Chem. Senses 24: 679-690,
1999
© Oxford University Press 1999
A Functional Map in Rat Olfactory Epithelium
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322-3030, USA
Correspondence to be sent to: John W. Scott, Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322-3030, USA. e-mail johns{at}cellbio.emory.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Multiple (four or eight) electrode arrays were placed for simultaneous electro-olfactogram (EOG) recordings of responses to a series of odors applied directly to the olfactory epithelium. Three different surfaces of the epithelium were exposed in rats immediately after death by anesthetic overdose. We tested three terpene compounds (carvone, limonene and 1,8-cineole) across the epithelium along the medial surface of the endoturbinate bones. Carvone, a ketone, evoked larger responses dorsally on the epithelium. The largest responses to 1,8-cineole (an ether) were seen in an intermediate-ventral region. The responses to limonene (a hydrocarbon) did not vary greatly across the regions, although they were often larger ventrally. The response distributions deviated from this simple pattern on the caudal part of endoturbinate IV, where the carvone responses were small and the limonene responses were larger. These differences were evident across a substantial concentration range. Similar distributions were seen for these three odors in tests along the dorsal-to-ventral direction across the nasal septum and in the medial-to-lateral direction across the dorsal aspect of one of the endoturbinate bones reaching out into the lateral recess. We argue that the spatial distributions of responses are correlated with the olfactory receptor gene expression zones.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The question of regional specificity of mammalian olfactory receptors is still an important one. Although axons from cells expressing the same receptor gene converge onto a single set of olfactory bulb glomeruli in rodents (Ressler et al., 1994
; Vassar et al., 1994; Mombaerts et al., 1996), this
convergence is limited by the organization of the olfactory epithelium. Receptor cells expressing
any
particular olfactory receptor gene are usually found within one of four longitudinal zones within
the
olfactory epithelium of rats and mice (Nef et al., 1992; Ressler et al., 1993; Vassar et al., 1993;
Strotmann et al., 1994; Wang et al., 1998). This convergence is consistent with physiological evidence suggesting that each
glomerulus may receive a different sampling of information about the olfactory environment (Leveteau and MacLeod, 1966; Lancet et al., 1981; Imamura et al., 1992; Guthrie et al., 1993; Mori and Yoshihara, 1995; Bozza
and
Kauer, 1998). Our laboratory has used multiple electrode electro-olfactograms
(EOGs) to
describe regional olfactory sensitivity in the rodent olfactory epithelium (Ezeh et
al.,
1995; Scott et al., 1996). We drew parallels between
the distribution of odor responses and the expression zones for olfactory receptor genes (Scott et al., 1997).
The regional localization of odor sensitivity suggests that there is a general pattern of
olfactory
information entering the olfactory bulb. This pattern is consistent with the broad regional
differences in
the sensitivity of neurons in the olfactory bulb (Mori and Yoshihara, 1995) and
with the projection patterns from the receptor cells to the bulb (Pedersen et al.,
1986; Astic et al., 1987; Schoenfeldet
al., 1994). However, other recent reports of recordings from the rat olfactory
epithelium (Mackay-Sim and Kesteven, 1994; Youngentob et
al., 1995) do not support response gradients that correlate with the gene
expression
zones. Therefore, we have tested a broader range of stimulus concentrations and of epithelial
surfaces
to carefully re-evaluate our earlier findings.
The recordings in the present study were taken from several surfaces of the olfactory epithelium. The primary set of sites was along the medial surface of three of the endoturbinate bones. Other recordings were from the nasal septum or the dorsal surface of one endoturbinate bone. These sites exposed regions corresponding to the four gene expression zones, but in different locations. For more precise localization, we increased the number of recording electrodes to eight for many of these recordings. With this eight-electrode array, we tested whether the response profile shifted systematically with concentration and tested for discontinuities between the expression zones. By charting the positions of the recordings in each animal, we made composite curves based on a large series of points in the epithelium. This allowed the combination of points from multiple animals to test whether the response profiles follow the gene expression zones. We tested a series of concentrations at each recording site and explored several classes of odorant compounds on some of these sites.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Surgical preparation
Male Sprague–Dawley rats (375–525 g) were killed with an overdose of
Nembutal
(20 mg/kg). We exposed the olfactory epithelium overlying the medial surface of the
endoturbinate
bones on the left side, as in our previous paper (Scott et al., 1997).
The endoturbinate bones are the protrusions of the ethmoid bones that extend closest 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. Room
temperature was kept at ~17°C to prolong the usefulness of the preparation. A constant flow
of
humidified air (1000 ml/min) was immediately established with an 8 mm tube positioned 2 cm
from the
epithelial surface. This tube was centered on the recording area at an angle of ~30°. In our
previous paper (Scott et al., 1997), we could detect no influence
of
the tube angle on the differential distribution of odor responses.
In four sessions, we also explored the epithelium on the nasal septum. In seven others, we explored the dorsal aspect of endoturbinate II after removing the roof of the nasal cavity and the overlying ectoturbinate bones. This allowed us to test whether the apparent correlation with the gene expression zones extended into the lateral recesses of the nasal cavity.
