PHYSIOLOGICAL REVIEWS   Vol. 78 No. 2 April 1998, pp. 429-466
Copyright ©1998 by the American Physiological Society

Transduction Mechanisms in Vertebrate Olfactory Receptor Cells

DETLEV SCHILD AND DIEGO RESTREPO

Physiologisches Institut, Universität Göttingen, Göttingen, Germany; and Department of Cellular and Structural Biology, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado

I. INTRODUCTION
II. GEOMETRY AND PASSIVE MEMBRANE PROPERTIES OF OLFACTORY RECEPTOR NEURONS
    A. Geometry
    B. Capacitance and Resting Membrane Resistance
    C. Resting Membrane Potential
    D. Membrane Time Constant
    E. Sensitivity and Single Odorant Molecule Detection
    F. Summary
III. CONDUCTANCES INVOLVED IN ACTION POTENTIALS
    A. Voltage-Gated Sodium Currents
    B. Voltage-Gated Calcium Currents
    C. Potassium Conductances
    D. Summary
IV. ODORANT RESPONSES
    A. Recording Methods
    B. Odorant-Induced "Cation" Current
    C. Optical Recordings of Odorant Responses
    D. Diversity of Transduction Mechanisms
    E. Responses of Olfactory Neurons in Culture
    F. Responses of Vomeronasal Neurons
    G. Summary
V. ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE-MEDIATED SIGNALING IN OLFACTORY CELLS
    A. Olfactory Receptors, G_olf, and cAMP Formation
    B. Olfactory Cyclic Nucleotide-Gated Conductance
    C. Calcium-Activated Chloride Current
    D. Summary
VI. INOSITOL TRISPHOSPHATE-MEDIATED SIGNALING IN OLFACTORY CELLS
    A. InsP₃ Formation and Receptors
    B. Inositol Polyphosphate-Regulated Conductance(s)
    C. Electrophysiological Studies of InsP₃-Mediated Odor Responses
    D. Summary
VII. CALCIUM AS A THIRD MESSENGER IN OLFACTORY TRANSDUCTION
VIII. GASEOUS MESSENGERS IN OLFACTORY NEURONS (CARBON MONOXIDE AND NITRIC OXIDE)
IX. IONIC CONCENTRATIONS IN MUCUS AND ION HOMEOSTASIS
X. MULTIPLE PATHWAYS: IMPLICATIONS FOR OLFACTORY CODING
XI. SUMMARY AND PERSPECTIVES
REFERENCES

	ABSTRACT

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Schild, Detlev, and Diego Restrepo. Transduction Mechanisms in Vertebrate Olfactory Receptor Cells. Physiol. Rev. 78: 429-466, 1998.  Considerable progress has been made in the understanding of transduction mechanisms in olfactory receptor neurons (ORNs) over the last decade. Odorants pass through a mucus interface before binding to odorant receptors (ORs). The molecular structure of many ORs is now known. They belong to the large class of G protein-coupled receptors with seven transmembrane domains. Binding of an odorant to an OR triggers the activation of second messenger cascades. One second messenger pathway in particular has been extensively studied; the receptor activates, via the G protein G_olf , an adenylyl cyclase, resulting in an increase in adenosine 3',5'-cyclic monophosphate (cAMP), which elicits opening of cation channels directly gated by cAMP. Under physiological conditions, Ca²⁺ has the highest permeability through this channel, and the increase in intracellular Ca²⁺ concentration activates a Cl current which, owing to an elevated reversal potential for Cl, depolarizes the olfactory neuron. The receptor potential finally leads to the generation of action potentials conveying the chemosensory information to the olfactory bulb. Although much less studied, other transduction pathways appear to exist, some of which seem to involve the odorant-induced formation of inositol polyphosphates as well as Ca²⁺ and/or inositol polyphosphate-activated cation channels. In addition, there is evidence for odorant-modulated K⁺ and Cl conductances. Finally, in some species, ORNs can be inhibited by certain odorants. This paper presents a comprehensive review of the biophysical and electrophysiological evidence regarding the transduction processes as well as subsequent signal processing and spike generation in ORNs.

