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The Journal of Neuroscience, June 15, 2000, 20(12):4721-4731
Chemical Determinants of the Rat Electro-Olfactogram
John W.
Scott,
Tracy
Brierley, and
Frederick H.
Schmidt
Department of Cell Biology, Emory University School of Medicine,
Atlanta, Georgia 30322-3030
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ABSTRACT |
The chemical properties that determine the distribution of the
electro-olfactogram were studied after exposure of a large area
of the rat olfactory epithelium. Multiple electrodes were placed along
the rostral border of endoturbinate IV on the midline of the nasal
cavity. This array of electrodes spanned a region containing the four
receptor gene expression zones described for the rat. The
response to a series of odorants containing only carbon, hydrogen, and
oxygen was strongly related to electrode position. For most
hydrocarbons, the responses were progressively larger toward the
ventral epithelium. The only exceptions were aromatic hydrocarbons,
which evoked nearly equal response sizes across the epithelium. Ketones
and aldehydes evoked relatively larger dorsal responses than did
hydrocarbons with similar structures. Aromatic ketones and aldehydes
evoked systematically larger responses from the dorsal part of the
epithelium. The response profiles for most odorants were well described
by a linear fit to the electrode position along the dorsal-ventral
position on the epithelium. However, a few bicyclic odorants and
carboxylic acids evoked significantly nonlinear profiles. It is
concluded that there is a systematic distribution of odorant
sensitivity across this part of the epithelium and that this
sensitivity is related to general chemical properties. Other evidence
suggests that these properties extend to other parts of the epithelium.
This spatial sensitivity of the epithelium to odorants probably
contributes to olfactory coding in parallel with the convergence of
axons from olfactory sensory neurons expressing the same receptor type.
Key words:
electro-olfactogram; odorant; rat; hydrocarbon; ketone; olfactory epithelium
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INTRODUCTION |
The understanding of olfactory
coding has been greatly changed by two recent sets of findings. The
first was the finding that rabbit mitral/tufted cells of a particular
part of the dorsomedial olfactory bulb are sensitive to aliphatic
acids, aldehydes, and esters (Mori and Yoshihara, 1995 ). Individual
cells in this part of the bulb also respond maximally to odorous
compounds of a particular size. Recent behavioral data (Linster and
Hasselmo, 1999) have demonstrated that generalization across a series
of aliphatic aldehydes is predictable from those mitral/tufted cell
responses. These experiments show promise of developing a series of
dimensions that will ultimately describe the olfactory stimulus space.
The second important set of findings stemmed from the discovery of the
olfactory receptor gene family (Buck and Axel, 1991), which led to the
ability to localize the expression of these genes by in situ
hybridization (Nef et al., 1992; Ressler et al., 1993, 1994; Vassar et
al., 1993, 1994; Strotmann et al., 1994; Kubick et al., 1997) and other
techniques (Mombaerts et al., 1996). These studies showed that receptor
expression is localized in the epithelium of rats and mice. In
addition, any receptor gene is associated with axons converging onto a
small subset of glomeruli, often one medial glomerulus and one lateral
glomerulus. This convergence pattern provides a potential explanation
for the functional specificity of mitral cell recordings (Buonviso and
Chaput, 1990; Mori and Yoshihara, 1995) and of metabolic
markers of glomerular function (Guthrie et al., 1993; Johnson et
al., 1998, 1999).
Our laboratory has attempted to integrate some of these findings with
electro-olfactogram (EOG) recordings from the olfactory epithelium. The
EOG is a slow potential recorded extracellularly from a population of
olfactory sensory neurons. We found a general correspondence between
the gene expression zones described for neonatal rat and mouse
olfactory epithelium (Ressler et al., 1993; Vassar et al., 1993) and
the distributions of sensitivities of response on the exposed adult
olfactory epithelium to a set of terpene odors (Scott et al., 1997;
Scott and Brierley, 1999). We investigated a wider series of odors in
an intact animal (Scott et al., 1996), but this preparation did not
allow direct comparison with the receptor gene expression zones. In
addition, the intact nasal cavity is subject to influences from
differential sorption of odorants that alters the concentration in
different parts of the epithelium (Ezeh et al., 1995; Kent et al.,
1996) (P. E. Scott-Johnson, D. Blakley, and J. W. Scott, unpublished
observations). In the present series, we have investigated the
distribution of responses to a series of aliphatic, terpene, and
aromatic compounds and related odorants. The series were chosen
to investigate the structural relationships with the terpene odors that
we had tested previously. That is, the effects of different chemical
structures such as open chains versus cyclic structures and of
different functional groups were explored where we could find
appropriate commercially available stimuli. These results show
systematic differences between saturated hydrocarbon odorants, which do
not have polar bonds, and structurally similar compounds containing
oxygen, which adds an asymmetric charged group to the molecule. These
predictable relationships in the responses may be important in sensory function.