Recording procedure
An indifferent electrode (a silver chloride electrode connected by an agar bridge to a
saline-soaked
cotton pad) was placed on the frontal bone overlying the left olfactory bulb. Recordings were
made
with eight glass micropipettes filled with agar made up in Ringer's solution. These
micropipettes
were broken to a resistance of <5 M
. The leads from these electrodes were connected
to
two four-channel AC coupled amplifiers (low-frequency time constant 0.1 Hz). The electrode
placements were usually along the rostral borders of the turbinate bones, but other placements,
particularly the caudal parts of endoturbinate IV, were also explored. Figure 1A shows the exposure of
the medial wall of the endoturbinate bones and illustrates the terms that we will use to describe
electrode orientation. 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. For this
reason, the eight-electrode configuration was less stable than the four-electrode configuration
that we
used previously (Scott et al., 1997). Figure 1B illustrates examples of
EOGs from eight electrode recordings and illustrates variation in response size that may have
arisen in
part from electrode movement during placement. Figure 1C shows peak
voltage responses to isoamyl
acetate across all the initial placements along the rostral border of endoturbinate II'. In
some of
these cases, repositioning of the electrode led to larger responses. Nevertheless, the great
variation
shows the necessity for the response standardization described below. On average the isoamyl
acetate
response was slightly larger ventrally, but the difference was not statistically significant. The
electrode
array was usually left in place during a complete recording session, but occasionally two sets of
placements were used during a single experiment. The lateral set of recordings was done on a
different
set-up with only four electrodes.
|
Odor stimulation
Odor concentration was generated by an air dilution olfactometer and concentration was expressed as a proportion of air saturated with odorant. Odor dilutions were generated by using a syringe pump to force air through the head space in glass bottles in which 2–5 ml of the pure odorant were used to cover the bottom. The four tubes were connected by Teflon tubes to ports in the glass tube next to the epithelium. The rate of this flow through the odorant bottle, divided by the 1000 ml/min flow in the stimulus tube, determined the dilution.
The output of this system was evaluated by drawing the stimulus into a gas chromatograph with a flame ionization detector and a DC-200 column (Gow-Mac, Bethlehem, PA). Odors were drawn into a gas sampling valve from the port of the olfactometer. The gas chromatograph was calibrated by diluting known amounts of the odorant chemicals in hexane, which came off the column very rapidly. The output of the flame ionization detector was fed into the A/D converter and area under the curve was measured with a statistical package. These tests showed that the estimated odor concentration was close to the value observed with the gas chromatograph (Table 1). Calculated concentrations in that table were estimated from the vapor pressure at 17°C by dividing that vapor pressure by the atmospheric pressure. That ratio tells the fraction of the mixture contributed by the odor vapor. That ratio was divided by the number of liters occupied by a mole of gas at standard temperature and pressure, and adjusted for the room temperature, to give the number of moles per liter. We also found that the system was reliable down to a dilution of 1 x 10–4. We can observe reliable isoamyl acetate responses in the EOG at that dilution. The data figures will express odor concentration in terms of dilution rather than the estimated moles per liter.
|
A BASIC language program controlled the pump rate and set valves to inject each odor. This program used a standard file to determine the odor sequence and their concentrations. Each set of three test stimuli at a particular concentration was preceded by a standard isoamyl acetate stimulus, and a blank was presented after each two-stimulus set. The odor was always presented for a 2 s period. Figure 1B shows that there was usually a peak response followed by a slow decline and a distinct inflection following odor removal. All odors were tested in groups of four odors: the standard odor (isoamyl acetate at a dilution of 10 –1) and three test odors over a series of descending dilutions from 1 x 10–1 to 1 x 10–4. One and a half min elapsed after each stimulus presentation. A typical experiment began with two presentations of limonene, carvone and cineole odors at a dilution of 1 x 10–1, followed by two descending sets of dilutions for these three odors. There was no effect of stimulus presentation order. In the text of this paper, we will refer to 1,8-cineole simply as cineole. The recording session usually lasted for 3–5 h. We sometimes ended an experiment earlier if the responses began to deteriorate badly on three or more electrodes. In some cases, the highest concentrations of the terpene compounds were tested before other odors were used that were outside the scope of this report. As a result, we have more extensive data for the higher concentrations than for the lowest concentrations.
The stimulus control differed from our previous papers in steps taken to improve the
sharpness of
the odor onset and to extend the intensity to lower dilutions. Odors were injected into a
humidified,
clean airstream flowing at 1000 ml/min. This airstream, 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 s to
allow the
buildup of the odor in the system. During this build-up period, the vacuum port was opened to
draw 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 build-up 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 previously (Kauer and Shepherd, 1975; Mackay-Sim and Kesteven, 1994). While some airflow transients occurred during
these
valve operations, the blanks usually produced responses of <0.4 mV. If the blanks became
larger,
the system was flushed with clean air to reduce contamination. Data were not used in the analysis
if the
standardized blank response was <1 mV or if the average response to the standard was <4
mV.
(Large blank responses were observed only when the standard response was very large.)