	I. INTRODUCTION

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The morphology of olfactory receptor neurons (ORNs) was first described by Schultze (314, 315; reviewed in Ref. 366) 140 years ago. How ORNs function, however, has remained a riddle that still is not completely solved. In 1985 and 1986, research on olfactory transduction took a decisive turn. Two reviews published by Getchell and Lancet described the functional properties of vertebrate ORNs (106) and the processes in olfactory perception ranging from biochemistry to systems theory (188). This wealth of knowledge had been accumulated using classical morphological and biochemical methods and classical electrophysiological tools, above all extracellular recordings. Anderson and Ache's study of 1985 (in the lobster) (7) and Trotier's study of 1986 (in the salamander) (343) were the first to report the use of the patch-clamp technique to provide a description of voltage-gated membrane currents, single-channel recordings, and odorant-induced currents in ORNs. The various patch-clamp recording configurations (115), other biophysical methods, and molecular biological techniques allowed a more detailed study of signal transduction in the ORN. Since then, a number of reviews and minireviews have appeared, each dealing with a specific topic in olfactory perception, e.g., perireceptor events (107, 270, 271), the structure and function of receptor proteins (52, 189, 283), ultrastructural studies (232, 233), second messenger signaling (2, 9, 10, 40, 43, 47, 74, 295, 298, 372), cyclic nucleotide-gated channels (151, 357, 369, 372), Ca²⁺-dependent Cl channels in ORNs (185), and adaptation and concentration coding (340, 344) as well as aspects of coding and information conveyance between olfactory epithelium and olfactory bulb (52, 149, 189, 247, 252, 304, 316, 322). However, a comprehensive review of cellular studies of olfactory transduction focused on electrophysiological and biophysical aspects is lacking.

Here we review the biophysical, electrophysiological, and some of the biochemical work done in ORNs over the past 10 years. Although we focus on the functional aspects of vertebrate olfactory transduction, we point out interesting parallels to chemosensory transduction mechanisms in invertebrate ORNs (2, 3, 137, 139, 140, 329), eukaryotic microorganisms (346), and vomeronasal receptor neurons (197). Molecular biological studies are mentioned where appropriate but are not covered comprehensively. The reader is referred to recent reviews covering biochemical and molecular biological aspects of olfactory transduction (2, 47, 52, 189, 247, 295, 298, 322). This review of electrophysiological and biophysical studies documents significant advances in our understanding of olfactory transduction in the last 10 years. More importantly, the comprehensive review of the literature makes a compelling argument that olfactory receptors are more complicated than heretofore imagined and reveals the limitations in our knowledge of the complexity of the mechanisms underlying olfactory transduction.

	II. GEOMETRY AND PASSIVE MEMBRANE PROPERTIES OF OLFACTORY RECEPTOR NEURONS

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Olfactory receptor cells in vertebrates are bipolar neurons with a small soma, a single dendrite, cilia or microvilli attached to the olfactory knob or vesicle formed at the dendritic end, and an axon projecting to the olfactory bulb (Fig. 1). The soma has a round or ellipsoidal shape with the short and long axes ranging from 4 to 15 µm and from 7 to 21 µm, respectively. Among the species studied using electrophysiological and biophysical techniques, the largest ORNs are found in newts (182) and salamanders [Ambystoma tigrinum (92) and Necturus maculosus (76)], whereas the smallest ORNs are found in zebrafish (Danio rerio) (65). The unbranched axon is ~200 nm in diameter, and its length, when assessed in isolated cell preparations, varies considerably. The dendrite is ~1-3 µm thick and between 5 and 120 µm long (253, 284). In a preparation of isolated receptor cells, the dendrites may shorten either due to the preparation procedure (307) or due to an excessive Ca²⁺ load (310). The dendrite ends in a dendritic knob (diameter 2-3 µm) from which several cilia issue. The number of cilia appears to vary approximately between 5 and 40 (76, 147, 148, 236, 253, 284). The diameter of a cilium is 100-250 nm (depending on the species), i.e., at or below the resolution limit of light microscopes, and its length is in the range between 5 and 250 µm (124, 166, 279, 284, 371). A cilium is thicker at its base and tapers down its length somewhat (231-233, 318). The typical ciliary 9 + 2 structure changes over the length of a cilium, because the number of microtubules is reduced. In frog, short and medium-sized (up to 90 µm) cilia move irregularly, whereas the long cilia (up to 250 µm) do not move (124, 166). In mammals, the cilia are immotile (231).

