Chem. Senses 27: 277-286,
2002
© Oxford University Press 2002
Amino Acid Odorants Stimulate Microvillar Sensory Neurons
University of Utah School of Medicine, Department of Physiology, Salt Lake City, UT 84108-1297, USA
Correspondence to be sent to: William C. Michel, University of Utah School of Medicine, Department of Physiology, 410 Chipeta Way, Room 155, Salt Lake City, UT 84108-1297, USA. e-mail: mike.michel{at}m.cc.utah.edu
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Abstract |
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The olfactory epithelium (OE) of zebrafish is populated with ciliated and microvillar olfactory sensory neurons (OSNs). Whether distinct classes of odorants specifically activate either of these unique populations of OSNs is unknown. Previously we demonstrated that zebrafish OSNs could be labeled in an activity-dependent fashion by amino acid but not bile acid odorants. To determine which sensory neuron type was stimulated by amino acid odorants, we labeled OSNs using the ion channel permeant probe agmatine (AGB) and analyzed its distribution with conventional light- and electron-microscope immunocytochemical techniques. Approximately 7% of the sensory epithelium was labeled by AGB exposure alone. Following stimulation with one of the eight amino acids tested, the proportion of labeled epithelium increased from 9% for histidine to 19% for alanine; amino acid stimulated increases in labeling of 2-12% over control labeling. Only histidine failed to stimulate a significant increase in the proportion of labeled OSNs compared to control preparations. Most amino acid sensitive OSNs were located superficially in the epithelium and immuno-electron microscopy demonstrated that the labeled OSNs were predominately microvillar. Large numbers of nanogold particles (20-60 per 1.5 µm2) were associated with microvillar olfactory sensory neurons (MSNs), while few such particles (<15 per 1.5 µm2) were observed over ciliated olfactory sensory neurons (CSNs), supporting cells (SCs) and areas without tissue, such as the lumen above the OE. Collectively, these findings indicate that microvillar sensory neurons are capable of detecting amino acid odorants.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fish detect olfactory signals such as amino acids, bile acids, prostaglandins and steroids with a mixed population of ciliated (CSNs), microvillar (MSNs) and crypt olfactory sensory ncurons (Zeiske et al., 1976
,
1986;
Theisen et al., 1980;
Moran et al., 1992;
Hansen and Zeiske, 1998;
Hansen and Finger, 2000).
Previous attempts to determine if any of these groups of receptor cells are
sensitive to particular odorants have relied on indirect evidence. In char,
for example, the largest responses to general feeding stimuli (amino acids)
were recorded from areas of olfactory epithelium (OE) enriched in MSNs, while
areas enriched in CSNs had larger responses to social stimuli—bile acids
(Thommesen, 1982,
1983). In the channel catfish,
no correlation was found between the distribution of MSNs and CSNs and
sensitivity to amino acid and bile acid stimuli
(Erickson and Caprio, 1984). In
the goldfish, MSNs appear to respond both to amino acids and bile acids. It
has been reported that sensitivity to bile acid and amino acid odorants
following olfactory nerve transection recovers with time courses similar to
the epithelial regeneration of MSNs and CSNs, respectively
(Zippel et al.,
1997). Another study (Speca
et al., 1999) concluded on the basis of the superficial
location of the cells expressing an arginine sensitive V2R receptor that the
MSNs were amino acid sensitive. However, location of the cell soma in the
epithelium may not unambiguously distinguish MSNs from CSNs. Morita and Finger
identified short (presumably MSNs) and long (presumably CSNs) OSNs and a
population of intermediate length OSNs that were morphologically similar to
the other cell types (Morita and Finger,
1998). A more direct approach to the identification of odorant
stimulated OSNs is required to determine conclusively if odorants stimulate
specific types of OSNs.
We have previously shown that a cation channel permeant guanidinium analog,
agmatine (AGB), labels amino acid stimulated OSNs in an activity-dependent
fashion (Michel, 1999;
Lipschitz and Michel, 1999;
Michel et al., 1999).