Data analysis
The outputs were fed by an A/D 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 for each record was printed and stored in a file for later analysis. We inspected the plots for quality control during data collection and before analysis. Responses to all stimuli were standardized to the isoamyl acetate stimulus. The response to each odor was divided by the previous response to the standard stimulus at that electrode and was multiplied by the overall average response to the first isoamyl acetate (1 x 10–1) responses from the first 10 experiments. This standard was chosen because it is a very commonly used stimulus and because it is very effective at all sites on the epithelium, even at relatively low concentrations. The isoamyl acetate response tended to be slightly larger in the ventral epithelium, but that increase was small relative to the variation. Therefore we have assumed an equal isoamyl acetate response at all sites, even though it may introduce a slight bias. Standardization helped minimize differences between recordings that might result from slight damage or drying at one site. It allowed us to adjust for the gradual run down of the response over the period of recording. As the tissue dried, one or more electrodes might lose contact with the tissue and the electrode would have to be advanced slightly to re-establish the recording. Response standardization allowed us to adjust for the resulting changes in response size.
It was not possible to visualize the receptor gene expression zones in adult animals. Therefore, we attempted to space the electrodes at equal distances across the turbinate bones in a direction that should cross the expression zones at nearly right angles. However, the difficulties of manipulating eight electrodes into a small space produced substantial variation in placement for different recording sessions on the same bone. Individual recording sessions could be analyzed by ANOVA to calculate the mean and confidence intervals. Response plots in single animals show the mean peak voltage at each electrode and concentration, and the 95% confidence intervals based on the standard error at that concentration and point. The mean blank response is illustrated for the highest flow rate through the clean odor bottle and is shown next to one of the sets of odor data. The blanks are not shown for composite curves for simplicity and because responses were selected to exclude any data with a large blank response.
Regression variables and contours
For statistical comparison of response variation across position and across recording
sessions, we
used stepwise polynomial 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 x–y coordinates was used to record the positions of the tips. For analysis
along the
length of a single turbinate bone edge, the Euclidian distance from the most dorsal point of the
slit
between turbinate bones was used [D = 
(x2 + y2)]. The first-, secondand third-order values of this distance were used in fitting
lines through
these points.
The mean peak voltage at each point was calculated and that value used as the response variable. Graphs of these inter-animal comparisons show individual points and the fitted line. The number of points is determined by the number of animals and the number of electrodes with good responses (isoamyl acetate response >4 mV and standardized blank response <1 mV).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Effect of position in eight-electrode recording experiments
We restricted recordings to three terpene odors—limonene, carvone and
cineole—because they gave distinctively different response profiles in earlier experiments
(Scott et al., 1997). Figure 2A,B shows
recordings along the rostral
edge of endoturbinate IV for one animal and endoturbinate II' for a second animal. The
figure
shows the small amount of intra-animal variability that occurred in the standardized peak
response as
well as the typical low response to blanks. Responses to all odors increased with concentration.
There
was a larger response from the most dorsal region for carvone at all dilutions and this response
fell
steadily from the most dorsal to most ventral position. The limonene response was often smaller.
When
there was a spatial gradient for limonene, it was in the other direction (i.e. larger ventrally). The
cineole
response peaked at ~60–80% of the distance from the most dorsal site on the turbinate.
This
peak was large relative to the confidence intervals at all dilutions. The changes between regions
of the
epithelium were gradual. Although the response sizes varied as much as twofold, the changes
were not
abrupt.
|
We explored the caudal part of endoturbinate IV in several rats because the description of gene expression zones in the rat predicts a rostral-to-caudal as well as a dorsalto-ventral pattern in that region (Vassar et al., 1993) (also see Figure 1A). Figure 2C shows results from one session. Carvone at the highest concentration evoked the largest responses on the rostro-dorsal region of endoturbinate IV, and there was a peak in the profile at this position for all concentrations of carvone. The same region had the smallest responses for all concentrations of cineole and most concentrations of limonene. We were never able to place electrodes to span the entire caudal responsive region of endoturbinate IV, but it was apparent that the cineole (and usually the limonene) response was smallest at the point of the peak carvone response.
These standardized results were generally stable despite the gradual rundown of the response over time. Figure 3 illustrates the first and last pairs of recordings from the rostral border of endoturbinate IV of a single animal, which were taken 3 h apart. These records are for the same three terpene odors at the highest concentration tested (10–1). This figure shows another example of the gradients illustrated in Figure 2 and shows that the changes in profiles were small over that time period. This stability was present even when the absolute response declined by several millivolts. The standardized responses do not decline because they are based on comparison with the isoamyl acetate response.