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MATERIALS AND METHODS |
Surgical preparation. Male Sprague Dawley rats
(375-525 gm) were killed with an overdose of Nembutal (20 mg/kg) and
placed in a stereotaxic frame. The medial surface of the olfactory
epithelium overlying the endoturbinate bones on the left side was
exposed as in our previous papers (Scott et al., 1997; Scott and
Brierley, 1999). The endoturbinate bones are the protrusions of the
ethmoid bones that extend close to the midline of the nasal cavity. The entire region from endoturbinate II to endoturbinate IV was exposed from the dorsal margin at the cribriform plate to the ventral margin at
the respiratory epithelium. The room temperature was kept cool
(~17°C) to prolong the usefulness of the preparation. A room
humidifier and plastic sheeting were used to maintain high humidity
near the preparation. A constant flow of humidified air (see below) was
immediately directed over the epithelium.
Recording procedure. Simultaneous recordings were made with
four or eight glass micropipettes filled with agar made up in Ringer's
solution. These micropipettes were broken to obtain resistances <5
M . The leads from these electrodes were connected to four-channel AC-coupled amplifiers (low-frequency cutoff of 0.1 Hz and
high-frequency cutoff of 30 Hz). The electrodes were placed with
independent manipulators, but we spaced them as equally as possible
along the rostral borders of endoturbinate IV. Placement of the
electrodes was monitored by listening to an audio monitor output from
the amplifiers to fix the electrodes at the point of first contact with
the tissue. This should place the electrode at the mucosal surface and
give the maximal response amplitude. It is likely, however, that some
electrodes advanced into the tissue slightly as others were being set.
The electrodes usually remained in place during a complete recording
session, but on rare occasions two sets of placements were used during
a single experiment. Electrodes were reset in the same position if
vibration or slight tissue drying resulted in loss of contact. An
indifferent electrode (a silver chloride electrode connected by an agar
bridge to a saline-soaked cotton pad) was placed on the frontal bone.
Odor stimulation and computer control. Odorants were
injected into a humidified, clean air stream flowing at 1000 ml/min. This air stream, in turn, flowed into a chamber with two large ports
for clean air input and vacuum. Before each stimulus, the odor was
turned on for 20 sec to allow the buildup of the odor in the system.
During this buildup period, the vacuum port was opened to wash the odor
from the stimulus port and prevent stimulation. The vacuum line flowed
in excess of 1000 ml/min. The actual rate was adjusted to give no EOG
response at the onset of buildup or no upward drift at the presentation
of blanks. Stimulation was applied to the epithelium by the closing of
the vacuum port. This procedure is similar to that described by Kauer
and Shepherd (1975) or by Mackay-Sim and Kesteven (1994). Although it
is likely that there were some transients of airflow during these valve
operations and some differences between the total flow in the buildup
and stimulation periods, the blanks usually produced responses of <0.4
mV. If the blanks became larger, the system was flushed with clean air
to reduce contamination. The odor port was placed 1 cm from the
epithelium and centered over the recording area. The port approached
the epithelium from below the bar of the stereotaxic frame at an angle
of ~30°. In a previous paper (Scott et al., 1997), we could detect
no influence of the tube angle on the differential distribution of odor responses.
Odor stimuli were generated with an air dilution olfactometer, and
concentration was expressed as a proportion of air saturated with
odorant. A syringe pump forced air through the head space in glass
bottles in which 2-5 ml of the odorant was used to cover the bottom.
The odorant sample bottles were connected by Teflon tubes to ports in
the glass tube next to the epithelium. A BASIC language program
controlled the pump rate and set valves to inject each odor. This
program used a set of standard files to determine the odor sequence and
concentration. Stimuli were presented in sets of seven, with each set
preceded by a standard isoamyl acetate stimulus at a dilution of
10 1 and followed by a blank. In addition
to the standard isoamyl acetate stimulus, each set began with the three
terpene odorants D-carvone, D-limonene, and
1,8-cineole. We have used these stimuli extensively before, and their
use ensured that consistent responses were obtained. Overall, results
with the eight electrodes were obtained from 98 animals for these three
odorants, with smaller numbers of animals for the other test odorants.
Odorants were presented in a descending series of dilutions from 1 × 101 to 1 × 103; 1.5 min elapsed after each stimulus
presentation. The recording session usually lasted 3-5 hr. We
sometimes terminated an experiment earlier if the responses began to
deteriorate badly on three or more electrodes. We were often able to
repeat the concentration series several times or to substitute a
different set of odorants. We always tested the terpene odorants at the
end of the session to be sure that the response to these had remained
stable. Because of this arrangement, we have much more extensive data
for the three terpene odorants than for most other stimuli.
Data analysis. The amplifier outputs were fed through an
analog-to-digital converter (digitized at a rate of 26 Hz) to a
computer that plotted the traces and computed the peak negative voltage relative to the baseline just before the stimulus. This peak voltage was printed for each plot and was also stored in a computer file for
later statistical analysis. The plots were inspected for quality control during data collection and before analysis. Data were not used
in the analysis if the standardized blank response was >1 mV or if the
average response to the isoamyl acetate standard was <4 mV. (Large
blank responses were observed only when the response to the isoamyl
acetate standard was very large.) If a preparation was excessively
noisy or, in rare cases, it failed to show the response patterns
normally evoked by the three terpene stimuli, the experiment was terminated.