View larger version (79K): [in this window] [in a new window]   FIG. 1.   Top: scanning electron micrographs of human olfactory receptor cells. Left: olfactory mucosa viewed from side. Cell with long, slightly prominent dendrite is a typical olfactory neuron. Scale bar, 10 µm. Right: view of surface of olfactory mucosa shows an olfactory knob with 8 cilia. Scale bar, 1 µm. [From Morrison and Costanzo (253).] Bottom: schematic diagram of an olfactory neuron showing transduction compartments (cilia and dendritic knob), dendrite, soma, and beginning of axon as well as some of the characteristic properties of compartments. InsP3 , inositol trisphosphate; NO, nitric oxide.

A comparison of morphological data (124, 284) with electrophysiological reports clearly indicates a tendency for isolated cells to exhibit shorter cilia, although cilia as long as 130 µm have been found and recorded from (166). Occasionally, isolated ORNs lose all the cilia during the dissociation procedure. Deciliated olfactory receptor cells of the newt do not respond to odors (182). For a survey of the electrical properties of olfactory cilia in frog, see References 159, 164, 166.

Often during recordings in the whole cell mode of the patch-clamp technique, the seal with the patch pipette is established on the soma, which is the part of the cell most distant from the cilia. Because of this, space-clamp effects due to the fact that the resistance of the plasma membrane is finite can come into play, particularly in large ORNs with long cilia during odorant stimulation. The effects of space clamp in ORNs have been described by various investigators (166, 213, 276).

The receptor cells in vomeronasal organs (6, 20, 171, 318, 319) and a number of receptor cells in the olfactory epithelium of fish (78, 128, 250, 254, 338, 358, 367, 368), reptiles (112, 352), and amphibia (171) bear microvilli instead of cilia. Microvillous cells have also been reported in the olfactory epithelium of mammals, in particular, in humans (242, 251, 253, 300). There are no reports on their function, and there is controversy on whether they project axons to the olfactory bulb. Thus one study found that when horseradish peroxidase (HRP) is injected into the olfactory bulb, HRP reaction products can be found with a certain delay in microvillous cells of the olfactory epithelium (300), whereas another study failed to find axonal processes stemming from putative microvillar cells in rat (54). In the vomeronasal organ of mouse and rat, two different subclasses of receptor neurons have been described that express two different G proteins and project differentially to the accessory olfacory bulb (131). In the goldfish, there is a correlation between the reappearance of microvillous cells after axotomy and the behavioral responses to pheromones (368). Interestingly, ORNs and axons from the different kinds of chemosensory neuroepithelia, i.e., olfactory proper versus accessory olfactory or vomeronasal mucosa, can be labeled differentially by lectin agglutinins (94, 95, 239).

The resting electrical properties including the capacitance (C), the resting membrane resistance (R_m), and the resting potential (u_r) of ORNs have been measured in various vertebrate and invertebrate species. The measured capacitance of ORNs (Table 1) is in the range from 0.7 to 35 pF. The smallest and the largest values have been found in zebrafish (65) and in newt (F. Kawai, personal communication), respectively. With the assumption of a standard value of 1 µF/cm² (corresponding to 1 pF/100 µm²), the membrane surface of ORNs is thus in the range between 70 and 3,500 µm².

