Department of Anatomy and Neurobiology and Program in Neuroscience, University of Maryland, Baltimore, Maryland 21201
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ABSTRACT |
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Chen, Shan, Andrew P. Lane, Roland Bock, Trese Leinders-Zufall, and Frank Zufall. Blocking Adenylyl Cyclase Inhibits Olfactory Generator Currents Induced by "IP3-Odors". J. Neurophysiol. 84: 575-580, 2000. Vertebrate olfactory receptor neurons (ORNs) transduce odor stimuli into electrical signals by means of an adenylyl cyclase/cAMP second messenger cascade, but it remains widely debated whether this cAMP cascade mediates transduction for all odorants or only certain odor classes. To address this problem, we have analyzed the generator currents induced by odors that failed to produce cAMP in previous biochemical assays but instead produced IP3 ("IP3-odors"). We show that in single salamander ORNs, sensory responses to "cAMP-odors" and IP3-odors are not mutually exclusive but coexist in the same cells. The currents induced by IP3-odors exhibit identical biophysical properties as those induced by cAMP odors or direct activation of the cAMP cascade. By disrupting adenylyl cyclase to block cAMP formation using two potent antagonists of adenylyl cyclase, SQ22536 and MDL12330A, we show that this molecular step is necessary for the transduction of both odor classes. To assess whether these results are also applicable to mammals, we examine the electrophysiological responses to IP3-odors in intact mouse main olfactory epithelium (MOE) by recording field potentials. The results show that inhibition of adenylyl cyclase prevents EOG responses to both odor classes in mouse MOE, even when "hot spots" with heightened sensitivity to IP3-odors are examined.
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INTRODUCTION |
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In ciliated olfactory
receptor neurons (ORNs) of the vertebrate main olfactory epithelium
(MOE), transduction of odorants into excitatory electrical signals
involves the activation of a G-protein-coupled adenylyl cyclase/cAMP
second messenger cascade leading to the sequential opening of
Ca2+-permeable cAMP-gated channels and
Ca2+-activated chloride channels (for reviews see
Firestein 1996; Frings et al. 2000
;
Reed 1992
; Ronnett and Snyder 1992
;
Schild and Restrepo 1998
; Zufall et al.
1994
). Several reports have shown that odors sometimes also
cause formation of inositol-1,4,5-trisphosphate (IP3) (Boekhoff et al. 1990
;
Breer and Boekhoff 1991
; Huque and Bruch
1986
; Ronnett et al. 1993
), and it has been
suggested that IP3, via the subsequent gating of
plasma membrane channels in the olfactory cilia, mediates an
alternative transduction pathway causing ORN depolarization
(Okada et al. 1994
; Restrepo et al. 1990
). Other studies, however, were unable to confirm a
significant contribution of IP3 to the excitatory
odor responses of ciliated ORNs (Belluscio et al. 1998
;
Brunet et al. 1996
; Firestein et al.
1991a
; Kleene et al. 1994
; Lowe and Gold
1993
; Lowe et al. 1989
).
Very few data are available for the electrical responses to
IP3-producing odors
(IP3-odors), especially at the level of single ORNs, making an assessment of these conflicting reports difficult (Schild and Restrepo 1998). Therefore a primary goal of
this study was to provide a biophysical and pharmacological analysis of
olfactory generator currents induced by
IP3-odors. Our results show that there are no
detectable differences between the electrical responses to
IP3-odors and those induced by direct activation
of the cAMP cascade. By disrupting adenylyl cyclase to block cAMP
formation using two potent antagonists of adenylyl cyclase, SQ22536
(Harris et al. 1979
) and MDL12330A (Guellaen et
al. 1977
), we show that this molecular step is necessary for
the transduction of a wide variety of odors including those that
produced IP3 in biochemical assays.
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METHODS |
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ORNs were acutely dissociated from the nasal epithelium of adult
tiger salamanders (Ambystoma tigrinum) as described
previously (Leinders-Zufall et al. 1996). Odor responses
were recorded under voltage clamp applying the perforated patch
technique with amphotericin B. If not otherwise stated, the holding
potential was
60 mV. Current recordings, data acquisition, and online
analyses were controlled by an EPC-9 patch-clamp amplifier controlled
by Pulse software (HEKA Electronic, Lambrecht/Pfalz, Germany). Focal
stimulation of olfactory cilia was obtained by pressure ejecting the
odor solutions from multibarrel glass pipettes (Leinders-Zufall
et al. 1996
). ORNs were continuously superfused with Ringer
solution containing (in mM) 115 NaCl, 2.5 KCl, 1.0 CaCl2, 1.5 MgCl2, 4.5 HEPES, and 4.5 Na-HEPES (pH 7.6, adjusted to 240 mOsm). The pipettes contained (in mM) 17.7 KCl, 92.3 KOH, 82.3 methanesulfonic acid, 5.0 EGTA, and 10 HEPES [pH 7.5 (KOH), adjusted to 220 mOsm]. Data analyses and calculations were performed as described previously (Leinders-Zufall et al. 1999
).