Responses to all stimuli were standardized to the isoamyl acetate
stimulus. This had two purposes. It helped minimize differences between
recordings that might result from slight damage or drying at one site.
It also allowed us to adjust for the gradual rundown of the response
over the period of recording. The response to each odor was divided by
the immediately previous response to the standard stimulus at that
electrode and was multiplied by the best estimate of the optimal
isoamyl acetate response. This estimate was established by averaging
the initial response to the standard concentration of this
stimulus (1 × 101) over 10 preparations with four electrode recordings. Although there is
considerable variation in the size of responses to the standard odor,
the average of those responses changes little across the
dorsal-to-ventral extent of endoturbinate IV (Scott and Brierley, 1999). The frequent presentation of the standard stimulus allowed us to
correct for changes over time at each electrode independently. A second
normalization was applied within each recording session to remove
variation that may have come from slight leaks in the system or
systematic differences between animals. In this normalization, the
response at each electrode to each odor was divided by the average
response to that odor for the particular session. This normalized
response was then multiplied by the average response across all
sessions for that odor and concentration.
For the statistical comparison of response variation across position
and across recording sessions, we used stepwise multiple regression
instead of ANOVA. A photograph of each epithelium was made with
the electrodes in place, and the positions of the electrode tips were
marked on this photograph. Photocopy enlargements were made of the
marked photographs, and a standard transparent overlay with rectangular
coordinates was used to record the positions of the tips. We used
regression analysis for these data because it allowed comparison of
odorants that were not recorded in the same preparation.
Response profiles (standardized EOG amplitudes obtained for given
concentrations and electrode positions) were plotted for each odorant
stimulus. In the simplest cases, the response profiles could be fit by
a simple linear equation:
|
(1)
|
where Y was the standardized response magnitude,
X was the position measured along the rostral border of
endoturbinate IV (see Fig. 1A), and
ci values were the intercept and slope of
the line. For other profiles, a higher order polynomial regression formula was necessary for estimating the curve:
|
(2)
|
where ci values were the
coefficients of the polynomial terms.
To test whether profiles had significant nonlinear components, we used
an F test based on the sum of squares in regressions with
linear and nonlinear terms (Kleinbaum and Kupper,
1978):
|
(3)
|
where the degrees of freedom for the numerator are the
difference in the number of parameters for the total model and the linear model. The degrees of freedom in the denominator have been reduced because of the normalization.
When we compared the linear slopes of two response profiles, we
used the formula:
|
(4)
|
where D = 0 or D = 1 is a dummy
variable representing a comparison of two odors. The dummy variable is
a standard regression technique that allows comparison of multiple
variables in a single analysis. In this case, it allowed us to test
whether separating the points from two odors allowed a significantly
better fit than fitting a single slope through all the points. The
F test for Equation 4 was:
|
(5)
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RESULTS |
Aliphatic odorants
Figure 1A shows
the general placement of recording electrodes across the rostral border
of endoturbinate IV, which was used in all the figures reported here.
This view is similar to the whole-mount display of olfactory expression
zones shown by Vassar et al. (1993). When we refer to electrode
numbers, the "number 1" electrode was the most dorsal, and the
"number 8" electrode was the most ventral. The scale shows the
measurement in millimeters obtained from photographic overlays as
described in Materials and Methods. Figure 1B shows
the distribution of standardized responses for the straight-chain
hydrocarbon heptane. This distribution had a steep positive slope, in
that the responses became larger as distance from the dorsal part of
the epithelium increased. At the highest concentration (1 × 101) the curve fit to the response
profile was linear, whereas at 1 × 102 there was a slight, but
statistically significant, curvature evidenced by significant second-
and third-order terms in the polynomial fit. The coefficients of
determination for the linear and nonlinear equations were 0.78 and
0.82, respectively, showing that the nonlinear terms account for less
than a 4% difference in the variance. Figure 1, C and
D, shows distributions for heptanol and heptanoic acid
(enanthylic acid). The addition of alcohol (OH) or acid (COOH) groups
to the heptane molecule significantly changed the profiles. The slopes
determined by linear regression of the heptanol and heptanoic acid
profiles were significantly different from the heptane slope at any
concentration. In addition, the stepwise regression analysis showed
significance (p < 0.01) for the second-
and third-order terms in the profile for heptanoic acid. We did not
extensively study a series of carboxylic acids in this series because
the responses were so small. However, tests of hexanoic acid at 1 × 101 (data not shown) also showed a
similar distribution that showed deviation from a linear profile with
p < 0.05.
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Figure 1.
