10-12 oocytes tested.

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however after injecting mRNA for Gb1 and Gg3,
responses were increased about 2.5 fold, thus indicating
expression of metabotropic glutamate receptors. Constructs containing Gb1 and Gg3 subunits in the
pcDNA3.1 vector were purchased from the UMR cDNA
Resource Center [49]. Oocytes expressing membranes
from the respiratory epithelium were challenged with
isoproterenol to confirm successful microtransplantation
of adrenergic GPCRs.
Statistics

A two-tailed t-test was used to test for statistical significance (GraphPad Prism 5 software).

Results
Our first goal was to verify the suitability of microtransplantation method. Plasma membranes from mouse
brain were isolated and injected into oocytes. Current
responses were recorded at a holding potential of -70
mV and 3 mM GABA was applied for 5 sec. The GABA
response was fast; reflecting the activation of GABAgated ion channels (Figure 1A, left panel). Control
oocytes without microtransplanted plasma membranes
challenged with 3 mM GABA did not produce any current (Figure 1A, right panel). Thus, we demonstrated
expression of functional GABAA receptors from mouse
brain via microtransplantation in Xenopus oocytes (Figure 1A) [57]. In addition, mouse brain membrane preparations injected in oocytes together with Gb1 and Gg3
were challenged with 1 mM glutamate. The representative trace is presented in the Figure 1A, right. No
response was seen in un-injected oocytes when challenged with 1 mM glutamate (Figure 1A, right side,
trace below).
The mouse olfactory epithelium is easily distinguishable by its yellow-brownish color and differs from the
respiratory epithelium, which is mainly transparent and
highly vascular. To confirm our ability to isolate the
dorsal region of the olfactory epithelium, we used a dorsal zone marker, O-MACS. O-MACS immunostaining
of the anterior, middle and posterior part of dorsal zone
(yellow, zone I) is presented in the Additional file 1:
Supplemental Figure S1.
In order to implement microtransplantation method
to study dorsal and ventral ORs responses to different
odorants, we injected plasma membranes from mouse
respiratory and olfactory epithelium. When using the X.
oocyte system for heterologous expression of olfactory
receptors, the olfactory-specific signal transduction Gprotein Ga olf and a reporter channel are required for
the detection of functional ORs [49]. Plasma membrane
preparations injected without Gaolf and CFTR yielded
no responses when tested against 10 mixtures (each
mixture contained 10 odorants, each at 300 μM, results

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not shown). This demonstrated the need for signal
amplification (Gaolf) and the reporter channel (CFTR).
Application of the odorant heptanal (100 μM) initiated a
current response in oocytes injected with olfactory
epithelium (Figure 1C), but not in oocytes injected with
respiratory epithelium (Figure 1B). However, the isoproterenol (1 μM), the b2 adrenergic agonist, initiated a
current response in oocytes injected with respiratory
epithelium (but not olfactory epithelium), indicating
successful and specific expression of membrane proteins
[58,59]. The presence of heptanal responses in olfactory
epithelium injected oocytes and their absence from
respiratory epithelium injected oocytes demonstrates the
specificity of the olfactory response and successful
expression of olfactory receptors via microtransplantation approach.
We do believe that if indeed explicit representation of
the location of olfactory receptors and their likely
ligands/odorants exist in the olfactory system, we should
have been able to detect it by studying these two regions
with distinct anatomy and air flow dynamics.
We decided to isolate plasma membranes from cilia in
combination with Gaolf and CFTR as signaling partners.
Expression of functional ORs was studied using twoelectrode voltage clamp against a panel of 100 odorants
at 300 μM concentration. These odorants show a broad