For the electro-olfactogram (EOG) recordings from MOE, CD-1 mice
(2-mo-old, either sex) (Charles River, Wilmington, MA) were killed
using CO2. Following decapitation, the nasal
septum was removed to expose the endoturbinate system. The apical
surface of the epithelium was superfused continuously with oxygenated saline (95% O2-5% CO2)
containing (in mM) 120 NaCl, 25 NaHCO3, 5 KCl, 5 BES, 1 MgSO4, 1 CaCl2, and
10 mM glucose. Odor stimulation was performed using the same methods as
described above. Field potentials were recorded using glass pipettes
(resistance, 1 M; tips filled with extracellular solution in 1%
agar) that were connected via an Ag/AgCl wire to a differential
amplifier (DP-301, Warner Instruments, Hamden, CT). A second Ag/AgCl
wire connected to an agar bridge served as indifferent electrode. The
output signal was digitized, low-pass-filtered (8-pole Bessel; corner frequency, 60 Hz), and analyzed using Pulse software.
The following odorants were used in this study: acetophenone
(1-phenylethanone, Sigma); n-amylacetate (Sigma); isoamylacetate (Sigma); cineole (eucalyptol),
1,3,3-trimethyl-2-oxabicyclo(2,2,2)-octane (Sigma); citralva,
3,7-dimethyl-2,6-octadienenitrile (Aldrich); ethylvanillin,
3-ethoxy-4-hydroxybenzaldehyde (Aldrich); 2-heptanol (Aldrich);
isovaleric acid (Aldrich); lilial,
4-(1,1-dimethylethyl)--methylbenzenepropanol (Givaudan Roure);
(+)-limonene, (R)-4-isopropenyl-1-methyl-1-cyclohexene (Sigma);
lyral
(cyclohexanal), 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxyaldehyde (Givaudan
Roure); menthone, 5-methyl-2-(1-methylethyl)cyclohexanone (Sigma);
octanal (Sigma); pyrazine (Aldrich); and 3-ethyl 2-methylpyrazine (Aldrich). Odorants were of the highest purity grade available. Odorant
stock solutions were prepared in dimethylsulfoxide (DMSO) and diluted
to the final concentration with extracellular solution. The final
concentration of DMSO was <0.1% (vol/vol).
The adenylyl cyclase inhibitors 9-(tetrahydro-2-furyl)-adenine
(SQ22536) and
cis-N-(2-phenylcyclopentyl)-azacyclotridecan-2-imine-hydrochloride (MDL12330A) were obtained from Research Biochemicals
International (Natick, MA). Both compounds were water soluble but stock
solutions were sonicated for several minutes.
3-isobutyl-1-methyl-xanthine (IBMX, Sigma) was prepared as described
previously (Leinders-Zufall et al. 1999).
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RESULTS |
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Lyral and lilial-induced generator currents in salamander ORNs
The first series of experiments was designed to analyze generator
currents induced by IP3-odors at the level of
single ORNs. The results are based on a total of 242 ORNs of which 97 were tested with the aldehydes lyral and lilial, two odorants that have
failed to produce cAMP in biochemical assays (Breer and Boekhoff 1991; Sklar et al. 1986
). Of these 97 cells, 25 responded to lyral, 9 to lilial, and 5 to both odorants. In the example
of Fig. 1A, lyral (300 µM)
induced the activation of a large transient inward current with an
overall amplitude, shape, and time course typical of excitatory odor
currents in amphibian ORNs (Firestein and Werblin 1989
).
Lilial was ineffective in evoking a detectable current in this cell.
The same neuron responded also to cineole (Fig. 1A), which
is known to stimulate CNG channel activation via cAMP (Firestein et al. 1991a
) and to limonene, another
odorant that has failed to affect adenylyl cyclase activity in
biochemical assays (Sklar et al. 1986
). Application of
the phosphodiesterase inhibitor IBMX, which bypasses odor receptor
activation and leads to CNG channel activation because of the high
basal activity of olfactory adenylyl cyclase (Firestein et al.
1991b
), produced a similar inward current as lyral, cineole,
and limonene (Fig. 1A).
