Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
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ABSTRACT |
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Olfactory chemotransduction involves a signaling cascade. In addition to triggering transduction, odors suppress ion conductances. By stimulating with brief odorant pulses, we observed a current associated with odor-induced suppression of voltage-gated conductances and studied its time dependence. We characterized this suppression current in isolated Caudiverbera caudiverbera olfactory neurons. All four voltage-gated currents are suppressed by odor pulses in almost every neuron, and suppression is caused by odors inducing excitation and by those inducing inhibition, indicating a nonselective phenomenon, in contrast to transduction. Suppression has a 10-fold shorter latency than transduction. Suppression was more pronounced when odors were applied to the soma than to the cilia, opposite to transduction. Suppression was also present in rat olfactory neurons. Furthermore, we could induce it in Drosophila photoreceptor cells, demonstrating its independence from the chemotransduction cascade. We show that odor concentrations causing suppression are similar to those triggering chemotransduction and that both suppression and transduction contribute to the odor response in isolated olfactory neurons. Furthermore, suppression affects spiking, implying a possible physiological role in olfaction.
olfactory transduction; odor excitation; odor inhibition; olfactory cilia
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INTRODUCTION |
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VERTEBRATE OLFACTORY RECEPTOR NEURONS respond to
odorants with changes in action potential firing. Excitatory responses,
consisting of spiking rate increases, begin with a depolarizing
receptor potential triggered by an inward transduction current
(IT). The mechanism underlying
IT involves a
cAMP pathway that increases two ionic conductances, a nonselective
cationic conductance gated by cAMP (20) and a
Ca2+-activated
Cl conductance (12), both
of which contribute to the net inward current. Inhibitory responses
have also been reported in some vertebrate species and begin with a
hyperpolarizing receptor potential responsible for a decrease in the
spiking rate (5, 19; for reviews, see Refs. 2 and 21). The mechanism
underlying odor inhibition remains largely unknown. In the Chilean toad
Caudiverbera caudiverbera it has been
studied in some detail (16-19) and shown to be due to
a Ca2+-activated
K+ conductance increase, which is
a consequence of the activation of a
Ca2+ conductance. A latency of
hundreds of milliseconds precedes the inhibitory transduction current,
revealing the participation of a second-messenger pathway. However,
details of the second-messenger signaling pathway remain unknown.
All transduction conductances are confined principally to the olfactory cilia (7, 10, 13, 17). In contrast, voltage-gated conductances reside in the cell body. In Caudiverbera, four conductances are triggered by depolarization: a TTX-sensitive, inactivating Na+ conductance; a voltage-dependent, noninactivating Ca2+ conductance; a delayed rectifier K+ conductance; and a Ca2+-activated K+ conductance (4). In other vertebrate species, a similar array of conductances is present, with an additional inactivating K+ conductance in some of them (see Ref. 21). When an olfactory cell is depolarized, either by an excitatory odorant or by current injection, all four voltage-gated conductances are activated and give shape to the response consisting of a train of action potentials.
It was previously shown that sustained bath odor exposure of isolated olfactory neurons suppresses the voltage-induced conductance changes (9). This effect was manifested as a reduction in both inward and outward currents triggered by depolarizing voltage steps applied in the presence of odorants. On the other hand, odorant pulses suppressed transduction currents with a very short latency (~20 ms) (11). In the present work we undertook a systematic study of the time dependence of odor suppression of the voltage-gated conductances, using brief odor pulses. Moreover, most electrophysiological studies on olfactory transduction have been conducted on isolated neurons using pulses of odorants. Because our results, like those of Kawai et al. (9), revealed that suppression and transduction share the same odor concentration thresholds, we expected that the effect of suppression should influence odor responses. Therefore, it seemed important to study odor suppression under experimental regimes similar to those typically used to study odor transduction. Here we applied the chemical stimuli onto the entire cell, rather than focusing them onto the olfactory cilia. Our experimental approach, which consisted of pulses of odorants [unlike bath applications that were used by Kawai et al. (9)], allowed us to quantify the contribution of odor suppression to the net odor-induced effect in those cells capable of transducing the applied odors. In this manner, we could evaluate with precision the contribution of both odor-induced effects (transduction and suppression) for every cell, allowing us to correct for suppression the measured current-voltage (I-V) curves and even the odor-triggered currents as a function of time. Our results indicate that suppression develops after a much shorter latency than that preceding the activation of IT, suggesting that it results from a direct effect of odorants on the voltage-gated ion channels. We show that odor suppression of voltage-gated currents takes place in virtually every olfactory neuron in the toad and in the majority of these receptor cells in the rat, at the odor concentrations used in the present study. However, at higher concentrations, we observed that also all rat olfactory neurons displayed suppression. We also document that suppression occurs in nonolfactory cells as well, as in Drosophila photoreceptors, indicating that suppression is independent of the olfactory transduction cascade. We also show that suppression may be physiologically relevant in olfactory neurons, since it alters action potential firing activity.