|
Combined data across animals
In order to test whether there was consistency across animals, we used photographic measurements of electrode positions to overlay response plots. Figure 4 shows combined data from sets of sites recorded along the rostrodorsal edges of endoturbinates IV and II'. In this figure, we have only used one dimension of the position, the distance from the most dorsal recording site on the particular endoturbinate. This figure shows that the curves representing the response profiles for the three odors are different. This is true for each of the two endoturbinate bones and for each concentration. The intermediate dilutions shown in Figure 2 were not included for clarity of the figure, but the curves were generally parallel to those shown. For the two dilutions shown for endoturbinate IV, there is a prominent inflection in the cineole curves showing a peak response somewhere above the most ventral recording sites. This peak is not clearly present in the group curves for endoturbinate II', although it is present in most of the individual curves for both turbinate bones, as in Figure 2. Most of the individual profiles for cineole were similar to those for Figure 2A,B in that electrode 8 or electrodes 7 and 8 showed smaller responses than the immediately dorsal electrode. This was true for 90% of the rats in which we recorded from the rostral edge of endoturbinate IV. It was true for 73% recorded along endoturbinate II' and 65% recorded along endoturbinate II.
|
The statistical significance of differences between curves was tested by making pairwise comparisons between the curves at each concentration on each of the two endoturbinate bones. Odor was used as a dummy variable (Kleinbaum and Kupper, 1978
). A
significant
interaction at P < 0.01 was found between the dummy variable and the position
variable in
every case except for the comparison of carvone and limonene at 10–3 on
endoturbinate II'. Response maps
We summarized the recordings over a larger region of the epithelium by constructing maps
from the
x–y coordinates of the recording sites. Figure 5A
illustrates
those maps for two
dilutions of the three terpene odorants. These maps show larger carvone responses on the dorsal
regions of all turbinate bones. On endoturbinate IV, most of the larger carvone responses were
found
on the rostro-dorsal tip, corresponding to the pattern of the dorsal expression zone in neonatal
rats
(Vassar et al., 1993). The limonene responses tend to be larger
on
the ventral parts of the turbinate bones and on the posterior part of endoturbinate IV, both regions
where the carvone responses are smaller. The response gradients for cineole are steeper than
those for
limonene and, at least at the higher dilutions, show a decrease at the extreme ventral and
posterior
regions. We limited the figure to two dilutions for simplicity, but the responses at other odorant
dilutions
showed the same tendencies.
|
As noted for Figure 2, the pattern of responses on endoturbinate IV is more complicated than on endoturbinate II', which is the other surface that was extensively sampled. The carvone responses became smaller for most points that were posterior and ventral to the rostro-dorsal edge. Limonene response generally became larger for the posterior ventral points. Cineole responses, particularly at the two highest concentrations, were larger on the center of the bone than on either the dorsal or ventral portions. This pattern follows that predicted from the receptor expression zone maps of Vassar et al. (Vassar et al., 1993
) summarized in Figure
1.
There is a suggestion in Figure 5 that the regions of maximal
response to
the three odors are not
proportionally equal on the different turbinate surfaces. There were larger responses to cineole on
endoturbinate II' than on IV. The region of maximal cineole response also seems to
extend
more ventrally on II', which is consistent with the difference between the cineole curves
in
Figure 6 and with the numbers of cineole profiles that show a prominent
peak
on the three turbinate
surfaces. These differences must be interpreted cautiously. They could result from differences in
the
extent of the response zones to these odors, or they might result from differences in the response
to the
isoamyl acetate standardizing stimulus. A further caution is that points on different endoturbinate
bones
were recorded from different animals. However, reanalysis of the data of Scott et al. (Scott et al., 1997) indicates a relatively larger cineole response
compared with the limonene response on the more rostral endoturbinate bones (data not shown).
|
Recordings on other surfaces of the olfactory epithelium
Figure 6 shows one of two successful eight-electrode recordings from the nasal septum with the three terpene odors (the experimental set-up is shown in Figure 5B). We did not attempt to plot combined data for the two recordings because it was difficult to find reliable landmarks for lining up the recording sites. For the same reason, it is not possible to align these sites with the recordings from the endoturbinate bones. Nevertheless, the response patterns for the higher concentrations are similar to that seen in the previous figures, with carvone responses largest on dorsal sites, limonene responses largest in ventral sites and cineole responses peaking at intermediate sites.
We also made recordings from the dorsal, lateral surface of endoturbinate II in five animals. These were made in a different set-up because of the convenience of manipulating the electrodes into the requisite positions, and the olfactometer for that set-up did not extend to the lower concentrations. The preparation is illustrated in Figure 5C. We were able to produce overlays of photographs relying on the positions of exposed bones. Figure 7 displays individual data and the regression plots for these data. The response distributions were similar to those in the previous figures in that carvone produced larger responses at the medial sites and cineole produced the largest responses at intermediate sites. The limonene response distribution was flat and, for this odor only, none of the position parameters were significant for this odor. The comparisons of each pair of odor curves produced significant odor-by-position interactions (P < 0.01) by multiple regression except at the lowest concentration, where the interaction for carvone versus limonene was significant at P < 0.5.