A, The positions of the eight
electrodes (filled circles) set up
in each recording session. These electrodes were equally spaced along
the rostral border of the midline of endoturbinate IV.
B-F, The response profiles for a set of aliphatic
odorants with six or seven carbons. The concentration
key in B applies to all the
panels in this figure. We plotted responses as dilutions
of 1 × 101 and 1 × 102 for most odorants in this figure and below
(see Figs. 3-5). In some cases in which more extensive data
existed, we also plotted the responses for 1 × 103. For heptanoic acid (C),
we have plotted responses only at 3 × 101
because they were very small at lower concentrations. All the plots of
response profiles are presented on the same scale. The response
profiles were fit with straight lines if
the comparison of regression by first-order and third-order equations
did not indicate a significant improvement in fit by the higher order
equation. The numbers of rats are indicated for
each panel. In D, the dashed line
indicates that the regression is not significant.
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The effect of adding double bonds to the unsaturated heptane molecule
was small (Fig. 1E,F) and similar to that of
the addition of oxygen-containing groups. The hexadiene and hexatriene
odorants both produced stronger responses than heptane. In the case of hexatriene, it appears possible that the response reached a maximum, causing a flattening of the profile. Therefore, it was more appropriate to compare the heptane profile at 1 × 101 with the hexadiene and hexatriene
profiles at 1 × 102. The slope for
hexadiene was significantly (p < 0.01) more
positive, but the slope for the hexatriene profile was not
significantly different from that for heptane.
Because there have been several studies of the effect of chain length
on response to aliphatic aldehydes or acids (Mori and Yoshihara, 1995;
Johnson et al., 1998, 1999; Krautwurst et al., 1998; Zhao et al., 1998;
Linster and Hasselmo, 1999), we compared a series of aldehydes and
alkanes in 11 rats using a four-electrode array along the same extent
of endoturbinate IV. These results are shown in Figure
2. Even though these recordings were
conducted by a different person on a different setup, the profiles for
hexane agree quite well with those collected with eight electrodes
(data not shown). The profiles were fit by linear equations for
simplicity of comparison. There were no significant differences in
slope within either series across the dorsal-to-ventral extent of
endoturbinate IV, but the slopes for each of the aldehyde profiles were
different from each of the corresponding alkane profiles.
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Figure 2.
Aliphatic odorants tested with the four-electrode
array. Top row, Three of the five
straight-chain alkanes, identified by the number of carbons, are shown.
Although both the hexane (C-6) and heptane
(C-7; data not shown) profiles had slightly steeper
slopes, none of the slopes in this panel were
significantly different from each other. The numbers of
animals are indicated in each panel.
Bottom row, None of the aldehyde profile
slopes were significantly different from each other, but each of the
aldehyde profile slopes was significantly different
(p < 0.05) from each of the alkane profile
slopes. The dashed lines indicate that the regressions are
not significant.
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Figure 3 illustrates a series of cyclic
compounds. In these cases, the profile for the saturated hydrocarbon
cyclohexane had a steep positive slope with larger ventral responses.
The response distributions of cyclohexane and 1,3-cyclohexadiene did
not have significantly different linear slopes at any pair of
concentrations tested, even when we attempt to match response size
(e.g., comparing the strongest concentration of cyclohexane with the
weakest concentration of cyclohexadiene). Thus the effect of two double
bonds was small, as in the straight-chain odorants. In contrast, the
response profile slope for benzene was significantly less positive than
was the slope for cyclohexane, indicating that the resonance associated with aromatic compounds has a significant effect on the profile.
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Figure 3.
Cyclic odorants. A, The response
profile for cyclohexane. B-F, The effect on the
response profile of adding double bonds or oxygen atoms to the ring. In
C-E, the dashed lines
indicate profiles for which the regression was not significant for
either a linear or curvilinear function. A dashed
line is used merely to indicate the central tendency of
the points. It is important to note, however, that even in these cases
there is a significant response indicated by a highly significant
constant in the regression analysis and that the points are closely
grouped around the line, indicating that the
line is a reasonable estimate of the response
profile.
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Adding an ester group (C-O-C=O) to either the cyclohexane or benzene
rings, as in methyl cyclohexane carboxylate or methyl benzoate,
respectively, produced a large change in the slope of the profile. The
slope for the methyl benzoate profile was significantly more negative
than a linear slope through the methyl cyclohexane carboxylate profile.
This may reflect the summation of the effects of the ester and of the
aromatic ring. Comparison of the aromatic ether (anisole) and aromatic
ester (methyl benzoate) profiles suggests that esters had more powerful
effects than ethers in altering the response profile. The comparison of
benzene and anisole slopes was significant when the two were compared
at the highest concentration but not when the responses were matched
for equal magnitude (e.g., benzene at 1 × 101 and anisole at 1 × 102).