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MATERIALS AND METHODS |
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Cell Dissociation
Toad. Animals (C. caudiverbera) were cooled down to 0°C, killed, and pithed before dissecting out their olfactory epithelia. Olfactory receptor neurons were dissociated from the olfactory epithelium, as described by Morales et al. (19).Rat. For obtaining dissociated olfactory neurons, Wistar rats were decapitated after exposure to CO2. Olfactory epithelia dissected from the turbinates were cut into small pieces and mechanically dissociated, without the use of enzymes.
Drosophila. Omatidia were isolated from the eyes of Oregon-R flies, as described by Bacigalupo et al. (1). Single photoreceptor cells in isolated omatidia were whole cell voltage clamped.
Electrical Recordings and Stimulus Application
Electrical recordings were obtained with the patch-clamp technique (amplifier by Dagan), in its whole cell modality, as in our previous work (19). Experimental protocols and data analysis were conducted using pCLAMP 6.0 (Axon Instruments).Odor stimulation was achieved with double-barreled puffer pipettes made of glass (tip diameter ~2 µm, Sutter Instruments). They were positioned 20 or 40 µm from the cell. Stimuli engulfed the entire cell, except in the localization experiments. Pressure pulses (range 2-14 lb/in.2) were given to the pipettes with a picospritzer. We estimated that the delay of the puffing system was ~20 ms (delay of the current change induced by a H2O pulse delivered ~5 µm from the recording pipette). Latencies were corrected for (see RESULTS). Odor concentrations at the cell level were estimated as in Firestein and Werblin (8).
Solutions
Toad. Extracellular solutions were normal Ringer (NR) solution containing (in mM) 115 NaCl, 1 CaCl2, 1.5 MgCl2, 2.5 KCl, 3 glucose, and 10 HEPES, pH 7.6; 0-Ca2+ Ringer solution containing 2 mM EGTA and enough Ca2+ to give pCa 8.0 (Win Max); and high-K+ Ringer solution containing 100 mM KCl, 17.5 mM NaCl, and all other components as in NR. The internal solution contained (in mM) 120 KCl, 1 CaCl2, 2 EGTA, 1 MgCl2, 0.1 Na2-GTP, 1 Mg-ATP, and 4 HEPES, pH 7.6, pCa 7.5.Rat. The external solution contained (in mM) 1.3 CaCl2, 1.0 MgCl2, 0.7 MgSO4, 5.4 KCl, 0.30 K2HPO4, 137 NaCl, and 1.2 Na2HPO4, pH 7.6. The internal solution was the same as for the toad.
Drosophila. The external solution contained (in mM) 20 NaCl, 10 HEPES, 8 MgSO4, 5 KCl, 25 proline, 2.5 sucrose, and 1.5 CaCl2, pH 7.5. The internal solution contained (in mM) 124 KCl, 10 HEPES, 2 MgSO4, 1.1 EGTA, 0.1 CaCl2, 0.5 GTP, and 2 ATP, pH 7.15, pCa 7.3.
Odorants
Mixture I contained (1 mM of each in the stimulus pipette) citralva (3,7-dimethyl-2,6-octadienenitrile), citronellal (3,7-dimethyl-6-octenal), and geraniol (3,7-dimethyl-2,6-octadien-1-ol). Mixture II contained (1 mM of each within the pipette) isovaleric acid (3-methylbutanoic acid), pyrazine (1,4-diazine), and triethylamine. Odors were prepared directly in Ringer solution, from 100 mM stocks prepared in distilled water. All chemicals were obtained from Sigma, except for citralva, which was kindly donated by D. Restrepo. All experiments were conducted at ~22°C. ![]() |
RESULTS |
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Suppression by Odors of the Voltage-Gated Outward Currents in Olfactory Neurons
Suppression of the net outward current. If odorant-induced suppression results from an unspecific effect of odorants on ion channels, it should affect both transduction and voltage-gated currents in olfactory receptor neurons (9, 11). To test this idea we investigated the time dependence of the suppression effect on the voltage-gated outward currents, applying brief odor exposures to these sensory cells during depolarizing voltage steps.Olfactory neurons are selectively sensitive to odors. When stimulated
with micromolar concentrations of mixtures
I or II, ~30% of
the dissociated receptor cells are able to transduce one of the odorant
mixtures (I or
II), and a much smaller fraction of
the cells (18%) responds to both of them (19). We examined the effect
of mixture I on the net voltage-gated
outward K+ currents
(Io) in olfactory
neurons that could not transduce this odorant mixture. Two examples are
presented in Fig. 1,
A and
C, corresponding to two separate
cells. Each was exposed to odorant puffs during a depolarizing step
from a holding potential of 70 mV initiated 1 s before the onset
of the chemical stimulus. The cell in Fig.
1A responded to a 1.5-s odor pulse
with a rapid 35% reduction in the magnitude of the net
Io, activated by
a step to 0 mV. Mixture I normally
induces an excitatory
IT in responsive receptor neurons, with a reversal potential of 0 mV, as determined by
local stimulation of the olfactory cilia (data not shown) (7, 10).