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Figure 1B shows response profiles from 21 ORNs that were stimulated sequentially with six different odorants (lyral, lilial, cineole, n-amylacetate, acetophenone, and limonene) and with IBMX. These profiles make two important points with respect to the mechanisms underlying the lyral/lilial-induced currents. The first is that all ORNs that reacted to lyral or lilial were also responsive to IBMX. This indicates that lyral or lilial responses occurred in ORNs that contained an intact cyclic nucleotide second messenger cascade. A second point made by the response profiles is that lyral or lilial can be recognized by the same neurons that also respond to general cAMP-producing odors such as cineole, n-amylacetate, or acetophenone. Thus molecular recognition and subsequent signal transduction of cAMP-odors and IP3-odors is not mutually exclusive in salamander ORNs.
These results point to the possibility that lyral and lilial are
transduced by these ORNs via the same molecular mechanism as cineole
and n-amylacetate. To investigate this further, we examined
the biophysical properties of the sensory currents in more detail.
First, we analyzed the response kinetics by superimposing the onset
phase of the lyral and cineole-induced current traces (Fig.
1C). Both responses exhibited identical activation time courses (n = 11). The onset time course of the
limonene-induced current also matched the responses induced by lyral
and cineole (Fig. 1C). Second, we elicited voltage ramps at
the peak of the odor-induced currents to examine the corresponding
conductance change (Fig. 1D). Figure 1E
illustrates that the current-voltage (I-V) curves induced by
lyral or lilial were almost indistinguishable from the curves obtained
with cineole or IBMX, yielding nearly identical reversal potential
values [lyral, 19.8 ± 7.0 (SD) mV, n = 6;
lilial,
18.9 ± 2.9 mV, n = 4; cineole,
19.2 ± 3.9 mV, n = 6; IBMX,
17.5 ± 5.3 mV, n = 4; LSD: P = 0.49-0.93]. This suggests that all four responses were mediated by the same or very
similar ionic mechanisms. Third, we bath-applied
6-anilino-5,8-quinolinedione (LY83583, 20 µM), a potent inhibitor of
ion fluxes through CNG channels (Leinders-Zufall and Zufall
1995
). LY83583 completely but reversibly inhibited the lyral
and lilial-induced current (n = 3, data not shown). All
of these results are consistent with the notion that lyral and lilial
caused the generation of electrical currents via the same second
messenger system as cineole and IBMX.
Reversible disruption of odor transduction by inhibition of adenylyl cyclase
To test directly whether lyral and lilial affected the activity of
adenylyl cyclase, we attempted to disrupt cAMP formation by applying
two widely used adenylyl cyclase inhibitors, SQ22536 (Harris et
al. 1979) and MDL12330A (Guellaen et al. 1977
).
Because the actions of the two antagonists have not been investigated systematically in ORNs, we first tested their usefulness and
specificity (see also Sato et al. 1996
). Figure
2A shows that in the presence of SQ22536 (300 µM, added to the bath solution) the cineole-induced response was completely abolished, an effect that was reversible. Figure 2B displays the fraction of unblocked odor current
(I/Imax, open symbols) as a function of
SQ22536 concentration. The curve is constructed by pooling several
individual measurements (n = 13). The data are
well-fitted with a Langmuir function
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(1) |
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We next examined whether the two inhibitors would also block the responses to lyral and lilial. The inhibition curves of Fig. 2, F and G show that this was the case. The curves could be fitted according to Eq. 1 using almost identical Kd values as in Fig. 2, B and C. Thus lyral and lilial-induced currents were inhibited by SQ22536 and MDL12330A with the same potency as the cineole and IBMX-induced responses. We also tested the effects of both inhibitors at much lower concentrations of lyral or lilial (10 µM each, n = 4) but there was no difference in their blocking potency (data not shown).
EOG-responses to IP3-odors in mouse main olfactory epithelium depend on adenylyl cyclase
To assess whether these results are also applicable to mammalian
ORNs, we examined the responses to IP3-odors in
mouse MOE by recording extracellular field potentials from the
epithelial surface. This measurement, the EOG (Ottoson
1956), registers summed local activities of ORNs in intact
epithelium. Lyral- and lilial-induced EOG responses were observed at
several locations on the endoturbinates IIa, IIb, and III, (1-11, 14, 16; Fig. 3A), although with
varying relative sensitivities. Initially, we focused on hot spots
exhibiting heightened sensitivity to lyral or lilial. At these
locations, we recorded large negative field potentials at relatively
low stimulus concentrations, ranging from 100 nM to 1 µM (Fig.