|
The lateral recordings confirm the differences between limonene and carvone response that were previously reported from intact animals (Scott et al., 1996
) and
show
that the limonene–carvone differences can exist even when the odor is directly applied to
the
exposed epithelium. This experiment also demonstrated a region of peak cineole sensitivity in
the lateral
region that was not obvious in the earlier recordings from intact animals. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this discussion, we will separate the empirical data about response distribution from the hypothesis of relation to receptor gene distribution by referring to ‘regions’ in describing the response data and ‘zones’ in discussing the published observations on gene expression patterns. Because we were unable to visualize the expression zones directly, it is important that we could test with a reasonable spatial resolution to evaluate whether there is a set of response regions that could correspond to the expression zones. We have previously argued that the response distributions may have their basis in chemical properties such as polarity or the presence of the carbonyl group (Scott et al., 1996
). By this logic carvone (a ketone) and
limonene (a hydrocarbon) should have different response patterns. Cineole is a bicyclic ether and
seems
to have a more complex response distribution (Scott et al., 1997). Response distribution and gene expression zones
These data confirm and extend our previous conclusion that the adult rat olfactory epithelial
responses to some odors are distributed in correlation with the neonatal olfactory receptor gene
expression zones (Scott et al., 1997). We had investigated the
response profiles by placing one electrode on each of four endoturbinate bones and moving them
sequentially from the ventral to dorsal position in seven steps. Thus all four electrodes were
expected to
correspond to a single expression zone. The responses to limonene were greatest in the ventral
placements for all electrodes, and the responses to carvone were greatest in the dorsal placements
for
all electrodes. The effect of the dorsal-to-ventral position was confounded with the sequential
electrode
movement, and only a single concentration was used for each odor.
In the present study we doubled the number of simultaneous recordings. Within a single
experiment, we arrayed the electrodes across the expected distribution of the gene expression
zones. In
single experiments, as well as in grouped analyses, we saw the regions of largest carvone
response on
the dorsal parts of the medial walls of the endoturbinate bones. The limonene response gradients
were
less pronounced, but there was a slight tendency for them to be largest ventrally. The cineole
response
was more complex, becoming largest in the ventral or intermediate ventral region. These results
were in
general correspondence with the gene expression zones described by Vassar et al. (Vassar et al., 1993). This correspondence existed even as the
stimulus concentration was reduced. The correspondence also extended to other epithelial
surfaces on
the septum and the dorsal surface of endoturbinate II, extending out into the lateral recess.
These data do not test the number of response regions or whether the regions are discrete.
The
comparison of the carvone and cineole profiles suggests that there must be at least three different
response regions. The observed profiles do not change in discrete steps. However, the gene
expression
zones do not have sharp boundaries (Vassar et al., 1993). In
addition, the length constant of the EOG recording from electrical stimulation of the epithelium
is
probably ~100 µm (Mackay-Sim and Kesteven, 1994). If the zones
were equally represented across the 4 mm of recording distance, this space constant would
represent
~10% of a zonal width. These two factors might account for the apparently gradual changes that
we
observed.
Other epithelial features are also distributed parallel to the receptor expression zones.
Supporting
cell morphology (Menco and Jackson, 1997), enzymatic activity in
sustentacular cells (Miyawaki et al., 1996), NADPH-diaphorase
activity (Dellacorte et al., 1995) and receptor guanylyl cyclase
(Fülle et al., 1995) have zonal distributions. Yoshihara et al. (Yoshihara et al., 1997) found a zonal
distribution for the
adhesion molecule OCAM. Other factors, such as odorant binding proteins, could be distributed
in a
similar fashion (Rama Krishna et al., 1995). Some of these
factors
might influence responses, even if the average specificity of odorant receptors did not change
with
expression zones. The fact that we have observed distinct spatial gradients for different types of
odors
makes this caution less convincing, but certainly does not remove it. On the other hand, the
regional
responses we find can be functionally important in determining the sensory input to the olfactory
bulb
irrespective of their origin.
Not all receptor genes are distributed according to the same zonal arrangement (Strotmann et al., 1994; Kubick et al., 1997;
Ring et al., 1997). Since only a few receptor genes have been
studied
by the in situ hybridization technique, it is possible that more deviations from the zonal
arrangement will be found. This underscores the cautions about drawing the conclusion that our
data
necessarily reflect the properties of the receptor genes themselves, but does not detract from the
fact
that we have observed physiological properties that follow an important set of boundaries within
the
olfactory epithelium. It is possible that non-zonal receptor distributions may account for
non-zonal
response patterns observed for some odorants (Mackay-Sim and Kesteven, 1994; Youngentob et al., 1995). There may be similar
explanations for the small but consistent rostral-to-caudal relative difference in the cineole versus
limonene responses.