Although IT
should be undetectable at 0 mV, one expects to observe an inward tail
current (It) on
repolarization to
70 mV if the cell is responsive to
mixture I. The fact that no
It developed in
the response indicates that this neuron did not chemotransduce. Rather,
the decline in Io
resulted from odor suppression of the voltage-gated current. We define
the suppression current
(IS) as the
difference between the net current and the control current measured
during a puff of odorant-free Ringer solution. In the voltage range
examined, the I-V relation for
IS (peak values) is a straight line that approaches 0 pA around
38 mV (Fig
1B). IS is zero at
greater negative voltages, a range at which all voltage-gated channels
are virtually closed. Figure 1C shows
another nontransducing olfactory neuron (note the absence of
It). This neuron had lost its chemosensory cilia, which are essential for chemotransduction (for a review, see Ref. 21). We used a similar protocol with a shorter odor pulse (0.3 s), and the voltage was stepped
to +30 mV in this case. At such voltage an outward current would be
expected if transduction channels were open. In this example peak
IS was ~15% of
Io. We
investigated the I-V relations for
IS in 12 olfactory neurons that were equally unresponsive to
mixture I. In all cases they could be
fitted to a straight line; for clarity, only five of them are
superimposed in Fig. 1D. The potential
value at which IS
departs from zero ranged from
60 to
22 mV, with an
average value of
39 ± 11 mV (mean ± SD). The percentage
of the suppressive effect on
Io varied from
cell to cell. This variation was likely due to differences in odorant concentration, in addition to other factors (see
DISCUSSION).
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The shape of the IS I-V curve differs greatly from both the excitatory (6, 7, 10) and the inhibitory transduction I-V curves (17, 19), and it does not resemble either of the I-V curves associated with the voltage-gated ionic currents present in C. caudiverbera olfactory neurons. Rather, the shape of the suppression I-V relation is consistent with a reduction in the net outward current.
Dose-response relation of suppression.
Suppression was dose dependent. Figure
2A
illustrates a family of whole cell currents induced in a nontransducing
olfactory neuron (note absence of It) by
identical voltage steps from 70 mV (holding potential) to
10 mV, each one associated with an odorant puff of different intensity (including a control consisting of a puff of odorant-free Ringer solution). The dose-response relation for this experiment is
presented in Fig. 2B. Odorant
concentrations
40 µM produced a maximal
Io suppression of
50% (Fig. 2B, closed circles). The concentrations at which mixture I
started to induce suppression varied from cell to cell, ranging from 5 to 40 µM. The percentage of the outward current suppressed by odors
varied widely, virtually from 0 to 100%, when measured at the moment
of maximal suppression. In the majority of cases, suppression reached
intermediate values. Figure 2B
includes another example, from a different cell, to illustrate this
variability (open circles). In this case, maximal reduction was 25% of
Io, at odorant
concentrations
120 µM.
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Suppression and transduction currents contribute to
the net odor response. The range of odorant
concentrations at which suppression operates overlaps with that at
which isolated olfactory neurons transduce odorants (Fig. 2) (7, 19).
This is best illustrated in cells that responded with an
IT to odorant
puffs that engulfed the entire neuron. Figure
3A shows
currents induced by an odorant puff at 30, 0, and +30 mV (from a
holding potential of
70 mV) after subtracting the control
currents. All three traces exhibited tail currents, revealing that the
cell transduced the odors. It is also clear that in this cell the odors
suppressed Io
because an inward current was observed during the stimulus at 0 mV (the reversal potential of
IT) and,
therefore, the reduction of the net
Io was due
entirely to suppression (Fig. 3A,
middle trace). It is expected,
therefore, that at voltages other than 0 mV,
IS and
IT should
overlap. In cases where the kinetics of
IS and
IT are not
substantially different and
IT >>
IS, as in this
particular example, the I-V curve for
the odor-induced current should be somewhat shifted with respect to
that of IT,
without a major distortion in its shape (Fig.
3B). In contrast, in cases in which
IT
IS and their time
courses are markedly different, the
I-V curve shape may be considerably
distorted (see below). The closed circles in Fig.
3B correspond to the measured peak
values of the odor-induced currents and the open circles correspond to
IT, after
correcting for
IS. In making
this correction, we reasoned that, since
IS is linearly
related to voltage (see Fig. 1B) and
the total value of the odor-induced current equals
IS at 0 mV, we
could estimate the value of
IS at each
voltage and make the appropriate correction to the experimental values
to obtain IT at
every voltage (method I; see legend to
Fig. 3).
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Two further examples are presented in Fig. 4. In both
cells it is possible to distinguish both
IT and
IS as two
coexisting, but clearly distinguishable currents. The presence of
IT was confirmed by It in both
cases. In each cell,
IS could be
observed in isolation at the reversal potential of
IT, 0 mV (Fig.