Terpene odorants
Terpene odors are of interest because they occur in so many
odorous materials. Figure
4A shows the profile
for isopropyl cyclohexane, which is very similar in structure to the
hydrocarbon backbone of the terpene compounds in the remainder of this
figure. The response profile for this odorant was not significantly
different in slope from that of the cyclohexane concentrations that
evoke responses of similar size. The addition of two double bonds in the  terpinene odorant did not produce a profile significantly different in slope from that of the isopropyl cyclohexane profile. However the profile for D-limonene, in which the
double bonds are in different positions, had significantly different
slopes from that of both the terpinene and isopropyl cyclohexane
profiles. The ketone group in menthone has more effect on the response
than do the double bonds of limonene and terpinene, so that
menthone has a positive slope. The addition of the double bonds and the ketone group in D- and
L-carvone produces a significantly more negative
slope than does the menthone response, although the profiles for the
two carvone forms were not statistically different.
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Figure 4.
Terpene odorants. A, The response
to isopropyl cyclohexane, which is very similar in structure to the
basic menthane backbone of the terpene compounds illustrated here.
B-F, The effect on the response profile of adding
double bonds and/or ketone groups to the terpene ring. The profiles for
the hydrocarbon odorants in A-C all have positive
slopes, i.e., successively larger responses on the more ventral
electrodes. Note that the enantiomers of carvone in E
and F produce nearly identical response profiles.
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Bicyclic odorants
As described in our previous publication (Scott and Brierley,
1999), the bicyclic terpene 1,8-cineole produced a strongly nonlinear
profile with a peak in the intermediate ventral region, in spite of the
presence of an oxygen atom. The third-order equation fit the profiles
for this odor significantly better at each concentration from 1 × 101 to 1 × 103. The ratio of the coefficients of
determination (R2 indicating
the proportion of the variance accounted for by the regression) ranged
from 1.25 for the lowest concentration to 1.57 for the highest
concentration. Figure 5 tests whether
that profile shape was common to all bicyclic compounds or to ethers
spanning the cyclohexane ring. The bicyclic hydrocarbon norbornane
evoked a response profile not significantly different from that of
heptane (Fig. 1) or cyclohexane (Fig. 3), and an ether spanning the 1,4 positions of the ring (Fig. 5B,C) had very little effect on
the response. Of these profiles, only 1,4-cineole at 1 × 102 was significantly nonlinear.
Addition of a ketone to other positions on the ring produced a
curvilinear response in the case of camphor (Fig. 5E),
although the curvature was small relative to that for 1,8-cineole (Fig.
5D). Norcamphor (Fig. 5F), with a more
exposed ketone, had no significant curvature but had a slight positive slope close to that of menthone (Fig. 4D). For none
of the other odorants was the nonlinear effect as great as that for
1,8-cineole. For example, the ratio of the coefficients of
determination for camphor was 1.40 at 1 × 101 but was 1.15 at 1 × 102.
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Figure 5.
Bicyclic odorants. A, The saturated
bicyclic compound norbornane produced responses with a positive slope
similar to that of other saturated hydrocarbon odorants.
B-F, Addition of ether or carbonyl groups altered that
slope. For 1,8-cineole and, to a much lesser extent, camphor, there is
a marked curvature to the profile with the peak response occurring
~3-4 mm from the top of endoturbinate IV.
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Comparison of other odorants
Linear profiles
To make an overall comparison of the response profiles of the 40 odors in our sample, we chose to use the linear slope as the major
comparison along the x-axes of Figure
6. On the y-axes, we
represented the proportion of the variance accounted for by a simple
linear regression as opposed to a regression model using first-,
second-, and third-order terms (see Materials and Methods). On these
plots, profiles that were nearly linear, including those for isopropyl
cyclohexane and menthone, are found at the top, whereas the
1,8-cineole profile lies close to the 50% level. Because most of the
profiles were essentially linear, it was appropriate to use the slope
for these summary figures.
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Figure 6.
Plots summarizing the response profiles for most
of the odorants in this paper. The x-axis shows the
linear slope for each profile. The y-axis shows the
ratio of the variance around a linear regression to the variance around
a third-order regression. This ratio was close to 1.0 for
D-carvone but was only ~0.6 for 1,8-cineole. The data are
presented by type of compound to avoid crowding. The
identification numbers indicating the
position of each odorant in this space are in order of the linear
slope, with odorant 1 (benzaldehyde) having the greatest negative
slope. The asterisks indicate profiles for which there
was a significant nonlinear response (p < 0.01) by an F test (Eq. 3) comparing the linear and
nonlinear regression analyses at the concentration illustrated.
A, All profiles to the left of the
dashed line labeled I were
significantly different from all others (with the exception of anisole
that was not different from toluene). All profiles to the
left of the dashed lines
marked II and III were significantly
different from those to the right of those
lines. B, Similar significant differences
were demarcated by the dashed lines in
this panel. C, The camphor profile was
significantly different from all other profiles (except that of
norbornane). D, The odorants to the left
of the dashed line were not significantly
different from each other but were significantly different from the
hydrocinnamaldehyde, phenylacetaldehyde, and benzyl acetate profiles.