In patch-clamp recordings from ORNs, the seal resistance and the resting membrane resistance are often in the same range (between 1 and 40 G). This can lead to problems in the interpretation of data, and it is therefore particularly important to make a clear distinction between the resting input resistance (R_i) and R_m . The R_i value is usually measured in the whole cell configuration using voltage clamp. Starting at a holding voltage of about 80 mV, a small voltage pulse u of typically 10 mV is applied, and the resulting asymptotic current shift I is measured. The u/I determined at u_r gives the R_i , which is the combined resistance of R_m and the seal resistance (R_seal)

(1)

whereby the series (or access) resistance (R_s) of the pipette is neglected here because it should be at least two orders of magnitude smaller than R_m or R_seal . From Equation 1 it follows that the R_m can be obtained from R_i and R_seal (as measured in the cell-attached mode)

(2)

Seal resistances as reported in recordings from ORNs are in the range between 1 G (182) and 53 G (219). Clearly, the approximation R_m ~ R_i is better the smaller the R_i/R_seal .

The resting membrane potential u_r in ORNs, measured as the zero-current potential in the whole cell configuration of the patch-clamp technique, has been reported in the remarkably large range between 90 mV (98) and 30 mV (305). These widely differing values might be explained by species differences. However, Firestein and Werblin (92) explained the deviation of u_r (in their study 54 mV) from the K⁺ equilibrium potential (E_K; in their study 98 mV) by the membrane being permeable for ions other than K⁺. A deviation of u_r from K⁺ potential (u_K) could be brought about by three other conductances.

First, a Cl conductance (76, 165) with Cl potential (u_Cl) > E_K as reported in ORNs of the mudpuppy (76), newt (184), and the clawed frog (361) would raise u_r . In the frog, this explanation might, however, be incorrect because the Ca²⁺-activated Cl conductance (g_Cl) is half-activated at intracellular Ca²⁺ concentrations of ~5 µM (165), which is almost two orders of magnitude higher than the resting Ca²⁺ concentrations in ORNs as obtained in imaging studies in frog (309-311). Hence, the Ca²⁺-dependent Cl conductance in frog is presumably not activated at rest.

A second conductance that necessarily affects the measurements of u_r is the seal between plasma membrane and patch pipette. With g_seal (=1/R_seal) and g_Ko (=1/R_Ko) being the seal conductance and the resting K⁺ conductance, and with the assumption that other conductances are negligible at rest, the resting membrane potential is given by the equation (113) where f_seal = g_seal/(g_seal + g_Ko), f_Ko = g_Ko/(g_seal + g_Ko) are the fractional conductances with u_seal and u_K being the corresponding equilibrium potentials. Because u_seal ~0 mV, u_r can be approximated by

(3)

This shows that a "good seal resistance" is a relative concept and that u_r is reliably determined only if R_seal >> R_Ko , a requirement difficult to meet in small neurons with a high resting resistance.

Frings and Lindemann (99), for example, have reported u_r approximately equal to 85 mV with R_i ~5 G and R_seal ~40 G. Indeed, with u_K = 91 mV, Equations 2 and 3 give R_Ko ~7.5 G and u_r = 79 mV, which is in the range of the measured value. Whenever R_i and R_seal happen to be of the same order of magnitude, the seal short circuits the membrane potential. For example, with R_i = 4 G, R_seal = 10 G, and u_K = 97 mV, Equations 2 and 3 yield u_r = 57 mV (90). The short-circuiting effect of the seal shunt has been modeled and analyzed in detail by Pongracz et al. (276).

An additional problem arises if the seal dependence of a measured resting membrane potential affects further steps of data evaluation. For example, to investigate the Cl equilibrium potential in mudpuppy ORNs, Dubin and Dionne (76) recorded the Cl channel blocker-dependent shift u of current-voltage (I-V) curves recorded in the cell-attached mode. The resulting value u added to the resting membrane potential as subsequently measured in the whole cell mode (in this case u_r was 40 mV) gave u_Cl = 45 mV. However, the seal-dependent inaccuracy in the measurement of u_r affects the resulting value for u_Cl so that u_Cl is more negative than 45 mV, the exact value depending on R_seal .