4A, bottom
trace and 4D,
middle trace). In the cell in Fig.
4A,
IS clearly
preceded IT in
such a manner that, at positive potentials, the inward
IS component was
followed by the outward
IT component
(Fig. 4A, middle trace). This situation was more dramatic at further
positive potentials, where the odor-induced currents became clearly
biphasic (Fig. 4A,
top trace). The
I-V curve, built from the maximal
values of the net current at each potential (which in this case
coincided with the end of the pulse), is depicted by the closed circles in Fig. 4B. The cell in Fig.
4D had a larger
IS relative to
IT. At 30
mV, IS preceded
IT, the latter
one expressed as a further inward current before the end of
depolarization. At +30 mV, voltage at which
IT is outward,
the magnitude of this current was insufficient in this case to overcome
IS, resulting in
a net inward current at the end of depolarization (Fig.
4D, top
trace). The net current I-V relation for this cell is shown in
Fig. 4E (closed circles). Both
experimental curves (closed circles in Fig. 4,
B and
E) are remarkably distorted from the
expected I-V relation shape, as a
result of suppression.
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The more complex odor-induced currents, apparent in the two cells, enabled us to validate our IS-correction procedure by comparison with another method (method II). Figure 4B plots the I-V built from the experimental points (closed circles) and the IT I-V curve after correcting for IS by method I, as explained above (open circles). Taking advantage of the fact that IS can be distinguished within the odor-induced current, because it developed earlier than IT, we attempted a different manner of correcting for IS (method II). We plotted the peak IS vs. voltage (triangles) and subtracted these values from those of the odor-induced currents at the end of the depolarization. This method yielded an I-V curve (squares) virtually indistinguishable from the one obtained by correcting with method I (open circles). Method I is of general use, because it can be applied to any olfactory neuron responding with a transduction current; whereas method II can only be applied to those cells in which IS had fully developed before IT became evident. In the present example, IS was sustained at 0 mV (Fig. 4A, bottom trace). Considering that IS had the same behavior at the other voltages (as we have observed in nontransducing olfactory neurons), we fitted a Boltzmann function to IS in each case (Fig. 4C, broken lines) and subtracted the calculated currents from the recorded odor-induced currents (IOD, same as in Fig. 4A) to obtain IT as a function of time in isolation.
For the second example (Fig. 4E), we corrected the experimental I-V curve (closed circles) by method I (open circles) and by method II (squares) using peak IS (triangles). The curve crosses 0 mV and displays the characteristic shape of the IT I-V curve. Despite the pronounced distortion exhibited by the experimental I-V curve, both methods corrected the curve quite well. The two methods gave slightly different values only on the two points taken at the most positive potentials. We attribute this small mismatch principally to the rundown of the voltage-dependent currents, which in this particular cell was larger than usual. Rundown principally affected the two positive values, because they were taken at the end of the experiment, when the rundown effect was more pronounced.
We examined a total of 17 transducing neurons in which odor stimuli were applied to the cilia. The two components, IT and IS, could be clearly discriminated in 15 of such cases; in all of them IS preceded IT. In the two remaining neurons, both currents had indistinguishable time courses (Fig. 3 and see DISCUSSION).
In conclusion, our results indicate that, no matter how anomalous the experimental I-V relation for odor-induced currents was, its shape and reversal potential became quite close to the expected after correcting for IS by either method.
Suppression Takes Place Independently of the Transduction Cascade
Suppression of the voltage-gated conductances takes place in 94% of isolated olfactory neurons (n = 121), including those missing their chemosensory cilia. Transduction currents (IT), on the other hand, are triggered only in a fraction of these sensory cells (30%) (19) and never in neurons missing their cilia. This suggests that suppression is independent of the transduction cascade, which is localized in the cilia (7, 10, 13, 17). An additional important distinction between transduction and suppression is that IT exhibits rundown until it eventually disappears, whereas IS persists for as long as there are voltage-gated currents. The voltage-gated currents are susceptible to rundown, although considerably less than transduction currents. The lower sensitivity to rundown of IS compared with IT is in agreement with the notion that IS is not dependent on second messengers, in contrast to IT. Three additional lines of evidence further support the view that suppression is independent of the transduction cascades: 1) the latencies of IS are much shorter than those of IT, 2) odors are more effective on activating IS in the nontransducing cellular regions, and 3) there is further evidence supporting the notion that IS activation is nonspecific.Suppression latency. If suppression
results from a direct effect of odorants on the voltage-gated channels,
unlike transduction, which is a cascade process, its latency (the time
between the puff onset and the moment at which the current induced by
the puff develops) should be substantially shorter than that of the transduction response. Figure 5 shows that
this was indeed the case. The olfactory neuron in Fig.
5A exhibited a typical transduction response to mixture I, with a latency
of 560 ms, when a localized stimulus was delivered 20 µm away from
the olfactory cilia. The cell in Fig.