At lowered criteria (p < 0.05) the methyl
benzoate profile was different from the phenyl acetate and phenethyl
acetate profiles. Note that the x-axis of
D corresponds to the left 40% of that of
the other panels. Odorant
codes, The numbers of rats tested are
indicated for each odorant in parentheses, followed by a
lowercase letter showing the
concentration code as follows: a, 1 × 102;
b, 3 × 102; and c, 1 × 101. 1, Benzaldehyde
(n = 9; b); 2, methyl benzoate
(n = 11; a); 3,
D-carvone (n = 78; b);
4, ethyl benzaldehyde (n = 12; b);
5, L-carvone (n = 6; b);
6, propyl benzoate (n = 8; b);
7, menthone (n = 8; b);
8, isopiperiterone (n = 5; a);
9, phenethyl acetate (n = 10; c);
10, hydrocinnamaldehyde (n = 10; c);
11, heptanoic acid (n = 11; c);
12, phenylacetaldehyde (n = 5; c);
13, phenyl acetate (n = 9; b);
14, benzyl acetate (n = 12; b);
15, methyl cyclohexane carboxylate
(n = 4; c); 16, fenchone
(n = 12; a); 17, limonene oxide
(n = 5; b); 18, anisole
(n = 10; b); 19, norcamphor
(n = 9; a); 20, heptanol
(n = 10; a); 21, cyclohexyl acetate
(n = 4; c); 22, toluene
(n = 11; a); 23, camphor
(n = 8; c); 24, benzene
(n = 13; a); 25,
D-limonene (n = 78; b);
26, ( terpinene (n = 7; b);
27, 1,4-cineole (n = 9; a);
28, isopropyl cyclohexane (n = 3;
c); 29, 1,8-cineole (n = 77; a);
30, 1,4-epoxy cyclohexane (n = 5;
a); 31, cyclohexane (n = 17; b);
32, norbornane (n = 10; c);
33, terpinene (n = 13; b);
34, hexane (n = 15; b); and
35, heptane (n = 8; b).
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The odorants were matched as closely as possible for average response
magnitude. For example, the average response size for norbornane at
1 × 101 (Fig. 5A) was
approximately equal to the average response magnitude for methyl
benzoate at 1 × 102 (Fig.
3F), even though the slopes were opposite. This match
was not perfect; for example the heptanoic acid (Fig. 1C)
response was a little smaller. The purpose of this matching was to
reduce the possibility that the profiles were differentially affected by saturation of the response at high concentrations. There was a
suggestion of such an effect in the profiles for 1,3,5-hexatriene (Fig.
1F), which had a steeper slope at 1 × 102 than at 1 × 101. Because there was no concentration
tested at which the average response magnitude for this odorant matched
those for heptanoic acid, we excluded this odorant from these plots.
For the same reason of inability to match response magnitude, we also
excluded 1,3-hexadiene and the cyclohexadienes. Of those excluded
compounds, 1,3,5-hexatriene and 1,3-hexadiene would have plotted at the
far right at the lowest tested concentration. The
cyclohexadienes would have plotted further left, near the
terpinene profile.
The characterization of odorant profiles in these plots by a single
parameter allowed clearer comparisons than were possible with Figures
1-5. These figures support the conclusion that hydrocarbon odors
evoked larger ventral responses (i.e., positive slopes), whereas
odorants with carbonyl groups (i.e., an oxygen double bonded to a
carbon) produced profiles with negative slopes. This is true among the
aliphatic and cyclic odorants (Fig. 6A), terpene odorants (Fig. 6B), and bicyclic odorants (Fig.
6C). This figure also shows that there were significant
differences among the hydrocarbons. These differences were assessed by
comparison of the linear slope through the profiles using Equations 4
and 5. The profiles for straight-chain odorants had significantly
steeper slopes than did those for cyclic odorants, and the aromatic
odorants had even flatter profiles (Fig. 6A). There
were also differences among the odorants with carbonyl groups.
Interposing carbon or even single-bonded oxygen atoms between the
carbonyl group and a ring significantly altered the slope (Fig.
6D). The slopes for most of the terpene ketones (Fig.
6B) were similar to those for benzaldehyde and methyl
benzoate (Fig. 6D), but cyclohexane ester profiles (Fig. 6A, odorants 15, 18) had
significantly less negative slopes. This suggests that the resonance
within the aromatic ring is important.
On the other hand, ethers did not usually have very strong effects on
the profile slopes. Although the profile slopes for benzene and anisole
were significantly different, the linear profile slopes for 1,4-epoxy
cyclohexane, 1,4-cineole, and 1,8-cineole were not significantly
different from the slopes for norbornane and cyclohexane. Limonene
oxide (Fig. 6B, odorant 17) was the ether with the greatest difference in profile slope from that of the
homologous compounds such as limonene or isopropyl cyclohexane. However, as noted above, the profile for limonene oxide was not as
steep as that for the ketones.
