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First published online July 14, 2008
Journal of Experimental Biology 211, 2417-2422 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018796
The elusive crypt olfactory receptor neuron: evidence for its stimulation by amino acids and cAMP pathway agonists
Centro de Neurociencia de Valparaíso, Universidad de Valparaíso, Avda. Gran Bretaña 1111, Casilla 5029, Correo 4 2360102 Valparaíso, Chile
* Author for correspondence (e-mail: oliver.schmachtenberg{at}uv.cl or oliver{at}cnv.cl)
Accepted 27 May 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: crypt cell, fish, olfaction, chemosensory, olfactory receptor neuron, odorant, transduction
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Hansen and Zeiske,
1998; Hansen and Zielinski,
2005). Recently, the presence of crypt-like ORNs was also
demonstrated in a cartilaginous fish
(Ferrando et al., 2006),
suggesting that they are a conserved feature among fishes. The crypt ORN has
an oval cell body that is almost completely surrounded by one or two
supporting cells. Mature crypt ORNs localize to the surface of the sensory
area of the olfactory epithelium, and their apical invagination (the `crypt')
opens to the exterior space. Both cilia and microvilli are found within the
crypt, and are thought to hold the olfactory receptor proteins and other
elements of the transduction machinery.
During recent years, considerable progress has been made in the
understanding of the molecular components involved in the transduction cascade
of vertebrate ORNs. As in mammals, the tall ciliated ORNs of teleost fishes
express OR-type odorant receptors and transduce signals through a cyclic AMP
(cAMP) cascade that includes the G-protein G
olf
(Cao et al., 1998;
Ngai et al., 1993). Homologous
to mammalian VNO neurons, the shorter microvillous ORNs in the fish olfactory
epithelium express V2R-like receptors molecules, the G-protein
Go and possibly others
(Belanger et al., 2003;
Hansen et al., 2004;
Speca et al., 1999), and the
transient receptor potential channel C2
(Sato et al., 2005), but the
identity of the intracellular messenger that activates these channels is still
unclear. Crypt ORNs apparently lack any homologous counterpart in terrestrial
vertebrates, and with the exception of the olfactory G-proteins
Go and Gq, which were identified by
immunohistochemistry (Belanger et al.,
2003; Hansen et al.,
2004; Hansen et al.,
2003), neither their olfactory receptors nor the downstream
elements of the transduction cascade have been established.
Based on the projection pattern of crypt ORNs in crucian carps and their
seasonal density variation, they were proposed to mediate chemical signaling
related to reproductive behavior (Hamdani
el and Doving, 2006; Hamdani el
et al., 2008). Yet, the responsiveness of crypt ORNs to fish
pheromones and the involvement of these cells in behavioral control have not
been clarified. Therefore, apart from their odorant specificity and
transduction process, the entire functional context of crypt ORNs is still
hypothetical.
Since neither the molecular nor the anatomical strategies of the cited
studies were successful in resolving the function of crypt ORNs, we decided to
pursue this matter with a physiological approach. In a previous article, we
described the basic electrophysiological properties of crypt ORNs isolated
from the Pacific jack mackerel and provided initial evidence for their
chemosensitivity towards amino acids
(Schmachtenberg, 2006). Here,
we further analyzed the odorant response properties of crypt ORNs and show
that they are excited by agonists of the cyclic cAMP pathway.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Briefly, the fish were sacrificed by fast decapitation on ice, the olfactory
organs were removed and transferred to ice-cold saline solution, containing
(mmol l–1): 150 NaCl, 3 KCl, 1 CaCl2, 1
MgSO4, 5 Hepes, 10 glucose, pH 7.4. The sensory area of the
olfactory lamellas was cut into small pieces, triturated with a fire-polished
Pasteur pipette and the dissociated cells were transferred to a cooled
(10–15°C) poly-L-lysine-coated recording chamber that was
mounted on an Olympus IMT-2 microscope. Cells were viewed with a 100x
oil immersion objective and identified by their characteristic morphology.
Minimum criteria for their identification were a comparatively large size,
their ovoid shape and a clear separation between the crypt ORN and the
supporting cell. Frequently, these features were complemented by the presence
of a supporting cell foot process and an apical brush border. Sometimes the
crypt was also visible.