5B, incapable of chemotransducing
mixture I, was used to test the delay
of the suppressive effect. Because
IS latency is
strongly dependent on stimulus strength (being longer for lower pressure pulses; Fig. 2A), we chose
a pressure value (10-14
lb/in.2) for which the latency
was minimal. We stimulated the neurons with a double-barreled pipette,
one barrel of which contained mixture
I while the other was filled with
high-K+ Ringer solution (100 mM
K+). The
high-K+-induced current is
presented superimposed on
IS in Fig.
5B. The odor puff induced
IS with a 30-ms
latency. A high-K+ puff applied
with the same pressure and from the same location also induced an
inward current, but with a somewhat shorter latency of 10 ms (Fig. 5,
inset). The
high-K+ puff is followed by an
inward tail current, reflecting the fact that the
high-K+ concentration favored the
influx of this cation after shifting the voltage back to 70 mV,
until the excess of K+ diffused
away.
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The latency of the suppression effect is one order of magnitude shorter than that typical of IT. In a total of 15 experiments in which an identical protocol was used (pressures 10-14 lb/in.2), we found similar results, with an average latency of 19 ± 13 ms (means ± SD). These results are consistent with the notion that suppression directly affects the voltage-gated channels without involving a signaling process.
Localization of IS.
Although transduction channels are mostly confined to the olfactory
cilia, voltage-gated channels are localized in the cell body and
dendrite of olfactory receptor neurons (7, 10, 13, 17). Using locally
applied odorant puffs in cells that did not respond to
mixture I (note the absence of
It in Fig.
6A), we
found that suppression of voltage-gated currents by local application to the cell body was significantly larger than that induced by application to the cilia (Fig. 6A).
In the typical example illustrated in Fig. 6 (n = 4), odors suppressed
the outward current by 8% when directed to the cilia and by 20% when
addressed to the cell body, while the membrane potential was held at
0 mV. Most likely, suppression observed when stimulating the
cilia was due to odorants that diffused to the dendrite and cell body.
The longer latency and slower time course of the effect when the odors
were applied to the cilia compared with the cell body support this
interpretation. This observation is contrary to what would be expected
if suppression had an effect on the transduction channels or on any of
the upstream cascade components. In a second example (Fig.
6B), from a neuron responding to the
odorants with a transduction current (note the presence of
It) also held
at 0 mV, Io was
reduced by 34% when odors were directed to the cilia and by 50% when
the cell body was stimulated. In contrast, it is worth noting that the
It of the
response obtained by cilia stimulation was larger than the It of the
response to the stimulus directed to the soma, in agreement with the
transduction conductance being localized to the cilia.
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Suppression by Brief Odor Pulses Affects All Voltage-Gated Conductances
We examined suppression on each of the four relevant voltage-gated currents in Caudiverbera, the delayed rectifier K+ current [IK(V)], the Ca2+-dependent K+ current [IK(Ca)], the Na+ current (INa), and the Ca2+ current (ICa). We showed above how the net outward current (Io) was suppressed by odors. To investigate whether each of the individual voltage-gated K+ current components in these cells was a target of suppression, we examined the direct effect of brief odorant pulses on each component in isolation. For this purpose, we temporarily abolished IK(Ca) by briefly exposing the olfactory neurons to 0-Ca2+ Ringer solution, as illustrated by Fig. 8A. Both superimposed currents were induced by a voltage step from
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INa and
ICa were both
odor suppressed as well. Because
INa rapidly
inactivates, we used a different protocol for this study. In this case,
the odorant puff was initiated 450 ms before the depolarizing step,
lasting to the end of it. In addition, the pipette was filled with
Cs+ internal solution to abolish
Io. It can be
observed that depolarization from 70 to
10 mV induced the
transient INa
followed by the sustained ICa. Both
currents were suppressed by odorants in a dose-dependent fashion (Fig.
9A). The
dose-suppression curve for
INa is presented in Fig. 9B. Suppression of
INa reached a
maximum of ~70% in this particular cell. In general, odorant
concentrations
5 µM were required to induce an observable effect,
and the average maximum suppression was 72 ± 18% (means ± SD;
n = 12). Although
ICa was also
suppressed in a dose-dependent fashion, it was difficult to build a
dose-suppression curve due to the small magnitude of this current and
to the possible contribution of other small current components
(possibly carried by Cs+ or other
ions; Madrid and Bacigalupo, unpublished results, and see Ref. 4). For
these reasons, we were able to clearly resolve ICa in only 3 of
the 12 cells examined, all of which exhibited suppression.
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Suppression by Odors in Nonolfactory Cells
If suppression is a nonspecific phenomenon on the voltage-gated ion channels of the olfactory receptor neurons, independent of the transduction cascades existing in this sensory cell (3), it might affect ion channels of cells other than these chemoreceptors. To test this prediction, we applied odorant puffs (mixture I) to Drosophila photoreceptor cells and examined their effect on voltage-dependent currents. Mixture I reduced the net outward current activated by depolarizing the membrane from a holding potential of
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Physiological Significance of Suppression
An important question regarding the phenomenon of suppression is whether it contributes to the odorant responses. To investigate the physiological effect of suppression on these receptor cells, we undertook current-clamp experiments and monitored the behavior of the membrane potential. For this study we only used olfactory neurons that did not transduce mixture I.Figure
11A
shows an olfactory neuron with a rather high spontaneous spiking rate.