3B). When we added SQ22536 to the bath solution (100 µM
for 10 min), activation of both lyral and lilial-induced responses was
completely prevented, an effect that could be reversed after washing
the tissue for 10 min with drug-free extracellular solution (Fig. 3B). As in the salamander, this inhibition depended on the
concentration of SQ22536, with an apparent
Kd of 1 µM but was independent of the odor concentration (data not shown). Almost identical results were
obtained using a panel of 14 structurally diverse odorants (Fig.
3C). SQ22536 disrupted transduction of odorants which have been reported to elicit increases in cAMP (amylacetate, cineole, citralva, and menthone) as well as those which have been reported to
elicit IP3 (ethylvanillin, isovaleric acid,
lilial, lyral, and pyrazine). There was no significant difference in
the effect of SQ22536 at the various recording sites. As a control, we
tested the effect of SQ22536 on field potentials caused by IBMX
application (100 µM, n = 5; Fig. 3C).
Application of MDL12330A (30 µM for 10 min) also eliminated the
responses to both lyral/lilial and n-amylacetate/cineole
(Fig. 3C). Finally, to rule out species differences, we
analyzed EOG responses in rat MOE using various concentrations of the
odors listed in Fig. 3C and found that these responses, too,
were abolished by SQ22536 (data not shown).
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DISCUSSION |
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Our data show that generator currents induced by
IP3-odors exhibit identical biophysical and
pharmacological properties as those activated by cAMP-odors or direct
activation of the cAMP cascade. Pharmacological blockade of cAMP
formation prevented the activation of the responses to both odor
classes in isolated salamander ORNs and intact mouse MOE. This blockade
was also seen when epithelial hot spots with heightened sensitivity to
IP3-odors were examined. Several tests were
conducted to show that the effects of the two adenylyl cyclase
inhibitors used here, SQ22536 and MDL12330A, are specific and
selective. Our results, using effective pharmacological blockade, and
those of previous gene knock out studies (OCNC-1: Brunet et al.
1996; Golf: Belluscio et al.
1998
) are conclusive evidence that in a majority of
ciliated ORNs of the MOE,
Golf-mediated adenylyl cyclase activation and CNG
channel opening are essential for the transduction of a wide variety of odors including those that produced IP3 in
biochemical assays.
We must emphasize, however, that we cannot rule out the possibility
that there are small subsets of ORNs in the MOE that use second
messengers other than cAMP for odor transduction. Two emerging possibilities should be noted here. First, there is mounting evidence that microvillous ORNs are present in the MOE of diverse
vertebrates, including most teleost fishes, salamanders, and some
mammals including man (see Morita and Finger 1998 and
references therein). Microvillous ORNs seem to express odor receptors
that share similarity with the vomeronasal receptors (Cao et al.
1998
; Speca et al. 1999
). Although the precise
transduction pathways in these cells remain to be determined, it seems
likely that they respond to chemostimulation with phosphatidyl inositol
turnover, resulting in the production of IP3 (see
discussion in Speca et al. 1999
). Isolated "cilia preparations" used for biochemical measurements of odor-induced second messenger formation may have contained both cilia and
microvilli. Second, given the identification of a subset of ORNs
lacking type III adenylyl cyclase but instead expressing a receptor
guanylyl cyclase (GC-D) in their cilia (Juilfs et al.
1997
), it seems highly likely that these cells, too, respond to
odors in a cAMP-independent manner. This notion is further supported by
the result that, in OCNC1-null mice, tyrosine hydroxylase expression in
the olfactory bulb, which reflects afferent activity, is reduced in the
majority of periglomerular neurons but retained in the "necklace"
glomeruli which receive afferent input from the GC-D expressing
receptor neurons (Baker et al. 1999
). The
pharmacological approach developed here should add an important tool
for the identification of ORNs exhibiting odor responses that are
independent of cAMP-formation. It should also be useful for examining
the role of activity-dependent processes in the development of the
olfactory system.
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ACKNOWLEDGMENTS |
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We thank Givaudan Roure for the generous gift of lyral and lilial and M. Shipley and F. Margolis for comments on earlier versions of this manuscript.
This work was supported in part by National Institutes of Health Grants NS-37748 to F. Zufall and DC-03773 to T. Leinders-Zufall. A. P. Lane was the recipient of NIH Training Grant DC-00054.
Present address of S. Chen: Synaptic Physiology Unit, National Institute of Neurological Diseases and Stroke, Bldg. 36, Rm. 2C-09, National Institutes of Health, Bethesda, MD 20892.
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FOOTNOTES |
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Address for reprint requests: F. Zufall, Dept. of Anatomy and Neurobiology, University of Maryland School of Medicine, 685 West Baltimore St., Baltimore, MD 21201. (E-mail: fzufa001{at}umaryland.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 March 2000; accepted in final form 7 April 2000.
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REFERENCES |
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