EDITORIAL FOCUS
Odor suppression of voltage-gated currents contributes to the odor-induced response in olfactory neurons

Magdalena Sanhueza and Juan Bacigalupo

Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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INTRODUCTION
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.


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INTRODUCTION
MATERIALS AND METHODS
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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Fig. 1.   Suppression of the voltage-gated net outward current (Io) by odorants of mixture I. A: this cell was under voltage clamp [holding potential (Vhold) = -70 mV]. An odorant stimulus was given (bar, 100 µM) during a step to 0 mV. Io rapidly decayed by 35% (peak) of control level. Superimposed to it is the control current, obtained with an identical protocol, except that the puff consisted of odorant-free Ringer solution. Note the absence of a tail current (It), indicative of the inability of this cell to transduce mixture I. B: current-voltage (I-V) relation for the suppression current (IS) was built with peak values of odor-induced currents after subtraction of control currents. Experimental points from -38 mV up were fitted with a straight line. C: suppression of net outward voltage-gated current in an olfactory neuron missing its cilia. Note that voltage was pulsed to +30 mV, and odor puff (50 µM) was shorter than in A. D: IS I-V relations from 5 cells. Curves obtained from other 7 cells were all similar to those shown here but were left out for clarity. Average 0-current potential: -39 ± 11 mV (means ± SD). Percent suppression of Io is indicated beside each line.

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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Fig. 2.   Dose-response relation of the suppression effect on Io for a nontransducing cell. A: family of currents displaying the suppression induced by odorant stimuli of increasing concentrations, obtained by incrementing the pressure on the stimulus pipette. B: dose-response curve built with the suppressed currents at the points of maximal effect, relative to the control current (). Experimental data were fitted to a Hill equation I/Io = (1 - min)/(1 + C/I50)n + min, where I50 = 17 µM, n = 5.5, min = 0.51 (min = asymptotic value of I/Io due to suppression). open circle , Another example (I50 = 64 µM, n = 3.2, min = 0.75).

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 approx  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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Fig. 3.   Suppression in an olfactory neuron transducing mixture I. A: odor-induced currents for 3 different voltages, after subtracting control currents. Odor stimuli are indicated by bar. B: I-V curve built from peaks of odor-induced (experimental) currents (). open circle , Obtained after correcting each of the experimental values for suppression. The correction considered the facts that at 0 mV the odor-dependent current corresponds solely to IS and that the IS I-V is linear (Fig. 1B). We calculated IS at any given voltage (V) by the equation IS = aV + b, where a = -b/39 (39 mV is average 0-current potential; see legend to Fig. 1D) and b is IS at V = 0 mV. The corrected I-V curve was obtained by subtracting IS from the experimentally measured current (method I).

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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Fig. 4.   Suppression in olfactory neurons having IS and transduction current (IT) with clearly distinguishable time courses. A: odor-induced currents at 3 different voltages. IS precedes IT. At 0 mV, IT is 0 (its reversal potential). B: I-V relations of odor-induced currents measured at end of voltage steps (), of peak IS values (triangle ), of IT after correcting for IS by method I (open circle ), and of IT after correcting for IS by method II (; see below). C: odor-induced current (IOD), IS, and IT in isolation. IS at 0 mV (left) was well fitted by a Boltzmann equation {dashed line; IOD = A/[1 + Bexp(t/q)], where A = 39 pA, q = 85 ms, B = 0.02 pA}. The same function (+30 mV: A = 72 pA, q = 141 ms, B = 0.03 pA; +50 mV: A = 112 pA, q = 283 ms, B = 0.18 pA) predicts an IS shape for the other 2 voltages (middle and right traces, respectively). Dotted line, zero-current level. D: current recordings from a separate olfactory neuron, using same protocol as in A. E: I-V curves for the cell in D; symbols are equivalent to those used in B for the first cell. Method II involves subtracting peak IS value (triangle ) from maximal value of net odor-induced current () at every voltage.

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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Fig. 5.   Latencies of transduction and suppression currents. A: representative recording of a transduction current triggered by mixture I. Latency = 560 ms. B: superimposed currents induced by puffs of odorant and high-K+ Ringer solution (100 mM K+, different cell to that in A). Inset: detail of both currents; arrowhead, stimuli onset. Latencies were 10 and 30 ms for high-K+ and odor stimuli, respectively.

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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Fig. 6.   Localization of suppression. A: an olfactory neuron unable to transduce mixture I was stimulated locally with identical odor puffs on its cilia, and subsequently on its cell body, during a step to 0 mV. B: a similar experiment in an olfactory neuron capable of transducing mixture I. Control traces consist of odorant-free puffs.

Nonspecificity of suppression. Suppression is not only induced by odorants that excite Caudiverbera olfactory neurons but also by odorants that inhibit these cells (mixture II) (2, 19). Figure 7A shows an example (n = 20) of suppression caused by mixture II, which was not different from suppression by mixture I, shown in the previous examples.


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Fig. 7.   Suppression by mixture II in Caudiverbera and by mixture I in rat olfactory neurons. A: a toad olfactory neuron exhibiting suppression by putrid odors (80 µM). Control: puff of odorant-free Ringer solution. B: mixture I also suppressed a rat isolated olfactory neuron (100 µM). Superimposed are 2 recordings obtained at 0 and +30 mV. Dotted lines correspond to single exponential fits to Io before odor stimulation.