The inset to Fig.
11A indicates that
mixture I, although producing
suppression, did not trigger a transduction response in this cell.
Under current-clamp conditions, we gave successive 1.5-s puffs of
odorant-free Ringer solution and of increasing concentrations
(60-80 µM) of mixture I. The
firing rate was not altered by a puff of Ringer solution. In contrast, the odorant puffs transiently depolarized the cell, increased the
firing rate, and decreased the size of the spikes
(middle traces). During the
strongest stimulus, the action potentials suddenly ceased
(bottom trace), presumably because
the Na+ channels were suppressed
or the level of depolarization reached the threshold for
Na+ channel inactivation, which
appears to be by itself modified by odors (9). After spiking resumed,
repetition of the same experimental protocol gave identical results.
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Another example, corresponding to a separate olfactory neuron, shows
that in some cases the end of firing induced by an odor stimulus
(similar to those used in Fig.
11A) appears to result from a
hyperpolarization rather than a depolarization (Fig.
11B). The same odorant stimulus that
ended spiking activity induced a hyperpolarization from 70 to
80 mV, when presented after the interruption of the action potentials.
We investigated the behavior of the receptor potential induced by
identical odorant stimuli applied at different membrane potential
values under current-clamp conditions. Odorant stimuli either
depolarized or hyperpolarized the cell, depending on where the membrane
potential was set. The voltage value at which the odor effect reversed
varied across cells, and it was usually different from the cell resting
potential. This is illustrated by Fig. 12 for two separate olfactory neurons. In one of the cells (Fig. 12A; same cell as in Fig.
11A), with a resting potential of
65 mV, the polarity of the effect reversed at
80 mV (Fig.
12B), and in the other cell (Fig.
12C) with a resting potential of
60 mV, it reversed at
55 mV (Fig.
12D).
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Our results indicate that odors affect the spiking activity of isolated olfactory receptor neurons not only by triggering the transduction cascade, but also by suppressing ion conductances.
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DISCUSSION |
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In this work we have studied odor-induced suppression of the voltage-gated conductances in voltage-clamped or current-clamped isolated olfactory neurons, using short odorant exposures. Suppression appeared as a transient reduction of the whole cell membrane currents. Our results indicate that it is a nonspecific effect on voltage-gated ion channels occurring in almost every tested olfactory neuron. Suppression contrasts with odor transduction, which is a rather specific physiological phenomenon. Only a small fraction of all olfactory neurons responds with a transduction current to a particular odorant; the underlying conductances are triggered by a signaling cascade (2, 21). We demonstrated that suppression and transduction are different phenomena, both of which contribute to shaping odorant-induced responses, at least in isolated olfactory neurons. Furthermore, our current-clamp measurements suggest that suppression is physiologically significant in olfactory neurons. Because odor suppression and odor transduction are induced by odor pulses of similar concentration and duration, it seems likely that both IS and IT effectively contribute to the electrical response to odors in the olfactory epithelium.
Characterization of Odor Suppression
Odor suppression of voltage-gated currents was previously investigated in the Japanese newt, by examining the effect of prolonged bath applications of odors on the voltage-gated currents (9). Chronic odor application reversibly suppressed all voltage-gated currents present in those sensory receptors. However, those results did not shed light over the time course of the suppression effect and they did not allow an evaluation of its possible contribution to the cell responses to brief odor exposures.Much of what we know about odor transduction comes from electrophysiological studies of isolated olfactory receptor cells, stimulated with odorant pulses. To determine whether suppression contributes to the odor-induced responses, it was important to characterize suppression under a similar experimental regime. The most straightforward approach was to study olfactory neurons that were incompetent to transduce the utilized odorant stimuli. The absence of transduction and tail currents was taken as an indication of the inability of a cell to chemotransduce.
For convenience, we characterized suppression of the net outward current because this is a large sustained current. The magnitude of suppression (percent reduction of Io) varied widely from cell to cell. We attribute this large variation to various factors, among which are the following: the different odor concentrations used in our experiments, unequal relative contribution of both K+ currents and their different sensitivities to odors (see Ref. 9), and geometrical problems associated with the way odors were applied to the cells. The latter factor relates to the fact that the puffer pipette was positioned on one side of the cell, making it likely that the concentration of odorants was not homogenous around the entire neuron. This lack of homogeneity depended on differences in size and shape of the cells (including the dendrite length, which is greatly variable), in the position of the pipette with respect to the cell, and slight differences in pipette shape. Despite these variations, the experimental conditions remained constant during each experiment, permitting us to draw important conclusions from the kinetics and voltage-dependence relation of suppression.