The EOG technique cannot tell whether a larger response for a particular odorant in one
region
comes from a small number of receptor cells that respond strongly to that odorant or from a large
number of cells that respond weakly. These are questions that will have to be answered by
techniques
that measure individual cells or study receptors in expression systems (Raming et
al., 1993; Sato et al., 1994; Rawsonet
al., 1997; Wellerdieck et al., 1997; Bozza and Kauer, 1998; Krautwurst et al., 1998; Zhao et al., 1998). So far, a complete answer to these questions
has
not emerged. Zhao et al. (Zhao et al., 1998) measured
EOGs and whole cell patch recordings of rat receptor cells transfected with a virus carrying the
I7
receptor gene. They found increased responses to octanal and closely related aldehydes, but not
to any
others of a series of 74 odors, including fatty acids and alcohols. The I7 receptor is expressed in
the
most ventral zone (Vassar et al., 1993). This is one contradiction
to
the expectation that more ventral receptors would respond to less polar odors (Scott et
al., 1996). Bozza and Kauer (Bozza and Kauer, 1998),
on
the other hand, found many examples of carvone responses in receptor cells projecting to the
dorsal
bulb, but no examples of limonene responses in the same cells. Krautwurst et al. (Krautwurst et al., 1998) tested a set of 80 chimeric receptors
containing the
regions expected to code odorant recognition. They found a high degree of specificity among the
26
odorants that they tested with a cellular expression system. They confirmed the high selectivity
reported
for the I7 receptor in EOG experiments (Zhao et al., 1998).
These
data suggest a narrow band of specificity for the olfactory receptors, at least in expression
systems.
This seems contrary to our previous speculation that many of the receptors in a particular
expression
zone might respond to a broad range of odorants with similar properties (Scott et
al., 1996).
Advantages and limitations of the EOG recording technique.
While the EOG recording procedure is limited in spatial resolution, it has distinct
advantages. The
recordings in situ avoid potential problems that may result from isolation of cells in
artificial
medium. Many odors can be tested over a series of concentrations. The preparation is simple and
the
responses in freshly killed animals are very similar to those in live animals (Scottet al., 1996). Several different surfaces of the epithelium can be approached to
test the
correspondence with the gene expression zones, as we have done for the terpene odors in this
paper.
We propose that any claim of full correlation with the expression zones ought to test several such
surfaces.
The EOG recording technique is very sensitive to tissue exposure, to mechanical damage, to
the
exact depth of the electrode and to drying. Some of these problems were accentuated in the
placement
of eight simultaneous electrodes. Standardization of the response is very useful for comparing
responses
across electrodes and across animals. The choice of the standardizing stimulus would alter the
shape of
the profiles, but would not alter the fact that the profiles are different for different odors. On
average,
the isoamyl acetate response has been a little larger ventrally than dorsally. Consequently, the
absolute
odor profiles collected under ideal conditions might differ from the standardized profiles shown
here.
We have implicitly assumed that there is no difference in the isoamyl acetate response from one
turbinate bone to another. The large response variation in Figure 1C
shows
how difficult the absolute
response is to assess. Other authors (Mackay-Sim and Kesteven, 1994;
Youngentob et al., 1995) presented data indicating that the
average
size of amyl acetate responses changes substantially across the rat epithelium, but those data
disagree
strongly about the regions of maximal response.
The use of multiple electrodes allows a comparison of odorant series or concentrations within a single animal. This is a faster procedure than exploring many sites with a single electrode (Mackay-Sim and Kesteven, 1994) and avoids introduction of variance from moving the electrode. The multi-animal regression plots show that the relationships seen in single animals are repeatable. The hypothesis of zonal expression is essential to making comparisons of responses to an odorant series in single animals, because it allows electrodes to be placed linearly without the necessity of sampling the entire epithelium. Therefore, hypotheses about the regional distribution of responses to a series of compounds can reasonably be tested in single preparations.
We propose that this preparation will be useful for screening the mass response to odors across the expression zones. Such data could show which odors are likely to be strong ligands for receptors expressed in particular zones. In addition, as noted above, studies on isolated receptor cells or on receptor genes expressed in non-olfactory cell lines will not be subject to the enzymes or other factors expressed locally in olfactory receptor cells or surrounding tissue. If these turn out to be significant in determining the response, then it will be necessary to study receptor cell processes in situ if we are to understand the pattern of sensory input to the olfactory bulb.
|
Acknowledgments |
|---|
Supported by National Institute of Deafness and Other Communications Disorders Grant DC00113. The authors thank F.H. Schmidt for aid with instrumentation and consultation about data analysis and D. Blakley who performed the lateral recordings. We thank D. Wellis, P. Ezeh, M. Singer, N. Buonviso and M. Chaput for reading earlier drafts of the manuscript.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Astic, L., Saucier, D. and Holley, A. (1987) Topographic relationships between olfactory receptor cells and glomerular foci in the rat olfactory bulb. Brain Res. , 424, 144–152.[Web of Science][Medline]
Bozza, T.C. and Kauer, J.S. (1998) Odorant
response properties of convergent olfactory receptor neurons. J. Neurosci., 18, 4560–4569.
Dellacorte, C., Kalinoski, D.L., Hugue, T., Wysocki, L. andRestrepo, D. (1995) NADPH diaphorase staining suggests localization of nitric oxide synthase within mature vertebrate olfactory neurons. Neuroscience, 66, 215–225.[Web of Science][Medline]
Ezeh, P.I., Scott, J.W. and Davis, L.M. (1995) Regional distribution of rat electroolfactogram. J. Neurophysiol., 73, 2207–2220.
Füclle, H., Vassar, R., Foster, D.C., Yang, R., Axel, R. andGarbers, D.L. (1995) A receptor guanylyl cyclase expressed specifically
in
olfactory neurons. Proc. Natl Acad. Sci. USA, 92, 3571–3575.