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range in water solubility and volatility, expressed as log
values in Additional file 2: Supplemental Table S1. In
addition, molecular weight, formula, octanol/water partition coefficient (log P) and polar surface area (PSA)
parameters for all odorants are presented. The water
solubility of the selected odorants ranged over one million times, from 1.70 mg/L for farnesol (log solubility =
0.23, compound 98) to 1 × 106 mg/L for pyrrolidine (log
solubility = 6, compound 50). Odorant volatility ranged
over 10 billion times, from 1.07 × 10-8 mmHg for nonanedioic acid (log volatility = -7.9, compound 51) to 538
mmHg for diethylether (log volatility = 2.73, compound
46).
ORs microtransplanted from the dorsal region of
mouse olfactory epithelium were challenged with a set
of 10 odorants in a single run (odorants 1-10, 11-20
etc.). At the end of each run 1 mM IBMX was applied
to verify expression of the reporter, CFTR. ORs from
dorsal region responded to the following odorants: neryl
acetate (compound 8 in Additional file 2: Supplemental
Table S1), putrescine (compound 40), caffeine (compound 44), ethyl guaiacol (compound 64), ethyl vanillin
(compound 65) and octanal (compound 87). All these
responses were detected in 3-8 separate recordings.
Representative traces are presented in Figure 2A. Summary of the results with significant differences in

Figure 2 Current responses to 100 odorants in oocytes injected with plasma membranes from the dorsal region of olfactory
epithelium. A. Representative traces. Odorants were applied at 300 μM, for 15 sec and at -70 mV holding potential. Odorant number is shown
at the indicated time of application. At the end of the each run, 1 mM IBMX was applied for 5 sec. Arrows indicate responses. B. Current
responses of dorsal region injected oocytes were normalized to the IBMX response in each oocyte (n = 3-8, mean ± SEM). Responses were
normalized to the IBMX response in each oocyte. As a control, M1 receptor was injected and tested for the same odorants (n = 8, mean ± SEM).
Significant differences when compared to M1 receptor responses were indicated as * for p ≤ 0.05, ** for p ≤ 0.001 and *** p ≤ 0.0001.

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odorant responses evoked by receptors from dorsal
regions were compared to the responses in control, M1
GPCR, Gaolf and CFTR expressing oocytes, and significant differences from M1, Gaolf and CFTR are indicated
with * (Figure 3B).
In parallel experiments, ORs from the ventral region
responded to: pentanethiol (compound 22), vanillin
(compound 23), eugenol (compound 24) hexanol (compound 31), decanol (compound 60), octanal (compound
87) and tridecanal (compound 93). Representative traces
are presented in Figure 3A. Significant differences in
odorant responses evoked by receptors from ventral
regions were compared to the responses in control, M1
GPCR, Gaolf and CFTR expressing oocytes and significant differences from M1, Gaolf and CFTR are indicated
with * (Figure 3B).
To control for non-specific responses, all odorants
were tested in the M1 muscarinic receptor expressing
oocytes. Responses in control oocytes expressing the M1
GPCR, along with Ga olf and CFTR to the odorants
listed in the Figure 2 and 3 are presented in the Figure
4. Control oocytes injected with M1 muscarinic receptor, Gaolf and CFTR responded to the following odorants: linalool (compound 35), lyral (compound 43),
isobutylphenyl acetate (compound 77) and lauric acid
(compound 79); indicating direct CFTR activation, independent of olfactory receptor activation. CFTR is known
to have many activators belonging to different chemical

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classes including flavones, xanthines, benzimidazoles,
triazines and thiazolidine like compounds [60].
The physico-chemical properties of odorants activating
olfactory receptors in dorsal and ventral regions of the
olfactory epithelium were analyzed by hierarchical cluster analysis (UPGMA algorithm, MolSoft LLC.) Kurtz et
al [61] showed that solubility of hydrophilic odorants in
the mucosa can be predicted by their air/water partition
coefficient. However, this cannot be applied to hydrophobic odorants, since they get dissolved in the lipophilic mucus and consequently show increased diffusion
[61]. Thus, as an additional predictor of odorant solubility we used the octanol/water partition coefficient.
Descriptors used in cluster analysis were: log of water
solubility and volatility (data obtained from SRC, Syracuse Research Corporation), PSA -polar surface area
and Log P were calculated by MolSoft software [62]
(Additional file 2: Supplemental Table S1). Hierarchical
clustering failed to detect separate clusters based on
these odorant descriptors and differential odorant
responses from dorsal and ventral region.
Despite the detection of different odorants by the two
regions, the significant overlap of odorant properties
made separation based on physico-chemical properties
into clusters impossible.
Water solubility is described by log P - the octanol/
water partition coefficient, where low water soluble
compounds have log P ≥1, while highly water soluble

Figure 3 Current responses to 100 odorants in oocytes injected with plasma membranes from ventral region, endoturbinates. A.
Representative traces. Odorants were applied at 300 μM, for 15 sec and at -70 mV holding potential. Odorant number is shown at the indicated
time of application. At the end of the each run, 1 mM IBMX was applied for 5 sec. Arrows indicate responses. B. Current responses of ventral
region were normalized to the IBMX response in each oocyte (n = 3-8, mean ± SEM). As a control, M1 receptor was injected and tested for the
same odorants (n = 8, mean ± SEM). Significant differences when compared to M1 receptor responses were indicated as * for p ≤ 0.05, ** for p
≤ 0.001 and *** p ≤ 0.0001.