The superficial location of the odor-stimulated OSNs and the inability of AGB
to permeate through open CNG channels
(Michel et al., 1999)
suggested the labeled cells were microvillar, but labeled knoblike dendritic
endings was consistent with the labeling of ciliated OSNs. Light microscopy
provided insufficient resolution to permit the discrimination of the ciliated
and microvillar apical processes needed for unambiguous identification. Using
electron microscopy, MSNs and CSNs can be distinguished by the presence of
numerous thin (0.04 µm diameter) microvilli, or thicker (0.2-0.25 µm
diameter proximally) cilia covering the apical surface
(Moran et al., 1992;
Hansen and Zeiske, 1998). In
the current investigation, we used activity-dependent labeling techniques in
combination with conventional immunocytochemical electron microscopy to
confirm the identity of the amino acid sensitive OSNs. The OE of adult
zebrafish was stimulated with amino acids previously shown to interact with
the acidic, basic and neutral amino acid receptor sites of fish
(Caprio and Byrd, 1984;
Ohno et al., 1984;
Bruch and Rulli, 1988;
Friedrich and Korsching, 1997;
Michel and Derbidge, 1997).
Results of the current study confirm that MSNs are stimulated by amino acid
stimuli. A portion of this study has been previously published in abstract
form (Lipschitz and Michel,
2000).
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Materials and methods |
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Animal maintenance
Zebrafish (Danio rerio) were purchased from a commercial supplier, housed in recirculating 40-80 1 aquaria (28.5°C) at the University of Utah and fed flake food (Tetramin) daily.
Activity-dependent labeling procedures
Zebrafish were stimulated in vivo after immobilization with an
i.m. injection of Flaxedil (60 µg/g body wt) and positioning in a recording
chamber. Immediately thereafter, the olfactory rosettes were provided with a
continuous flow of fish Ringer's solution (FR, see solutions). The gills were
irrigated with 3 ml/min of artificial freshwater (AFW) containing a general
anesthetic (MS-222, 20 mg/l). The anesthetic was prevented from contacting the
olfactory organs in order to minimize the loss of afferent sensory activity
associated with topical application of the anesthetic
(Spath and Schweickert, 1977).
After a 5 min FR wash, both olfactory rosettes were stimulated for 10 s/min
for 10 min with either 5 mM AGB (control) or 5 mM AGB plus amino acid odorant
(100 µM). Thus, during the 10 min stimulation period, the OE received 10
discrete stimulations each consisting of 10 s of odor stimulation each minute
followed by a 50 s FR rinse to minimize desensitization before the next
odorant stimulation. After a 5 min FR wash, the fish was decapitated and the
head immersed in cold fixative solution (see solutions) and stored at 4°C
overnight to several days. Fixed tissue was washed in 0.1 M phosphate buffer
(PB) for 30 min and dehydrated through a graded series of methanol and
absolute acetone, then embedded in Eponate resin (Pella Inc.). The University
of Utah Institutional Animal Care and Use Committee approved all experimental
procedures used in the current study.
Tissue processing
Light microscopy
Semi-thin sections (250-500 nm) were prepared using a diamond knife and an
American Optical Ultracut ultra-microtome. Individual sections were placed in
a well of a teflon-coated spot slide (Erie Scientific, Portsmouth, NH),
deplasticized using sodium ethoxide (NaEtOH, a saturated solution of sodium
hydroxide in ethanol diluted 1:4 with ethanol before use), rinsed, dried and
treated overnight in an anti-AGB IgG antibody. The rabbit anti-AGB primary
antibody (1:100 dilution, Signature Immunologics, Salt Lake City, UT) was
raised against a glutaraldehyde-conjugated AGB—albumin complex.
Immunoreactive sites were visualized with a nanogold-conjugated goat
anti-rabbit secondary antibody (1 nm, Amersham) and silver intensification
using previously described methods (Marc,
1999a,b).
Electron microscopy
Single ultra-thin sections (50 nm) were collected on formvar-coated gold
grids and the adjacent serial sections were individually mounted in wells of
teflon-coated spot slides for light microscope immunocytochemistry (see
above). Sections were treated with either 10% NaEtOH for 1-2 min or 10%
H2O2 for 10 min, rinsed, dried, incubated with 3% goat
serum in 0.1 M PB for 1 h to block non-specific labeling and then incubated
overnight in anti-AGB antibody. After several washes in 0.1 M PB, the tissue
was placed in nanogold-conjugated goat anti-rabbit secondary antibody for 1 h
(1:10 dilution, 15 nm gold particle size, Amersham), washed, stained with
uranyl acetate for 45 min, rinsed and dried. Viewing and photography employed
a Hitachi Model No. H-7100 transmission electron microscope.