Guthrie, K.M., Anderson, A.J., Leon, M. and Gall, C. (1993) Odor-induced increases in c-fos mRNA expression reveal an
anatomical ‘unit’ for odor processing in olfactory bulb. Proc. Natl.
Acad. Sci. USA, 90, 3329–3333.
Imamura, K., Mataga, N. and Mori, K. (1992) Coding of odor molecules by mitral/tufted cells in rabbit olfactory bulb. I. Aliphatic
compounds. J. Neurophysiol., 68, 1986–2002.
Kauer, J.S.and Shepherd, G.M. (1975) Olfactory stimulation with controlled and monitored step pulses of odor. Brain Res., 85, 108–113.[Web of Science][Medline]
Kleinbaum, D.G. and Kupper, L.L. (1978) Applied Regression Analysis and Other Multivariable Methods. Duxbury Press, North Scituate, MA.
Krautwurst, D., Yau, K.-W. and Reed, R.R. (1998) Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell, 95, 917–926.[Web of Science][Medline]
Kubick, S., Strotmann, J., Andreini, I. and Breer, H. (1997) Subfamily of olfactory receptors characterzed by unique structural features and expression patterns. J. Neurochem., 69, 465–475.[Web of Science][Medline]
Lancet, D., Kauer, J.S. and Greer, C.A. (1981) High resolution 2-deoxyglucose localization in olfactory epithelium. Chem. Senses, 6, 343–349.
Leveteau, J. and MacLeod, P.L. (1966) Discrimination des odeurs par les glomérules olfactifs du lapin (étude électrophysilogique). J. Physiol. (Paris), 58, 717–729.
Mackay-Sim, A. and Kesteven, S. (1994) Topographic patterns of responsiveness to odorants in the rat olfactory epithelium. J.
Neurophysiol., 71, 150–160.
Menco, B.P. and Jackson, J.E. (1997) A banded topography in the developing rat's olfactory epithelial surface. J. Comp. Neurol., 388, 293–306.[Web of Science][Medline]
Miyawaki, A., Homma, H., Tamura, H. and Mikoshiba, K. (1996) Zonal distribution of sulfotransferase for phenol in olfactory sustentacular cells. EMBO J., 15, 2050–2055.[Web of Science][Medline]
Mombaerts, P., Wang, F., Dulac, C., Chao, S.K., Nemes, A., Mendelsohn, M., Edmondson, J. and Axel, R. (1996) Visualizing an olfactory sensory map. Cell, 87, 675–686.[Web of Science][Medline]
Mori, K. and Yoshihara, Y. (1995) Molecular recognition and olfactory processing in the mammalian olfactory system. Prog. Neurobiol., 45, 585–619.[Web of Science][Medline]
Nef, P., Hermans-Borgmeyer, I., Artieres-Pin H., Beasley, L., Dionne, V.E.and Heinemann, S.F. (1992) Spatial pattern of receptor
expression in
the olfactory epithelium. Proc. Natl. Acad. Sci. USA, 89,8948
–8952.
Pedersen, P.E., Jastreboff, P., Stewart, W.B. and Shepherd, G.M. (1986) Mapping an olfactory receptor population to a specific region in the rat olfactory bulb. J. Comp. Neurol., 250, 93–108.[Web of Science][Medline]
Rama Krishna, N.S., Getchell, M.L., Margolis, F.L. and Getchell, T.V. (1995) Differential expression of vomeromodulin and odorant-binding protein, putative pheromone and odorant transporters, in the developing rat nasal chemosensory mucosae. J. Neurosci. Res., 40, 54–71.[Web of Science][Medline]
Raming, K., Krieger, J., Strotmann, J., Boekhoff, I., Kubick, S., Baumstark, C. and Breer, H. (1993) Cloning and expression of odorant receptors. Nature, 361, 353–356.[Medline]
Rawson, N.E., Gomez, G., Cowart, B., Brand, J.G., Lowry, L.D., Pribitkin,
E.A. and Restrepo, D. (1997) Selectivity and response
characteristics of human olfactory neurons. J. Neurophysiol., 77, 1606–1613.
Ressler, K.J., Sullivan, S.L. and Buck, L.B. (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell, 73, 597–609.[Web of Science][Medline]
Ressler, K.J., Sullivan, S.L. and Buck, L.B. (1994) Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell, 79, 1245–1255.[Web of Science][Medline]
Ring, G., Mezza, R.C. and Schwob, J.E. (1997) Immunohistochemical identification of discrete subsets of rat olfactory neurons and the glomeruli that they innervate. J. Comp. Neurol., 388, 415–434.[Web of Science][Medline]
Sato, T., Hirono, J., Tonoike, M. and Takebayshi, M. (1994) Tuning specificities to aliphatic odorants in mouse olfactory receptor neurons
and
their local distribution. J. Neurophysiol., 72, 2980–2989.