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compounds have log P ≤ -1. Plasma membrane preparations from dorsal region of olfactory epithelium
responded to odorants that have log P in the range
from -1.25-3.9, while preparations from ventral region
responded predominantly to more hydrophobic compounds with log P ranging from 1.36-5.31. However, the
difference in the mean log P value of odorants between
these two membrane preparations was not significant
(two-tailed t-test, P = 0.4507, confidence level a = 0.05)
(Additional file 3: Supplemental Figure S2A). Our set of
100 odorants included some highly volatile odorants like
hexane, acetone and diethyl-ether. We did not detect
responses to these odorants in our plasma membrane
preparations. The volatility of odorants detected by ORs
from the ventral domain ranged from 1.18-4 mmHg for
vanilin, compound 23 (log volatility = -3.9) to 13.8
mmHg for 1-penthanethiol, compound 22 (log = 1.13).
The volatility of odorants detected by ORs from the
dorsal domain ranged from 1.04-5 mmHg (ethyl vanilin,
log = -4.983) to 41.2 mmHg (putrescine, log = 0.61).
The difference in the mean volatility values of odorants
between dorsal and ventral membrane preparations was
not significant (t-test, two tailed, P = 0.929, confidence
level a = 0.05) (Additional file 3: Supplemental Figure
S2B). There were no significant differences in the mean
water solubility (expressed as mg/L and presented as log
values) of odorants detected from dorsal and ventral
regions (t-test, two tailed, P = 0.248, at confidence level
a = 0.05, Additional file 3: Supplemental Figure S2C).

Discussion
Our ciliary plasma membrane preparations, enriched
with olfactory receptors were isolated and fused with
the X. oocyte plasma membrane by direct injection
[63-66].There are many advantages of this microtransplantation approach [66] when compared to cRNA
injection of known OR sequences. First, microtransplantation of ORs allows us to study receptors from defined
regions within the olfactory epithelium (dorsal and ventral). The second advantage is that we can study a number of ORs simultaneously. Microtransplantation has
also been successfully applied to study neurotransmitter
receptors from postmortem brains in the context of Alzheimer’s disease [57], autism [55] and epilepsy [67]. In
the abovementioned references, receptors under study
were ligand-gated ion-channels (e.g. ionotropic GABA
and glutamate receptors) and the voltage-gated Ca +2
and Na+ channels.
We confirmed our ability to identify dorsal region
using OMAS-immunolabeling (Additional file 1: Supplemental Figure S1). The precise function and significance
of OMACS-exclusive expression in this region of olfactory epithelium remains unknown; however, few possible
roles have been indicated. One possibility is that O-