Quantification of AGB immunoreactivity
Light microscopy
Digital images (8-bit gray scale) were obtained using a Zeiss Axioplan 2
microscope equipped with bright field illumination and a CCD camera (Axiocam,
Carl Zeiss Inc., Thornwood, NY) and Axiovision software. For each image, a
region of interest (ROI) was drawn around a portion of the sensory epithelium
that contained no labeled neurons. The mean pixel intensity and standard
deviation of this ROI was used to calculate a background staining value with a
95% confidence interval (CI). The lower 95% confidence interval limit was used
to discriminate labeled pixels from background staining in a second ROI of the
entire sensory epithelium. The number of labeled pixels below the cutoff was
divided by the total pixels in this ROI and multiplied by 100 to obtain the
percentage of labeled sensory epithelium. For each olfactory rosette, the
average proportion of labeled epithelium was calculated for four to six ROIs
from each of four planes of section, each separated by a minimum of 10
µm.
Electron microscopy
Following electron-microscope immunocytochemistry, analysis of AGB labeling
involved digitization of 7000x photographic negatives (resolution
600-1200 p.p.i., Scanmaker 5, Microtek, CA) and calculating the number of gold
particles in a standard rectangular area (1.5 µm2) placed at
least 2 µm below the apical surface of identified cells. Levels of labeling
were established from measurements taken in the lumen, the mucous and fluid
filled region above the OE and between adjacent olfactory lamellae.
Determination of centroid length
An Excel (Microsoft) macro estimated each cell's centroid length as the shortest distance from the centroid of the cell body to a straight line overlaying the mucosal surface. For both light- and electron-microscope images, an ROI outline was drawn around each labeled area or cell to be measured and the coordinates of the cell centroid were applied to the macros. For light-microscope samples, measurements were carried out only on those cells with cell bodies that could be easily separated or individually distinguished. Measurements of cells in electron-microscope images were carried out only for cells in which both identifiable apical processes and cell bodies were visible.
Statistical analysis
For light-microscope immunocytochemistry, the percentage labeling of
sensory regions under the control condition (AGB only) was statistically
compared to labeling with a binary mixture of AGB plus an amino acid odorant.
A one-way ANOVA for unbalanced data determined the overall significance for
the odorant main effect, while a post hoc Tukey's multiple comparison
of means test was used to determine individual differences between the control
and each binary mixture. The mean cell centroid length for the control
condition (AGB only) was compared to the mean cell centroid length for each
binary mixture of AGB and odorant using a one-way ANOVA and a post
hoc Tukey's multiple comparison of means test. For electron-microscope
immunocytochemistry, the numbers of gold particles were compared across
odorants for each cell type and the lumen using a two-way ANOVA. Individual
comparisons within odorants and within cell types were made using a one-way
ANOVA, while a Tukey's multiple comparison of means test was used to establish
individual differences within each variable. Further analysis determined
whether any significant differences existed among cell types in the
percentages of cells with labeling greater than background staining in the
lumen. A 99% CI was calculated for the lumen of each odorant condition and the
upper limit was used as a cutoff to calculate the percentage of OSNs and SCs
containing significantly greater numbers of nanogold particles than the lumen.
A 
2 Tukey-type multiple comparison test of proportions
(Zar, 1984) was used to
compare the percentages of labeling among cell types for each odorant
condition.