Nonlinear profiles
Some of the profiles, notably that of 1,8-cineole and
heptanoic acid, had significantly nonlinear response profiles, as shown by their position significantly below the top of
Figure 6. Those for which the nonlinearity of the profiles was
highly significant (p < 0.01 by F
test) are marked with asterisks in Figure 6. The y-axis of the plot is intended solely to illustrate those
profiles for which the linear slope was not a good fit and not as a
valid descriptor of the profiles. For example, the cyclohexyl acetate profile was linear at other concentrations, whereas the 1,8-cineole profile was strongly nonlinear at all concentrations. For the bicyclic
odorants, it appears that the nonlinear profiles were associated with
the presence of methyl groups that provide some steric hindrance of the
ether or carbonyl groups. Testing this hypothesis will require
additional data.
| |
DISCUSSION |
There is a long series of observations [Mustaparta
(1971); Kauer and Moulton (1974); for review of other reports, see Ezeh et al. (1995); Scott et al. (1996, 1997)] showing preferential activation of parts of the olfactory epithelium by particular odorants.
The present study proposes some consistent relationships that hold
across a series of chemical structures. It is premature to try to state
a general rule for all of these stimuli, but it is clear that for any
of the homologous sets of stimuli (i.e., aliphatic, terpene, cyclic,
bicyclic, and aromatic) that the more polar compounds (represented by
carbonyl groups compared with hydrocarbons or ethers) are shifted in
the direction of having larger dorsal responses. It is also clear that
there are important differences between the hydrocarbons of these
different odorant sets, especially for comparison of the aromatic and
alkane odorants.
These results expand on our previous results by detailing the
effects of hydrocarbons and polar compounds across a range of chemical
structures and applying those odorants to an opened epithelium where we
could observe regions where the responses were quite different. There
is an important difference from our previous study of odorant structure
(Scott et al., 1996), which was conducted by penetrating the olfactory
epithelium from the outside surface, that is, through the lamina
propria and nerve layer. That approach had two important consequences:
the pattern of odorized airflow was different from that in the present
case, and access was restricted to the most medial and most lateral
parts of the epithelium. The airflow pattern is significant because of
the possibility that some odorants might be more extensively sorbed by
the lining of the nasal cavity, thus removing them from the air stream
before they reached the receptor surface (Hahn et al., 1994;
Kent et al., 1996) (Scott-Johnson, Blakley, and Scott, unpublished
observations). Our subsequent reports (Scott et al., 1997; Scott
and Brierley, 1999) used direct application of odorant to the exposed
mucosal surface of the olfactory epithelium to reduce the influence of this effect. We showed that differential responses could be obtained with very simple airflow patterns and that the direction of airflow did
not determine the response pattern in that preparation. However, some
of the results reported here may differ from those with the intact
nasal cavity (Scott et al., 1997), in that responses to some highly
polar odorants were proportionally larger in the ventral part of the
exposed epithelium than they were in the lateral part of the intact
epithelium in the previous study. Thus the data of the present study
may give a more realistic appraisal of the direct response of the
epithelium but may underestimate the selectivity in the intact nose.
Our subsequent reports using the opened epithelium (Scott et al., 1997;
Scott and Brierley, 1999) explored a much larger surface of the
epithelium but concentrated on a small number of terpene odorants. One
significant finding with that approach was that 1,8-cineole evoked a
more complicated response profile than did the other stimuli. That fact
could not be appreciated using the intact nose preparation because of
restricted access to the epithelium. That observation with 1,8-cineole
has been confirmed here and extended to show that other bicyclic ethers
also evoke larger ventral responses than dorsal responses, although
their profiles do not exactly match that of 1,8-cineole. This
observation shows that the larger ventral responses are not restricted
exclusively to hydrocarbons.
Some of the profiles were significantly nonlinear. These included
most notably the 1,8-cineole and carboxylic acid responses, but also
the camphor and fenchone (profile not shown) responses. For the
bicyclic compounds, there is a suggestion that the steric hindrance of
the ether or carbonyl group by methyl groups might be an important
factor in whether these profiles showed an increased response in the
intermediate region of endoturbinate IV. The case of the carboxylic
acids was different. For those stimuli, the pattern was nearly
opposite, with a slight decreased response in the intermediate region
compared with either the dorsal or ventral regions. Although it is
possible to imagine a single process that would determine the major
slope of most of the profiles shown here, the nonlinear profiles
suggest that there are several processes involved in some cases.
The issue of the mechanism(s) producing these profiles is not addressed
by these data. We have suggested that they may result from the
olfactory gene expression zones as described by Vassar et al. (1994)
and Ressler et al. (1993). In that case, there would be a
preponderance of particular types of receptors in each different zone.
Interestingly, the nearly linear profiles seen here and even in data
from individual animals (cf. Scott and Brierley, 1999) do not suggest
discrete regions of sensitivity as we might expect from the strictest
interpretation of the zonal hypothesis. Nevertheless, these recordings
may not have the resolution to test whether the boundaries of the gene
expression zones are functional boundaries. Although direct odorant
application greatly reduces the chance of differential odorant sorption
along the pathway to the receptor sheet, it does not remove the
possibility of differential rates of entry of stimuli into the tissue.