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MACS activates medium chain fatty acids (which can be
perceived as odorants) by addition of coenzyme A, thus
producing acyl-CoA esters that are essential for many
diverse metabolic processes including fatty acid synthesis, phospholipid synthesis and fatty acid oxidation.
Another possibility is that O-MACS may have a role in
zonal segregation of the OE during development, since
its expression precedes OR expression [10].
This is a first time that the olfactory receptors have
been successfully expressed using the microtransplantation approach. The total number of odorant responses
from both dorsal and ventral regions was 14. This represents about 14% of all tested odorants probably reflecting the fact that by using microtransplantation
approach, the responses we detect are from the most
abundant receptors.
We detected octanal responses in both the dorsal and
ventral regions of the olfactory epithelium (Figure 2 and
3). Recently, octanal was detected both as a volatile
released from mouse body, as well as a significant component of mouse urine [68]. Our results confirm the
importance of this aldehyde in the mouse world. In
agreement with our results, Igarashi and Mori showed
that octanal induced glomerular activity from both dorsal and ventral surface of rat olfactory bulb [69].
In this study, olfactory receptor proteins were microtransplanted with other membrane proteins from the
olfactory epithelium in their “microenvironment”, thus
our system does not mirror heterologous expression in
X. oocytes where cRNA of particular OR was injected.
As mentioned earlier, we used 300 μM concentration of
odorants. This value is high when compared with the
odorant concentration used to elicit responses in glomeruli during in vivo experiments [70,71]. As summarized and explained by Oka et al. odorant sensitivity
when tested in isolated olfactory sensory neurons or in
the heterologous expression system is much lower than
the sensitivity seen in the in vivo experiments in the
olfactory bulb, due to the absence of factors like olfactory mucosa and air-flow dynamics [72].
We did not find evidence supporting correlation
between the solubility and volatility of odorants and the
functional expression of olfactory receptors in the dorsal
or ventral region of the olfactory epithelia. Thus, no
simple clustering or relationship between these parameters could be associated with the different regions.
Odorants detected by ORs from the dorsal and ventral
regions showed overlap both in volatility and water solubility, indicating that the location of the ORs within
olfactory epithelium is not related to the physicochemical properties.
Odor coding is a result of the interplay of many different factors, and we are just beginning to understand
the role of some of them. For example, as discussed in

Abaffy and DeFazio BMC Research Notes 2011, 4:137
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Scott et al. paper [34] an increase in carbon chain
length (in case of hydrocarbons) correlates with the
responses in the ventral region. In addition, the airflow
pattern along the nasal cavity has a significant effect
on odor coding [73] as well as selective projection of
axons related to the particular olfactory receptor to
specific glomeruli [74].
A comprehensive review of current knowledge regarding spatial organization of the odorant receptor maps
was presented by Mori et al. [75], while Johnson and
Leon recently summarized the importance of the chemotopic odorant coding in olfactory perception [76].
Mori et al. summarized their previous work and mapped
glomerular responses to 72 odorants divided into 12
structural classes in the dorsal surface of an olfactory
bulb. We used 18 compounds that were the same (from
10 structural groups listed in Mori’s paper, the numbers
in parenthesis are numbers of volatile compounds from
Additional file 2: Supplementary Table 1): aliphatic
ketones (compound 72), cyclic ketones (compounds 4
and 15), hydrocarbons (compound 5), cyclic alcohols
(compound 7), aldehydes (compounds 10, 17 and 29),
aliphatic alcohols (compounds 12, 26, 31 and 60), aromatic aliphatic ketones (compound 14), phenyl ethers
(compounds 1, 16 and 24), phenols (compound 37), aliphatic acids (compounds 30 and 32). Two other structural groups were diketones and ethers. We used
different representative of diketones: butanedione (compound 3) and different representative of ethers: diethyl
ether (compound 46).
In summary, all the structural classes in Mori et al. are
represented in our paper, however some compounds are
different. We do want to point out that our primary reason in selection of volatile compounds was their broad
range in volatility and solubility. In contrast to this previous work, we directly evaluated the hypothesis that
olfactory receptors are spatially organized within the
olfactory epithelium based on the chemical properties of
their ligands.
We compared the volatility and water solubility parameters for odorants that activate rat I7 olfactory receptor (including C7-C11 aldehydes, trans-2-octenal, citral
and citronellal [77]) and found that water solubility
among these odorants differed over a 67-fold range
(20.2 for undecanal to 1340 mg/L for citral). Volatility
differed 57-fold, (0.06 for undecanal to 3.52 mmHg for
heptanal). Compared to the ranges detected in our
study, these results indicate a relatively small range in
volatility and solubility of the preferred ligands for this
particular receptor. It would be interesting to see
whether this applies to other olfactory receptors, particularly those considered broadly tuned. Whether our
results, showing the broader range in odorants volatility
from dorsal region (over 3,900 000 times compared to