Chemicals and solutions
The composition of artificial freshwater (AFW) was (in mM): NaCl, 3; KCl, 0.2; CaCl2, 0.2; MgCl2, 0.2; HEPES, 1; pH 7.2. The composition of fish Ringers was (in mM): NaCl, 140; KCl, 10; CaCl2, 1.8; MgCl2, 2; HEPES, 5; pH 7.2. AGB Ringers was prepared by substituting 5 mM NaCl in the fish Ringers with 5 mM AGB. Fixative consisted of 11.2 ml of 18.3% paraformaldehyde, 10 ml of 50% glutaraldehyde, 80 ml of 0.2 M PB, 12 ml of 0.02% CaCl2, 6 g sucrose and sufficient distilled water to bring the final volume to 200 ml, yielding a final concentration of 2.5% glutaraldehyde and 1% paraformaldehyde. AGB and the eight L-amino-acid odorants, L-alanine (Ala), L-arginine (Arg), L-cysteine (Cys), L-glutamate (Glu), L-glutamine (Gln), L-histidine (His), L-lysine (Lys) and L-methionine (Met), were of the highest purity available from Sigma Chemical Co. (St Louis, MO). All amino acid odorants were tested at 100 µM.
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Results |
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Throughout the results, the term `OSNs' designates AGB labeled cells that have not been examined by transmission electron microscopy to identify definitively microvillar or ciliated processes. The terms `MSNs' and `CSNs' are reserved for cells definitively identified by electron microscopy.
Relative stimulatory effectiveness of amino acid odorants
A total of 18 adult zebrafish (two per odorant) were used to analyze
responses to each of the amino acid odorants. One olfactory rosette from each
fish was used to measure the percentage labeled OE and the lengths of the
amino acid stimulated OSNs. As previously reported
(Michel et al.,
1999), AGB immunoreactivity was observed in the sensory epithelium
and olfactory nerve layer but not in the non-sensory epithelium
(Figure 1). All of the amino
acid stimuli (100 µM) increased the percentage of sensory epithelium
labeled above that observed in the AGB control preparations of 
7%
(Figure 2A). The percentage of
labeled sensory epithelium ranged from 9% for L-histidine to 19% for
L-alanine. The ANOVA indicated that these increases were significant (ANOVA,
F = 7.98; d.f. = 8; P < 0.0001), while the Tukey's
comparison of means showed that, with the exception of histidine, all the
odorants tested significantly increased the percentage of labeled sensory
epithelium compared to the AGB control.
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Location of odor-stimulated OSNs
As shown in the light micrographs in Figure 1, the majority of cells labeled by either AGB alone or by AGB and amino acid stimulation were closer to the apical than the basal surface of the OE. The average length of labeled cells from the apical surface to the somal centroid for all measured OSNs was 6.5 ± 3.2 µm (n = 6168), while the average epithelial thickness at the same locations was 20.0 ± 3.6 µm (n = 6168). Frequency distributions for each of the odorants revealed a small population of labeled cells that were 15-20 µm long and whose somata were located near the basal surface (see Figure 2B). Although the cell centroid length distributions for the different odorant conditions overlapped considerably, mean cell centroid lengths for several amino acid combinations were significantly different (ANOVA, F = 87.611, d.f. = 8, P < 0.001). The Tukey's multiple comparison of means test indicated that the mean cell centroid length for AGB stimulated preparations was significantly longer than for histidine, arginine, glutamate, cysteine and alanine, but significantly shorter than for methionine.
Lengths of identified MSNs and CSNs
The lengths of identified CSNs and MSNs were measured in electron micrographs from five olfactory rosettes from five different adult zebrafish. Uranyl acetate staining alone permitted identification of cells on the basis of their apical surface morphology. Large differences in average diameter distinguished cilia (0.20 ± 0.03 µm, n = 8) from microvilli, (0.06 ± 0.01 µm, n = 9) and thus provided a reliable means of establishing the identity of MSNs and CSNs; ciliary bodies in the CSNs were also common (Figure 3; also see Figure 5). All measured cells were required to have an attached apical process and cell body to be included in the data set. The mean cell centroid length of the 36 MSNs was 4.9 ± 1.2 µm and is consistent with the hypothesis that MSNs occupy a superficial location in the sensory epithelium. The mean length of the 45 CSNs was 10.2 ± 2.5 µm, indicating a displacement at least one cell diameter below the MSNs. However, inspection of the lengths of the MSNs and CSNs reveals some overlap in the distribution of the two OSN types (Figure 4A). The average epithelial thickness in the regions used to measure OSN length was 15.4 ± 3.2 µm (n = 81, range, 9.7-23.3 µm). Epithelial thickness was positively correlated with cell centroid lengths for both MSN (coefficient of correlation, R = 0.66, P < 0.001, slope = 0.24) and CSN (R = 0.70, P < 0.001, slope = 0.52) distributions (Figure 4B).