We have discussed previously the possibility of regional differences in
sorption (Scott and Brierley, 1999). We have not seen differences in
the kinetics of the responses that would support this interpretation,
but this has not been formally studied.
If the general pattern of response seen across endoturbinate IV
is general on the olfactory epithelium, then there would be a very
definite pattern of input into the olfactory bulb related to a set of
odorant chemistry because of the pattern of projection of axons of
olfactory sensory neurons (Astic et al., 1987; Schoenfeld et al.,
1994). Such a pattern would be consistent with physiology indicating a
larger response to carboxylic acid stimuli in the dorsal part of the
olfactory bulb (Mori and Yoshihara, 1995; Johnson et al., 1999) and a
larger alkane response in the ventral part of the bulb (Mori and
Yoshihara, 1995). If our data are borne out on other surfaces of the
epithelium, it would suggest that there is a very general pattern of
these inputs that extends far beyond the aliphatic odorants considered
in those reports. Such a relationship, taken together with recent
evidence of a rostral-to-caudal response gradient for the length of
acid and aldehyde odorants in the olfactory bulb, suggests a map of
chemical properties on the olfactory bulb (Johnson et al., 1999; Rubin
and Katz, 1999). So far, we have seen no evidence of a regional
variation of response to the chain length of aliphatic aldehydes or
alkanes in our preparation, and those gradients in the bulb response
probably arise from the pattern of selective projection of axons
related to particular receptor genes to specific glomeruli
(Ressler et al., 1994; Vassar et al., 1994; Mombaerts et al.,
1996). One of those receptor types has been shown to be associated with
high-chain length specificity among the aliphatic aldehydes (Krautwurst
et al., 1998; Zhao et al., 1998). Such high specificity has not yet
been demonstrated in olfactory sensory neurons (Duchamp-Viret et al.,
1999; Malnic et al., 1999).
The pattern of response demonstrated here would certainly not
account for all the variance in the response to odors. For example, we
know that in some cases enantiomers of the terpene odorants smell
different (Laska and Teubner, 1999) and seem to bind different receptors (Krautwurst et al., 1998); yet at least in the case of
carvone, these enantiomers have indistinguishable profiles. Thus the
pattern of the responses in the different regions of the epithelium is
certainly refined by the pattern of projection from olfactory sensory
neurons expressing particular receptor genes onto specific glomeruli.
Yet the EOG patterns are probably in register with the projection
patterns from the mucosa to the bulb (Astic et al., 1987; Schoenfeld et
al., 1994) and thus determine a significant portion of the spatial
pattern of the bulbar response to odor. Such observations make it clear
that there is a strong spatial pattern in this input. On the other
hand, the fact that responses to all odorants can be found in all parts
of the epithelium may help us to understand why removing large parts of
the olfactory bulb does not remove the response to particular odors, as
demonstrated by Slotnick and colleagues (Slotnick et al., 1997; Lu and
Slotnick, 1998). Laurent (1999) has written recently a provocative
challenge to the idea that the pattern of spatial representation of
odorants can have a significant effect in odor coding. Observations
such as ours coupled with the demonstration of a spatial localization in the acid or aldehyde response measured with 2-deoxyglucose (Johnson
et al., 1999), optical imaging (Rubin and Katz, 1999), or
electrophysiology (Yokoi et al., 1995) provide strong evidence of such spatial representation. The spatial localization of response within the olfactory bulb is probably produced by several factors: (1)
the regional distribution of sensitivity described here, (2) the
effects of sorption along the airway that probably acts to accentuate
the separation of polar and nonpolar odorants (Hahn et al., 1994; Ezeh
et al., 1995; Kent et al., 1996) (Scott-Johnson, Blakley, and Scott,
unpublished observations), (3) the convergence pattern of
olfactory receptor neuron axons expressing the same receptor onto small
populations of glomeruli (Ressler et al., 1994; Vassar et al.,
1994; Mombaerts et al., 1996), and (4) interneuronal interactions within the olfactory bulb (Yokoi et al., 1995).
Therefore, it is likely that discrepancies will be evident when
different levels of the system are compared. It is too early to state
the degree to which any or all of these spatial constraints are
essential for odor discrimination.
| |
FOOTNOTES |
Received Jan. 27, 2000; revised April 4, 2000; accepted April 4, 2000.
This work was supported by the National Institute of Deafness and Other
Communications Disorders Grant DC 00113. We thank Dr. M. Chaput for
reading a previous draft of this manuscript and Dr. D. Goldsmith for suggestions.
Correspondence should be addressed to Dr. John W. Scott, Department of
Cell Biology, 1648 Pierce Drive, Emory University School of Medicine,
Atlanta, GA 30322-3030. E-mail: JohnS{at}cellbio.emory.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