Page 8 of 11

100 000 times from ventral region) are consequence of
increased diversity of ORs in that region or are the
result of the presence of more broadly tuned receptors,
remains to be tested.
Because each odorant is delivered to the oocyte in an
identical fashion, the different kinetics (rise time, decay
time, and width) of responses evoked by different odorants both in dorsal and ventral regions may indicate different receptor density, sensitivity and/or different
modes of desensitization (see traces, Figures 3 and 4). A
plethora of GPCR signaling components is known to be
endogenously expressed in X. oocytes [78-81]. A recent
study by the Lefkowitz group [82] demonstrated ligand
bias towards different desensitizing pathways. This offers
an attractive explanation for the variability of the
kinetics of the responses in Figures 3 and 4. It would be
interesting to study whether similar mechanisms exist in
olfactory sensory neurons. This would add an additional
dimension in our understanding of activation/de-activation (desensitization) of olfactory receptors. A huge
odorant space, a huge number of olfactory receptors
and now the possibility that the odorants/ligands of the
same receptor differentially desensitize the receptor,
evokes even more complexity not just in the temporal
dimension of odorant detection, but also in the whole
process of olfactory perception.

Figure 4 Confirmation of OR specificity using M1 receptor as a
control. Representative traces to the selected odorants in X.
oocytes injected with a control, M1 receptor (A and B). Odorant
number is shown at the indicated time of application. At the end, 1
mM of IBMX and 10 μM of ACh was applied for 5 sec.

Abaffy and DeFazio BMC Research Notes 2011, 4:137
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Conclusions
We did not detect a significant correlation between the
physicochemical properties of odorants (solubility and
volatility) and the functional expression of olfactory
receptors between the dorsal and the ventral region of
the olfactory epithelia. A differential sorption of odorants in the mouse peripheral olfactory system is likely to
be mediated by air flow dynamics and physico-chemical
properties of olfactory mucosa.
Additional material
Additional file 1: Supplemental Figure S1. Scheme of mouse
olfactory epithelium and dorsal immunolabeling. A. OMACS
immunolabeling of the dorsal region (green). Blue indicates nuclear
staining. The images were obtained by two-photon microscopy (Zeiss/
BioRad Radiance 2100MP coupled with a Coherent Chameleon Ultra) of
the intact olfactory epithelium at 955 nm excitation and using standard
blue and green emission filter sets. Images are maximum Z-projections
of 10-20 images at 5 micron steps. Each image is a Kalman average (n =
4) acquired at 16-bit resolution. Post-processing was accomplished with
NIH ImageJ. B. The scheme of the sagittal view of the left hemisphere of
mouse olfactory epithelium. A dorsal region, zone I is colored in yellow
and ventral region (endoturbinates (IIa, IIb, III and IV) in orange. OBolfactory bulb. Dashed lines indicate sites of immunostaining images of
anterior (A), middle (M) and posterior (P) part of dorsal region (A).
Additional file 2: Supplemental Table S1. 100 odorants with structures
and all physico-chemical parameters used in Cluster analysis.
Additional file 3: Supplemental Figure S2. Overlapping physicochemical properties of odorants detected by dorsal and ventral
region. A. log P (octanol/water partition coefficient) B. log volatility
(mmHg) C. log water solubility (mg/L) and D. log PSA (polar surface area
in Å2). Dotted lines indicate the range in log P, water solubility, volatility
and PSA of all 100 odorants used in the experiment. Each dot represents
a single physico-chemical value from each odorant that evoked
responses from either dorsal or ventral region.

Acknowledgements
We would like to thank Mrs. Vanessa F. Santos for her assistance with the
electrophysiology.
This work was supported by National Institutes of Health, grant R21
CA132046-01A1 (to T.A.) and by University of Miami Scientific Awards
Committee Research Grant, (to T.A.). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Author details
Department of Molecular and Cellular Pharmacology, Miller School of
Medicine, University of Miami, 1600 NW 10thAve, Miami, 33136, Fl, USA.
2
Department of Neurology, Miller School of Medicine, University of
Miami,1420 NW 9thAve, Miami, 33136, Fl, USA.
1

Authors’ contributions
TA conceived and designed the study, set up the experiments, performed
immunostaining, analyzed the data and wrote the paper. RAD performed
microscopy and critically revised the manuscript. Authors read and approved
the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 2 December 2010 Accepted: 6 May 2011
Published: 6 May 2011

Page 9 of 11

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doi:10.1186/1756-0500-4-137
Cite this article as: Abaffy and DeFazio: The location of olfactory
receptors within olfactory epithelium is independent of odorant
volatility and solubility. BMC Research Notes 2011 4:137.

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