Recording pipettes were drawn from borosilicate electrode glass (WPI,
Sarasota, Fl, USA) or hematocrit glass capillaries (Hirschmann, Eberstadt,
Germany) in a horizontal puller (P-97; Sutter, Novato, CA, USA) to a tip
resistance of 5–10M
. The pipette solution contained (mmol
l–1): 120 KCl, 4.6 MgCl2, 10 glucose, 10 Hepes, 10
EGTA, 4 Na-ATP and 0.4 Na-GTP, pH 7.4. Cell-attached recordings were obtained
with a Dagan 3900 amplifier (Dagan Corp., Minneapolis, MN, USA; generously
provided by John Caprio), low-pass filtered at 5 kHz with a four-pole Bessel
filter, and digitalized with pClamp software (Axon Instruments, Union City,
CA, USA) at a sampling rate of 20 kHz. Odorants and reagents were supplied to
the cells with a custom-built picospritzer connected to a triple-barreled
stimulus pipette (WPI), as described previously in detail
(Schmachtenberg and Bacigalupo,
2004). Amino acids were applied as a mixture of the L-isomers of
alanine, arginine, asparagine, glutamate, glycine, methionine, phenylalanine
and tyrosine at a concentration of 1 mmol l–1 each in saline
solution. A mixture of lithocholic acid, sodium taurocholate and
taurolithocholic acid (`bile salts'), reportedly strong odorants to fish
(Rolen and Caprio, 2007), was
applied alternatively. Two candidate fish pheromones, prostaglandin F2
(PGF2) and 4-pregnen-17a,20b-diol-3-one (17,20βP) were tested
together at two concentrations of 5 and 50 µmol l–1 each.
8-Bromo cyclic AMP (8Br-cAMP), a membrane-permeant agonist of cyclic
nucleotide-gated ion channels and 3-isobutyl-1-methylxanthine (IBMX) together
with forskolin, which elevate intracellular cAMP levels, were used at a
concentration of 1 mmol l–1 in the stimulus pipette. Most
cells were tested with two or three stimuli. We did not measure or calculate
the agonist concentrations reaching the cell, since too many variables were
involved. The criterion for a response to a stimulus was a >30% increase in
the spike rate compared to pre-pulse levels over an interval of 5 s during or
immediately after the stimulus.
Calcium imaging
For calcium imaging experiments, crypt ORNs were isolated as described
above and were loaded with Fluo-4 AM (5 µmol l–1 in 0.1%
pluronic acid) at 4°C for 1 h. Images were recorded with a Sensicam QE
cooled digital camera (Cooke Corp., Romulus, MI, USA) attached to a Nikon
Eclipse 2000 (Nikon Corp., Japan) inverted microscope at intervals of 1 s, and
analyzed with the NIH ImageJ program. Additional experiments were performed on
an upright Nikon FN-1 microscope (objective 40x water immersion, NA 0.8)
with a Nikon DS-2MBWc camera controlled by the program NIS Elements (Nikan
Corp., Japan). Cells were perfused continuously with cooled saline solution
(at 
10°C) and stimulated with a picospritzer, as in the patch clamp
experiments. To obtain the relative fluorescence ratio, a bleaching curve was
calculated for every experiment and subtracted from the image sequence.
Relative pixel intensities were obtained by dividing all images through the
initial (prepulse) image of the series. The criterion for a significant
response was a >10% increase in fluorescence intensity compared to
pre-pulse levels.
Immunochemistry
For western blots, the olfactory epithelia of one jack mackerel and one
adult rat were homogenized in ice-cold lysis buffer containing 1% SDS, 50 mmol
l–1 Tris (pH 8.3), 5 mmol l–1 EDTA and a
protease and phosphatase inhibitor cocktail. The homogenates were centrifuged
at 13 000 g for 15 min at 4°C. The supernatant was
collected and the total protein contents was determined using the Bradford
method. The samples were diluted in Laemmli buffer (62.5 mmol
l–1 Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01%
Bromophenol Blue, 300 mmol l–1 dithiothreitol), and denatured
by heating to 95°C for 5 min. The wells of a 10%
acrylamide–bisacrylamide gel were loaded with 90 µg of protein and a
molecular mass standard (Kaleidoscope, Bio-Rad, Hercules, CA, USA) was used
adjacently. Proteins were subsequently transferred to a polyvinylidene
difluoride (PVDF) membrane (Amersham Biosciences, Buckinghamshire, UK),
blocked in 8% non-fat milk with 0.1% Tween 20 in Tris buffer for 1 h at room
temperature, and incubated overnight at 4°C with the polyclonal
anti-adenylate cyclase III antiserum (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), diluted 1:500. After three washes, the horseradish
peroxidase-coupled secondary antibody (anti-rabbit-HRP; Jackson
ImmunoResearch, West Grove, PA, USA; diluted 1:5000), was applied for 1 h at
room temperature. Bound immunoglobulins were visualized with the SuperSignal
West Pico chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA)
on Kodak BioMax Light film.