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) are correlated with epithelial thickness. Linear regressions
were significant for both MSNs (R = 0.66, P < 0.001,
slope = 0.24) and CSNs (R = 0.70, P < 0.001, slope =
0.52).
Direct AGB labeling of OSNs
For AGB electron-microscope immunocytochemistry, cells from one olfactory rosette were analyzed following either 5 mM AGB stimulation alone or exposure to a binary mixture of 5 mM AGB and 100 µM glutamine, arginine or glutamate (Figure 5, Table 1). Immunogold particles were quantified in all cell types and compared to their incidences in the lumen (Figure 6): 0-14 gold particles/1.5 µm2 for lumen; 0-62 gold particles/1.5 µm2 for MSNs; 0-10 gold particles/1.5 µm2 for CSNs; and 0-15 gold particles/1.5 µm2 for SCs. The labeling in MSNs was up to four to six times higher than in other cell types (Figure 6). A significant relationship between cell type and odorant variables was noted (ANOVA, F = 7.1, d.f. = 9, P < 0.001). Comparisons of mean numbers of gold particles within cell types and the lumen were significant for lumen (ANOVA, F = 19.22, d.f. = 3, P < 0.001) and MSNs (ANOVA, F = 3.27, d.f. = 3, P < 0.025) only. Arginine stimulated labeling in the lumen was significantly greater than the corresponding AGB, glutamine or glutamate stimulated labeling (Tukey's test, P < 0.05). Within all preparations, the mean number of gold particles in MSNs was significantly greater than in the lumen and in other cell types (AGB, F = 13.38, d.f. = 3, P < 0.001; glutamine, F = 61.16, d.f. = 3, P < 0.001; arginine, F = 18.57, d.f. = 3, P < 0.001; glutamate, F = 49.90, d.f. = 3, P < 0.001). Neither CSNs nor SCs had elevated gold particles relative to control values for the lumen.
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The upper limit of the 99% CI for labeling in the lumen was used as a
cutoff to determine the percentages of OSNs and SCs with significantly
increased labeling for each of the odorants
(Table 2). Comparisons among
cell types were significant overall for each odorant condition (AGB,
2 = 65.5, d.f. = 2, P < 0.001; Gln,
2 = 90.7, d.f. = 2, P < 0.001; Arg,
2 = 64.5, d.f. = 2, P < 0.001; Glu,
2 = 49.9, d.f. = 2, P < 0.001). For pairwise
comparisons, percentages of MSNs with gold particles above background were
significantly elevated compared to both CSNs and SCs for all odorstimulated
conditions, while percentages of SCs were significantly greater than CSNs for
glutamine and glutamate stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Sensory neurons in the OE of fishes detect a broad spectrum of chemically diverse olfactory stimuli, ranging from amino acids, serving as general feeding stimulants, to bile acids, acting as social stimuli, and prostaglandins and steroids, serving as specific pheromones. This investigation addressed the issue of whether particular OSN types mediate the detection of a specific class of stimuli, the amino acids. Light microscope activity-dependent immunocytochemistry demonstrated that all amino acid odorants (except histidine) elicited an increase in the proportion of labeled OE compared to the AGB control preparations, and it revealed that most labeled OSNs were superficially located. Immuno electron microscopy identified the majority of labeled cells as MSNs. Thus, stimulation with three amino acid odorants presumed to activate distinct odorant receptors (Michel and Derbidge, 1997
)
resulted in AGB labeling and a high incidence of nanogold particles over many,
but not all MSNs. We previously suggested that the amino acid stimulated
labeling of cells with a prominent dendritic knob was consistent with amino
acid sensitive ciliated sensory neurons
(Michel et al.,
1999). In the current investigation, electron microscopy reveals
knoblike terminations on both CSNs and MSNs, but only a few weakly labeled
CSNs (and SCs). Too few crypt cells were observed to determine if amino acids
stimulated these cells.
Activity-dependent permeation of AGB into weakly stimulated OSNs produced
levels of electron-microscope labeling only slightly greater than background,
while heavily labeled MSNs had signal-to-noise ratios comparable to, or better
than, other nanogold-based immuno electron microscopy studies
(Yang et al., 2000).