Schoenfeld, T.A., Clancy, A.N., Forbes, W.B. and Macrides, F. (1994) The spatial organization of the peripheral olfactory system of the hamster. Part I. Receptor neuron projections to the main olfactory bulb. Brain Res. Bull., 34, 183–210.[Web of Science][Medline]
Scott, J.W., Davis, L.M., Shannon, D. and Kaplan, C. (1996) Relation of chemical structure to spatial distribution of sensory responses in rat
olfactory epithelium. J. Neurophysiol., 75, 2036–2049.
Scott, J.W., Shannon, D.E., Charpentier, J., Davis, L.M. andKaplan,
C. (1997) Spatially organized response zones in rat olfactory epithelium. J. Neurophysiol.,77, 1950–1962.
Strotmann, J., Wanner, I., Helfrich, T., Beck, A., Meinken, C., Kubick, S. and Breer, H. (1994) Olfactory neurones expressing distinct odorant receptor subtypes are spatially segregated in the nasal neuroepithelium. Cell Tissue Res., 276, 429–438.
Vassar, R., Ngai, J. and Axel, R. (1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell, 74, 309–318.[Web of Science][Medline]
Vassar, R., Chao, S.K., Sitcheran, R., Nuñez, J.M., Vosshall, L.B. and Axel, R. (1994) Topographic organization of sensory projections to the olfactory bulb. Cell, 79, 981–991.[Web of Science][Medline]
Wang, F., Nemes, A., Mendelsohn, M. and Axel, R. (1998) Odorant receptors govern the formation of a precise topographic map. Cell, 93, 47–60.[Web of Science][Medline]
Wellerdieck, C., Oles, M., Pott, L., Korsching, S., Gisselmann, G. andHatt, H. (1997) Functional expression of odorant receptors of the
zebrafish Danio rerio and of the nematode. C. elegans in HEK293 cells. Chem. Senses, 22, 467–476.
Youngentob, S.L., Kent, P.F., Sheehe, P.R., Schwob, J.E. andTzoumaka, E. (1995) Mucosal inherent activity patterns in the rat,
evidence
from voltage-sensitive dyes. J. Neurophysiol., 73, 387–398.
Yoshihara, Y., Kawasaki, M., Tamada, A., Fujita, H., Hayashi, H.,
Kagamiyama, H. and Mori, K. (1997) OCAM: A new member
of
the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and
vomeronasal axons. J. Neurosci., 17, 5830–5842.
Zhao, H., Ivic, L., Otaki, J.M., Hashimoto, M., Mikoshiba, K. andFirestein, S. (1998) Functional expression of a mammalian odorant receptor. Science, 270, 237–242.
Accepted June 24, 1999
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. W. Scott and L. Sherrill Effects of Odor Stimulation on Antidromic Spikes in Olfactory Sensory Neurons J Neurophysiol, December 1, 2008; 100(6): 3074 - 3085. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Zaghetto, S. Paina, S. Mantero, N. Platonova, P. Peretto, S. Bovetti, A. Puche, S. Piccolo, and G. R. Merlo Activation of the Wnt {beta}Catenin Pathway in a Cell Population on the Surface of the Forebrain Is Essential for the Establishment of Olfactory Axon Connections J. Neurosci., September 5, 2007; 27(36): 9757 - 9768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Scott, H. P. Acevedo, L. Sherrill, and M. Phan Responses of the Rat Olfactory Epithelium to Retronasal Air Flow J Neurophysiol, March 1, 2007; 97(3): 1941 - 1950. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. C. Yang, P. W. Scherer, K. Zhao, and M. M. Mozell Numerical Modeling of Odorant Uptake in the Rat Nasal Cavity Chem Senses, March 1, 2007; 32(3): 273 - 284. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Scott, H. P. Acevedo, and L. Sherrill Effects of Concentration and Sniff Flow Rate on the Rat Electroolfactogram Chem Senses, July 1, 2006; 31(6): 581 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Mainland and N. Sobel The Sniff Is Part of the Olfactory Percept Chem Senses, February 1, 2006; 31(2): 181 - 196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Scott Sniffing and Spatiotemporal Coding in Olfaction Chem Senses, February 1, 2006; 31(2): 119 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Igarashi and K. Mori Spatial Representation of Hydrocarbon Odorants in the Ventrolateral Zones of the Rat Olfactory Bulb J Neurophysiol, February 1, 2005; 93(2): 1007 - 1019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Xu, N. Liu, I. Kida, D. L. Rothman, F. Hyder, and G. M. Shepherd Odor maps of aldehydes and esters revealed by functional MRI in the glomerular layer of the mouse olfactory bulb PNAS, September 16, 2003; 100(19): 11029 - 11034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.A. Chaput EOG Responses in Anesthetized Freely Breathing Rats Chem Senses, December 1, 2000; 25(6): 695 - 701. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.E. Scott-Johnson, D. Blakley, and J.W. Scott Effects of Air Flow on Rat Electroolfactogram Chem Senses, December 1, 2000; 25(6): 761 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Scott, T. Brierley, and F. H. Schmidt Chemical Determinants of the Rat Electro-Olfactogram J. Neurosci., June 15, 2000; 20(12): 4721 - 4731. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||