For immunocytochemistry, olfactory epithelium was dissociated as for the physiological experiments, and the cells were fixed in 2% paraformaldehyde for 25 min on poly-L-lysine-coated slides at room temperature. After permeabilization with 0.2% Triton-X-100 for 5 min, the preparation was blocked in 5% BSA for 1 h. The primary anti-adenylate cyclase III antiserum was applied at a dilution of 1:100 overnight at 4°C. As a control, the antiserum was preadsorbed overnight at 4°C with excess antigen (1 µgml–1; Santa Cruz). After three washes, the preparation was incubated in the secondary antiserum, donkey anti-rabbit Cy-3-conjugated IgG (Jackson ImmunoResearch, diluted 1:1000) for 1 h at room temperature. Finally, the cells were counterstained with DAPI (Invitrogen, Carlsbad, CA, USA) for 5 min at 5 µgml–1 and mounted in Fluomount (Dako Industries, Carpenteria, CA, USA). Photographs were taken with a CoolSnap-Pro digital camera on an Olympus BX-51 microscope operated by Image-Pro Express software (Media Cybernetics, Silver Spring, MD, USA).
The experimental procedures were approved by the Bioethics Committee of the University of Valparaiso and in accordance with the bioethics regulation of the Chilean Research Council (CONICYT). Reagents were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise indicated.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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To investigate the possibility that a cAMP transduction pathway is expressed and operative in crypt ORNs, we applied 8Br-cAMP through a puffer pipette aimed at the crypt of the cells. Pulses of 8Br-cAMP triggered transient excitation under patch clamp and a rise of intracellular calcium under fluorescence imaging with an intensity and time course similar to the responses to amino acids (Fig. 1A, Fig. 2C). A significantly stronger and longer-lasting effect was observed after the application of IBMX plus forskolin, which triggered larger calcium signals than amino acids or 8Br-cAMP, even more than the positive control KCl (Fig. 2B,D). The moderate effect of KCl might be due to the fact that the crypt ORN membrane is largely covered by the supporting cell(s). Under patch clamp, IBMX alone elicited prolonged excitation in a dose-dependent manner, which was frequently preceded by an initial suppression of spike activity during the stimulus application (Fig. 1B).
The prostaglandin PGF2 and the steroid hormone 17,20βP were
tested to find evidence for the hypothesis that crypt ORNs act as pheromone
detectors (Hamdani el et al.,
2008). Although isolated effects were observed in one cell under
patch clamp, these were just above the defined response threshold and
irreproducible in other cells (Table
1). No responses were obtained under calcium imaging
(Fig. 2F). Therefore, a direct
activation of crypt ORNs by PGF2 and 17,20βP remains to be
demonstrated.
In an attempt to complement our physiological evidence for cAMP odor transduction in crypt ORNs with immunocytochemical data, we labeled isolated crypt ORNs with an antibody against adenylate cyclase type III (Fig. 3). This antiserum detected a protein with the appropriate molecular mass on western blots of fish olfactory epithelium, which was completely abolished by preadsorption with excess AC-III antigen. Crypt ORNs treated with the antiserum displayed cytoplasmic labeling that tended to be concentrated in the apical part of the cells and around the crypt. This suggests that AC-III is expressed in crypt ORNs and could be a part of the odor transduction pathway as in the ciliated ORNs of fish and terrestrial vertebrates. Together, these results confirm a chemosensory function of crypt ORNs and support the presence of a cAMP transduction pathway.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Hansen and Zielinski, 2005).
Evidence is mounting that each ORN type is tuned towards distinct odorant
classes, which are transduced through different pathways and activate specific
regions of the olfactory bulb (Friedrich
and Korsching, 1998; Hansen et
al., 2004; Hansen et al.,
2003; Nikonov and Caprio,
2001; Sato et al.,
2005; Sorensen and Caprio,
1998). However, the precise match between ORN and odorant types is
still unclear, and there are evidently more chemical odorant classes than
morphologically distinguishable ORN types in fish, as pointed out previously
(Hansen et al., 2004).
Here, we demonstrate that an amino acid mixture excited a significant
percentage of isolated crypt ORNs and induced a transient rise of
intracellular calcium in these cells. This demonstrates some degree of
chemosensory selectivity as other odorant classes tested in the present and a
previous study (Schmachtenberg,
2006), bile salts, polyamines and pheromone candidates, did not
trigger any reproducible responses with both approaches.
However, theoretical considerations and indirect evidence suggest that
crypt ORNs may function as detectors of conspecific odorants or pheromones in
the aquatic environment. Crypt ORN density in the olfactory epithelium is low
compared to other ORN types, may vary throughout the year and reach a peak
during the spawning season (Hamdani el et
al., 2008). Furthermore, crypt ORNs project to small specific
areas of the olfactory bulb (Hansen et
al., 2003), which have been associated with reproductive behavior
(Hamdani el and Doving, 2006;
Lastein et al., 2006).