Given an average background labeling of three to five nanogold particles per
1.5 µm2 (Table 1)
and 62 nanogold particles per 1.5 µm2 in the most heavily
labeled MSN, the calculated signal-to-noise ratio ranges from 12:1 to 20:1.
Though not systemically investigated, antigen exposure with either
H2O2 (glutamine preparation) or NaEtOH (glutamate,
arginine and AGB preparations) yielded similar patterns of immunostaining
without obvious differences in either background or maximal labeling.
The amino acids used in the current study were selected because they
interact with at least partially independent odorant receptors
(Friedrich and Korsching, 1997;
Michel and Derbidge, 1997;
Lipschitz and Michel, 1999).
In general, the relative proportion of OE labeled by each of the amino acid
stimuli is in good agreement with the relative stimulatory effectiveness
established in earlier electrophysiological studies
(Michel and Lubomudrov, 1995)
and neutral amino acids were found to be more potent than basic and acidic
amino acids. Labeling during AGB stimulation alone is also not unexpected,
since we have previously shown AGB to be a relatively potent odorant
(Lipschitz and Michel, 1999;
Michel et al., 1999).
The proportions of electron-microscope-identified MSNs labeled by each of the
amino acid odorants were substantially higher than the corresponding
proportions of total OE labeled (glutamine, 71 versus 18%; glutamate, 59
versus 12%; arginine, 36 versus 11%; AGB, 51 versus 7%). In part, this
discrepancy can be accounted for by our bias of selecting lamellae previously
shown to have robust labeling for electron-microscope examination. If,
however, we assume that MSNs occupy only 30-50% of the total area in the OE
and that the majority of activity-dependent labeling is associated with this
cell type, then a high proportion of the MSNs would have to be labeled to
account for the proportions noted for the entire epithelium. The relatively
low numbers of labeled CSNs and SCs (maximum 7 and 23%, respectively) are
consistent with this interpretation; however, the labeling noted in these two
cell groups warrants further comment.
Only a few, very weakly labeled CSNs were noted. Failure to label CSNs was
anticipated in light of the inability of AGB to permeate through
cyclic-nucleotide-gated channels (Michel
et al., 1999), the ion channels thought to mediate
olfactory transduction in the CSNs of fish
(Ngai et al., 1993;
Speca et al., 1999).
Many odorants, such as bile acids and polyamines, elicit robust
electrophysiological responses yet fail to stimulate activity-dependent
labeling, presumably due to their activation of the CNG pathway of CSNs
(Ma and Michel, 1998;
Michel, 1999). Considering our
inability to stimulate robust labeling of CSNs with any stimulus, we are
unable to conclude that amino acid odorants do not stimulate CSNs.
Available evidence suggests that amino acid odorants activate an
IP3-mediated transduction cascade rather than the
cyclic-nucleotide-mediated transduction pathway
(Bruch and Teeter, 1990;
Restrepo et al.,
1993). In zebrafish, drugs thought to perturb
IP3-mediated transduction affect amino acid elicited responses to a
greater extent than bile acid elicited responses
(Ma and Michel, 1998) and
eliminated AGB and amino acid stimulated labeling
(Michel, 1999;
Michel et al., 1999).
In goldfish, an L-arginine-sensitive V2R receptor couples to phospholipase C
and IP3 production when heterologously expressed and is
superficially located in the OE in a position consistent with the MSNs
(Speca et al., 1999).
The goldfish L-arginine-sensitive receptor was shown to be insensitive to AGB
(Speca et al., 1999),
the arginine analog which served as the activity probe in the current
investigation. Thus, if a zebrafish homolog of this receptor exists it is not
likely to be mediating background AGB labeling. Consistent with this
interpretation is the electro-physiological observation that AGB and
L-arginine interact with largely independent odorant receptors in the
peripheral olfactory system of zebrafish
(Lipschitz and Michel,
1999).
An unexpected finding was the labeling of SCs above background levels in
the lumen in three of four preparations. Much of the labeling appears to be
very apically located and associated with intracellular vesicles (see Figures
3 and
5). The mechanism of AGB entry
into SCs is unknown, but may be associated with the endocytosis of secretory
vesicular membrane or the activity of an amino acid transporter.