Suppression by mixture I also takes place in rat isolated olfactory neurons. In this case, depolarizing steps, from -70 to +30 or to 0 mV, induced pronounced early transient outward currents (14), followed by a slowly decaying outward current (Fig. 7B). The puff of mixture I reduced the outward current by ~38% in a manner virtually identical to that in the toad. Within the concentration range of 1-150 µM, only 45% of all the rat olfactory neurons (n = 66) were suppressed by mixture I. However, 500 µM odors suppressed ~80% of the cells. Somewhat higher concentrations suppressed nearly every olfactory neuron, supporting the notion that the mechanism underlying suppression is nonspecific. Mixture II also suppressed the voltage-gated currents in the rat, although requiring two- or threefold higher concentrations than mixture I (not shown).

The fact that suppression is induced by odorants that excite Caudiverbera olfactory neurons as well as by those that inhibit those cells (mixture II) and that it also occurs in rat olfactory receptor neurons strongly supports the notion that suppression is a nonspecific effect of odorants on the voltage-gated ion channels.

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 -70 to +30 mV, under normal and 0-Ca2+ Ringer solution. The I-V curves for the families of currents measured at the end of the steps are presented in Fig. 8B (normal Ringer solution, closed circles; 0-Ca2+ Ringer solution, open circles). In this cell, suppression of Io under normal Ringer solution was of 700 pA (Fig. 8C). During exposure to 0-Ca2+ Ringer solution, we were able to measure odor suppression of IK(V) in isolation, using 0-Ca2+ Ringer solution as the carrier solution in the pipette. This current was suppressed by 500 pA (Fig. 8C). If rundown of the currents is ignored, the difference between suppression of Io and suppression of IK(V) corresponds to suppression of IK(Ca). Accordingly, for the cell in Fig. 8C, suppression of IK(V) accounted for ~70% of the suppression of the net outward current, the other ~30% corresponding to suppression of IK(Ca). Similar results were obtained in four other cells, although the percentage of suppression, as well as the relative contribution of each component to the net outward current, was different in every case. Figure 8D illustrates an extreme case in which odors completely suppressed both components of Io, IK(V), and IK(Ca).


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Fig. 8.   Suppression affects both Io components, delayed rectifier K+ current [IK(V)] and Ca2+-dependent K+ current [IK(Ca)]. A: whole cell current under normal Ringer solution (Io) and under 0-Ca2+ Ringer solution [IK(V)], induced by steps from -70 mV (Vhold) to +30 mV. B: I-V curves built from families of currents (same experiment as in A), induced by a series of voltage steps (20-mV increments), and measured at the end of the steps. , Normal Ringer solution; open circle , 0-Ca2+ Ringer solution. C: identical odor stimuli (mixture I) suppressed Io in normal Ringer solution and under 0-Ca2+ Ringer solution. D: another example, illustrating complete suppression of both Io components.

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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Fig. 9.   Suppression of voltage-gated inward currents. A: because of Na+ channel inactivation, odor exposures were initiated 450 ms before the depolarizing step, ending after the presented traces. In addition, Cs+ replaced K+ in the internal solution. Superimposed are currents obtained by 6 steps from -70 (Vhold) to -10 mV, accompanied by puffs of increasing concentrations of mixture I. B: dose-response curve for peaks of Na+ current in A. Data were fitted to same function as in Fig. 2, with I50 = 92 µM, n = 0.8, and min = 0.16.

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 -70 to 0 mV (in 2 out of 2 cells tested), as in olfactory neurons (Fig. 10). This result suggests that odor suppression is a nonspecific effect of odor molecules on voltage-gated channels, regardless of cell type. Moreover, it demonstrates that suppression is independent of olfactory transduction cascades.


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Fig. 10.   Odor suppression of voltage-gated ion conductances of Drosophila photoreceptor cells. An odor stimulus (30 µM mixture I) was applied (bar) to voltage-clamped Drosophila photoreceptor cells in isolated omatidia, during a step to 0 mV (Vhold = -70 mV). Control: odorant-free puff.

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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Fig. 11.   Suppression affects action potential firing by depolarization or hyperpolarization. A: spiking olfactory neuron not transducing mixture I (inset). Subsequent traces, under current clamp, show firing behavior in response to puffs of odorant-free Ringer solution and of increasing mixture I concentrations (60, 70, and 80 µM). B: a separate olfactory neuron, also incapable of transducing mixture I and with a resting potential (Vrest) of -70 mV, was given an 80 µM puff of mixture I during spiking and again during the silent period, inducing a hyperpolarization (time interval between traces approx  20 s).

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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Fig. 12.   Suppression as a function of membrane potential. A: responses at 4 different membrane potentials (indicated beside each trace) under current clamp. Membrane potentials were set by current injection (same cell as in Fig. 11A). B: odor-induced voltage changes (peaks) as a function of membrane potential (measured before stimulus application) for cell in A. C: another example, from a separate cell. D: odor-induced voltage responses vs. membrane potential for the cell in C.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bacigalupo, J., D. M. Bautista, D. L. Brinck, J. F. Hetzer, and P. M. O'Day. Cyclic-GMP enhances light-induced excitation and induces membrane currents in Drosophila retinal photoreceptors. J. Neurosci. 15: 7196-7200, 1995[Abstract].

2.   Bacigalupo, J., B. Morales, P. Labarca, G. Ugarte, and R. Madrid. Inhibitory responses to odorants in vertebrate olfactory neurons. In: From Ion Channels to Cell-to-Cell Conversations, edited by R. Latorre, and J. C. Saez. New York: Plenum, 1997, p. 269-284.

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5.   Dionne, V. E. Chemosensory responses in isolated receptor neurons from Necturus maculosus. J. Gen. Physiol. 99: 415-433, 1992[Abstract].

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