IS is a linear function of voltage. The voltage range in which IS is detectable coincides with that where voltage-gated currents were observed, as expected if IS is due to a suppression of these currents. The I-V curve for IS differs from that of any of the known conductances from these cells. Rather, its sign and shape (a straight line with a negative slope), in addition to the common origin of the IS and Io I-V curves, are consistent with a reduction of the net K+ conductance (Fig. 8B).
The slope of the IS I-V curve changed from cell to cell, most likely due to the same factors that explain the differences in the magnitude of suppression (see above), in addition to the important variations in the size of the ionic currents across cells.
IS latency was also highly variable among neurons due to differences in stimulus strength (pressure and distance) and to the geometrical factors described above. When stimulating by pressure pulses, as in our case, the time it takes to reach the minimal odor concentration needed for causing suppression is longer for low pressures (Fig. 2A). Because we used different pressures, we attribute the variability of the latency to this reason. In the cases (2 of 17) in which IS and IT latencies were similar (Fig. 3), the cells had long dendrites, and odor puffs were applied to the cilia with particularly low pressures (3-4 lb/in.2). We therefore used relatively high pressure stimuli (typically 10-14 lb/in.2) in those experiments addressed to investigate suppression latency. The minimal IS latency was shorter than IT latency by one order of magnitude or more, and it was comparable to the latency of the inward current induced by a high-K+ puff. Together, the results indicate that, unlike transduction, odors suppress Io by a direct action on the underlying ion channels, without mediation of second messengers. This action seems likely to take place within the membrane, due to the hydrophobic nature of odorant molecules. Consistent with this notion, the mechanism of suppression is voltage independent, as indicated by the linearity of the IS I-V curve. The fact that IS latency is somewhat longer than that preceding the high-K+-induced current may be related to the fact that volatile odors are liposoluble, and it may take longer for these molecules to partition into the membrane to cause their effect than for replacement of K+ at the extracellular side of the membrane. Alternatively, it is possible that odors cause suppression by acting on a separate molecule intimately related to the ion channels, thus increasing the latency. However, this possibility seems unlikely, because suppression is a nonselective process, and the putative intermediary molecule would have to be associated with all ion channels suppressed by odors and to present an extremely wide spectrum of interactions with odorants.
Localization studies suggest that the suppression mechanism is entirely independent of the transduction cascade. IS was larger and faster when the odor pulses were directed to the cell body, where the voltage-gated channels reside, than when directed to the cilia, which contain the transduction channels. On the contrary, the magnitude of It, which is associated with transduction, was larger when odors were applied to the cilia.
The observation that IS was considerably less affected by rundown than IT is in agreement with the view that suppression involves a direct effect on ion channels, in contrast to transduction, which is mediated by a cascade mechanism.
The dose-response relation of suppression indicates that odor concentrations causing suppression are similar to those that trigger transduction in olfactory neurons (7, 19). We applied chemical stimuli to the entire cell to examine to what extent IS interfered with IT by performing studies on neurons capable of transducing mixture I. We corrected the odor-induced response by subtracting the effect of suppression. After this correction was done, the shape of the I-V curve became virtually indistinguishable from that reported for the odor-induced current in various species, where the chemical stimuli were focused on the olfactory cilia (Refs. 6, 7, 10, and see Refs. 16 and 21). According to our study, variations on the reversal potential of the transduction current are expected to occur, depending on the extent that odors reach the nontransducing plasma membrane in each particular experiment (soma and dendrite). When the differences in time courses between both odor effects were not large enough to allow a clear distinction between the two current components (IS and IT), we were able to correct the odor-induced currents only by method I. The validity of method I is supported by the application of method II, which could be used only in cases in which IS and IT were clearly separated in time. In such cases, both methods gave virtually identical results. In some cases, rundown accounted for small differences between the corrected I-V curves. Furthermore, we were able to separate the two components (IS and IT) of the net odor-induced current as a factor of time (Fig. 4E).
The degree of distortion in the I-V curve depends, to a large extent, on the relative magnitudes of IS and IT, being more pronounced the closer IS was to IT.
Localization of the chemical stimulus to the cilia of isolated olfactory neurons is, therefore, important for diminishing the interference of odor suppression when studying transduction currents at potentials at which the voltage-gated channels are open.
We show that brief odor pulses suppress all four voltage-gated conductances in Caudiverbera. Suppression is induced by both odorant mixtures, I and II, in Caudiverbera and in rat olfactory neurons, including those receptor neurons incapable of transducing such odors. We observed that the fraction of olfactory neurons suppressed by odor mixture I was smaller in the rat than in C. caudiverbera, in the concentration range of 1-150 µM (45% in the rat, compared with 94% in Caudiverbera). This difference may be due to the fact that the magnitude of the voltage-gated K+ currents is considerably smaller in the rat than in the toad, making the resolution of the suppression effect more difficult. Indeed, we observed that, at higher odor concentrations, the percentage of suppressed rat olfactory neurons increased significantly. Additional reasons for the lower suppression effect observed in the rat, as possible differences in the nature of the K+ channels of the olfactory neurons from both species, cannot be ruled out.