Electron-lucent SC nuclei are located deeper in the OE, near the basal lamina
(Moran et al., 1992;
Byrd and Brunjes, 1995;
Hansen and Zeiske, 1998) in
areas corresponding to or deeper than CSNs. Thus, regardless of the mechanism
of labeling, we must conclude that SCs account for some of the labeling
observed at the light-microscope level and probably account for more of the
basally located labeling than the CSNs.
Differences in the epithelial thickness might contribute to differences we
noted in the lengths of electron-microscope identified OSNs and
activity-labeled OSNs from light-microscope measurements. The average length
of the labeled OSNs in silver-intensified light-microscope images was 6.5
µm, while the average lengths of identified MSNs and CSNs from
electron-microscope images were 4.9 and 10.2 µm, respectively. The average
epithelial thicknesses for the measured OSNs and the identified MSNs and CSNs
were 20 and 15.4 µm, respectively. As shown in
Figure 4B, both CSN and MSN
cell centroid length is correlated with epithelial thickness, thus the
intermediate location of OSN lengths relative to the measured lengths of MSNs
and CSNs may, in part, be a result of the epithelial thickness of each of the
preparations. Normalizing average cell centroid length to the average
epithelial thickness for both the light- and electron-microscope preparations
eliminates this effect. Odor-stimulated light- and electron-microscope OSN
cell centroid lengths normalized to epithelial thickness resulted in values of
0.325 µm for light-microscope OSNs, 0.318 µm for identified MSNs and
0.662 µm for the identified CSNs. Comparison of the cell centroid length
data obtained from light-microscope immunocytochemistry in this study for five
other amino acid odorants and for a series of arginine analogs tested in an
earlier study (Lipschitz and Michel,
1999) suggests that the amino acid stimulated cell centroid
lengths most closely match the distribution of identified MSNs. Nonetheless, a
shoulder on each of the OSN length distributions indicates the presence of a
small population of longer cells. These cells may correspond to the relatively
long SC population shown to be labeled under three of four odor-stimulated
conditions. Alternatively, we noted that the average cell centroid length of
methionine stimulated OSNs was significantly longer than the AGB stimulated
OSNs and the average lengths of histidine, arginine, glutamate, cysteine and
alanine stimulated OSNs were significantly shorter than the AGB stimulated
OSNs. Perhaps there are subclasses of OSNs of varying lengths that respond to
specific stimuli. It has been reported
(Morita and Finger, 1998) that
focal DiI injections into the channel catfish olfactory bulb labeled short,
intermediate and long OSNs in the OE.
Odorants (and drugs) eliciting electrophysiological responses from the OE
of the zebrafish can be divided into two groups: those that stimulate
activity-dependent labeling and those that do not. Most amino acids and
several guanidinium analogs (Lipschitz and
Michel, 1999) stimulate labeling of many of what we now assume to
be MSNs. Other odors, such as polyamines and bile acids, failed to stimulate
labeling of MSNs, CSNs or crypt receptor cells. Although we cannot exclude the
possibility that MSNs support a second transduction cascade impermeant to AGB,
it seems more likely that some of these odorants are specifically detected by
the CSNs (or crypt cells). At this time, we cannot exclude the possibility
that CSNs also serve a role in the detection of amino acids. Information from
activity maps in the olfactory bulb suggests that there is a chemotopic
separation of input in the OB (Friedrich and Korsching,
1997,
1998). Amino acids and
nucleotides preferentially activate regions of the lateral OB, while bile
acids activate the medial bulb. It will be interesting to determine if the two
transduction pathways map specifically to MSNs and CSNs and whether there is a
subsequent mapping to distinct areas in the OB.
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Acknowledgments |
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We thank Drs Mary Lucero and Larry Stensaas for critically reviewing the manuscript, Fatemeh Sahlolbei, Ping Deng, Dimitry Elkin and Jared Olsen for their assistance in processing the olfactory tissue, Nancy Chandler for assisting with the electron microscopy and Signature Immunologics for the gift of the anti-AGB antibody. This research was supported by National Institute of Health grants DC-01418 and NS-07938.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Accepted December 26, 2001
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