Finally, the restricted access to the crypt will limit the exchange rate of
odorant molecules at the receptors, which rather suggests a slow, highly
sensitive labeled-line type odorant detection than a fast and combinatorial
operation.
Unfortunately, our attempts to demonstrate the responsiveness of crypt ORNs
to PGF2 and 17,20βP, both reportedly strong odorants with
pheromonal function in some fishes
(Lastein et al., 2006;
Sorensen et al., 1988;
Stacey et al., 1989), were
unsuccessful. Although a large number of chemicals released by fish may act as
reproductive pheromones (Stacey et al.,
2003), it is possible that crypt ORNs of T. symmetricus
are tuned to detect only few specific compounds not tested here, or that their
group comprises a variety of odorant receptors responsive to multiple
substances acting as conspecific chemosignals, but were under sampled in our
experiments. The only odorant class that consistently triggered responses were
amino acids, which confirms previous observations
(Schmachtenberg, 2006).
In this regard, it is interesting to note that the axons of crypt ORNs
terminate in a region of the olfactory bulb that globally responds to amino
acids in the channel catfish (Hansen et
al., 2003; Nikonov and Caprio,
2001). However, the methodology used in these studies, local
extracellular recordings and retrograde labeling, has a limited resolution and
may not resolve restricted olfactory bulb zones of different odorant
sensitivity and physiology.
Interestingly, amino acids have already been shown to be detected by both
ciliated and microvillous ORNs in fish. Why should several receptor cell types
be employed for that task? Perhaps each ORN type is the starting point for a
different signaling pathway with separate analytical function. On the other
hand, the relatively high concentrations necessary to elicit responses in
isolated ORNs compared to intact olfactory epithelium in live animals may also
activate neurons that are tuned to different substance classes, possibly with
similar sterical groups. Finally, there is a theoretical possibility that some
putative crypt ORNs from our experiments were confounded with microvillous
ORNs whose dendrites retracted during the dissociation process. Yet, this
seems highly unlikely, since average crypt ORNs are at least twice as large as
microvillous ORNs in T. symmetricus [see
Fig. 1F
(Schmachtenberg, 2006)], and
cells of doubtful identity were excluded from the analysis.
The present study also aimed at providing evidence for the components of
the transduction pathway(s) operating in crypt ORNs. The most significant
result is that crypt ORNs that responded to odorants were also excited by
agonists of the cAMP cascade, 8Br-cAMP and IBMX plus forskolin. The latter
compounds frequently caused initial spike suppression, followed by
post-stimulus excitation. This might reflect a non-specific blocking action of
the agonists on ion channels (Sanhueza et
al., 2000), or result from excessive depolarization of the cell
(`excitation block'). A different phenomenon is post-stimulus inhibition,
which was often observed after agonist or odorant stimulation (cell 1 in
Fig. 1A) and appears to be an
adaptational consequence (Schmachtenberg,
2006).
The patch clamp data were complemented by calcium imaging experiments
testing the same compounds, altogether suggesting that a cAMP transduction
cascade may operate in crypt ORNs to transduce chemosignals. Further evidence
for this hypothesis is provided by the labeling of crypt ORNs with an
antiserum against adenylate cyclase III. This enzyme is an integral component
of the cAMP transduction pathway of ciliated ORNs of terrestrial vertebrates,
and presumably also operates in the ciliated ORNs of fish
(Hansen et al., 2003;
Schmachtenberg and Bacigalupo,
2004). Crypt ORNs have both cilia and microvilli in their crypt,
and may also express two different G-proteins, Go and
Gq (Hansen et al.,
2004; Hansen and Zielinski,
2005). This opens the possibility that more than one odorant
transduction pathway is operational in crypt ORNs. The fact that over 50% of
the crypt ORNs were unresponsive to the applied cAMP pathway agonists suggests
that these cells did not express that pathway, possibly because they were
immature or because they expressed another transduction cascade instead.
Alternatively, they might have been rendered unresponsive by the dissociation
process. While the cells maintained a healthy aspect for up to 6 h in primary
culture, tissue dissociation is clearly an invasive procedure that may affect
the response properties of isolated cells. This problem could be overcome with
the development of a genetic crypt ORN marker, that should allow the study of
these cells in intact tissue or even in live animals, and thus help to resolve
the mystery of these peculiar receptor neurons.
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Acknowledgments |
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References |
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F/F) and an overlay of both. The corresponding plots
display the time course of the normalized fluorescence average of the whole
crypt ORN and its supporting cell. Amino acids (A), IBMX plus forskolin (B)
and 8Br-cAMP (C) induced a transient rise of intracellular calcium in the
crypt ORN. Some supporting cells, which are coupled to the crypt ORN by gap
junctions (Schmachtenberg,
2006