Suppression can also be induced in nonolfactory cells, e.g., Drosophila photoreceptor cells, where it reduced the voltage-gated currents, showing that odor suppression is independent of chemotransduction.
Our results demonstrate that, in contrast to odor transduction, suppression is a nonspecific phenomenon directly affecting ion channels, without requiring a signaling cascade. This possibility was previously suggested by Kawai et al. (9), who reported suppression of voltage-gated currents activated by depolarizing pulses applied during odor exposure. Based on the rapid suppression of the transduction currents by odor pulses reported by Kurahashi et al. (11), Kawai and co-workers proposed that a similar direct mechanism may underlie suppression of the voltage-gated currents. In the present work, we directly measured the latency of the suppression effect on the voltage-gated currents and demonstrated its independence of the transduction cascade.
Physiological Role of Suppression
The observation of the dual effect of volatile odorants, of triggering transduction and nonspecifically suppressing ion channels, raises the question of the actual significance of suppression in the normal physiology of olfactory neurons. Previously, Kurahashi et al. (11) demonstrated that odorants had a dual effect on odorant-induced transduction currents. One such effect was to trigger the transduction cascade, leading to the activation of the transduction current. The other effect was to suppress the transduction current, apparently by directly affecting the underlying ion channels. Suppression took place in 20 ms, comparable to the latency that we found for suppression on the voltage-gated channels. Those authors attributed the remarkably long latency (hundreds of milliseconds) that normally precedes the odor-induced transduction current to this suppressive effect. It is likely that the same mechanism underlies odor suppression in both the transduction and the voltage-gated channels.The response induced by excitatory odorants on olfactory receptor
neurons consists of a depolarizing receptor potential accompanied by an
increase in action potential firing. The shape of this response is
determined by the orchestration of the transduction conductances, a
nonselective cationic cAMP-gated conductance and a
Ca2+-activated
Cl conductance, and the
voltage-gated conductances. Therefore, to understand the physiological
role of suppression, it is essential not only to consider suppression
of the transduction conductances but also to establish how suppression
alters the voltage-gated conductances in a time-dependent fashion.
To gain insight into whether odor suppression of the voltage-gated conductances may influence the response to excitatory odorants, we examined whether odorant pulses affected action potential firing in current-clamped olfactory neurons. We found that suppression can induce either a depolarizing or a hyperpolarizing membrane potential change, depending on the particular neuron, and that there was no apparent correlation between the polarity of the suppression-induced voltage change and the value of the cell resting potential. The type of effect of odor suppression in a given neuron depends on its particular pool of voltage-gated ion channels, whose relative densities vary from cell to cell (Ref. 15 and Madrid and Bacigalupo, unpublished observations), and on the membrane potential at the time of stimulation.
Our results indicate that suppression of voltage-gated channels is an important factor in determining the response of an olfactory neuron to odor stimulation and has to be taken into account to fully characterize the odor-induced response. It should be kept in mind, however, that our experiments could not address this question thoroughly, because in isolated olfactory neurons the odor stimuli reach the soma to an extent that depends on how the stimulus is applied. In situ, however, olfactory receptor neurons form part of the olfactory epithelium and odorants are presented by nature to the mucosal surface of this tissue. Tight junctions among the epithelial cells constitute a diffusion barrier for odorants toward the basolateral membranes. Nevertheless, the fact that volatile odorants can partition into the lipid bilayer of the plasma membrane and diffuse through it, as shown by Lowe and Gold (13), opens the possibility that, also in vivo, odorants may suppress ion channels present in the basolateral membrane of olfactory neurons. If this were the case, suppression would have a physiological role in olfaction that deserves to be properly evaluated. A definitive answer to this problem demands further studies that are beyond the scope of the present paper.
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ACKNOWLEDGEMENTS |
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We are indebted to Gonzalo Ugarte for his help with the photoreceptor cells experiments, to Oliver Schmachtenberg for help with the rat olfactory neurons, and to Rodolfo Madrid for providing the data on the effect of mixture II toad olfactory neurons. We also thank Drs. Mario Luxoro, Peter O'Day, and Francisco Sepúlveda for critical reading of the manuscript and Dr. Osvaldo Alvarez for his help in multiple aspects of our work. We are also indebted to Dr. Diego Restrepo for advice on dissociation of rat olfactory neurons.
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FOOTNOTES |
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This work was supported by Fondo Nacional de Ciencia y Tecnología Grants 1960878 and 1990938, a Presidential Chair for Science (J. Bacigalupo), and a graduate fellowship from Fundación Puelma (M. Sanhueza).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Bacigalupo, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile (E-mail: bacigalu{at}abello.dic.uchile.cl).
Received 29 March 1999; accepted in final form 23 July 1999.
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