A third conductance that can influence the measurement of R_m and u_r is an inward rectifying cation conductance (g_h), which is permeable for K⁺ and Na⁺ (P_K/P_Na = 5) and activates at hyperpolarized potentials. This conductance has been reported in ORNs of catfish (246), frog (343, 344), mouse (227), and rat (222). In other systems, a similar conductance is crucial for the generation of membrane potential oscillations (230). Whether or not this conductance underlies the membrane potential oscillations observed in ORNs (98, 205) is not known. Interestingly, however, Tokimasa and Akasu (339) have shown in sympathetic neurons that a conductance of the same type can be activated by increased levels of cAMP.

The product of the resistance R_m and the cell capacitance C determines the membrane time constant (_m). Average values of R_m × C are ~60 ms. The membrane can be regarded as an RC element or first-order low-pass filter with corner frequency (f_c) of ~16 Hz that dampens odor-induced current components for frequencies f > f_c . It thus rejects "high"-frequency noise (214). The time constants of ORNs were measured in various species using current injections into ORNs through a patch pipette. The values obtained with gigaohm seals are at least one order of magnitude larger than those obtained with sharp microelectrodes [e.g., 4.2 ms in the tiger salamander (226)]. Owing to the high resting resistance and, at least in some species, a relatively high capacitance, the time constant _m can be as long as ~100 ms [e.g., in squid (217), or salamander (92)]. This implies that, because of the high R_m , ORNs can be excited by very small currents but that the depolarization is relatively slow because of the long time constants. This was nicely demonstrated by Lynch and Barry (219) as well as by Maue and Dionne (227), who observed that the current supplied by the opening of a single ion channel was able to trigger an action potential. The underlying membrane biophysics have been analyzed in detail by Lynch and Barry (219). The delay of the action potential after channel opening depended on the current amplitude. For the smallest current that elicited an action potential, it was in the range of the membrane time constant.

The above-mentioned evidence that the current through a single channel can excite an olfactory neuron poses the question of whether a single odorant molecule binding to an odorant receptor can induce an action potential. Menini et al. (238) suggested this. These authors applied an odorant at concentrations well below the K_1/2 for the dependence of odor-induced steady-state current on odor concentration. The resulting current fluctuations were stereotyped, "quantal-like" in amplitude ranging from 0 pA (failure) to 9.5 pA, with an average response of ~1 pA (holding potential, 50 mV). Based on these data, Menini et al. (238) concluded that odors induce quantal-like current fluctuations presumably triggered by the binding of a single odorant molecule to its receptor. These data do, however, not prove that binding of one odorant molecule induces a quantal current. To show this, it would be necessary to demonstrate that the frequency of occurrence of the current fluctuations is equal to the frequency of binding of odor molecules to the receptor (23, 317). Moreover, the detection of a single molecule by a receptor cell would require that a quantal current could induce an action potential. Although this is not excluded, particularly in the case when amplification of channel activity occurs through opening of Ca²⁺-activated conductances (see sects. V and VI), the effect of quantal currents on membrane potential was not analyzed by Menini et al. (238).

On the other hand, Lowe and Gold (215) have shown that low concentrations of odorants elicit random current fluctuations in olfactory receptor cells from rat. These authors argue convincingly that these current fluctuations are not because of single molecular events. They show that the power spectrum for odor-induced currents is identical to the power spectrum of the current induced by increases in the concentration of the second messenger cAMP in the absence of odorants. They conclude that the current fluctuations induced by odorants are due to noise intrinsic to the transduction mechanism (intrinsic noise) and suggest that quantal detection is not a common property of olfactory receptor cells. Under basal conditions, the intrinsic noise may not be apparent because the steep dependence of current on cAMP concentration (Hill coefficient of 2-4) acts like a threshold. Addition of odorant would then elevate the level of activity above the threshold, thereby unmasking the intrinsic noise signals.

However, because of the differences in the experimental designs of the experiments carried out by Menini et al. (238) on the one hand, and Lowe and Gold (215) on the other, the data provided by Lowe and Gold do not rule out the possibility that the current fluctuations in the Menini study may be due to single molecular events (see also Ref. 295).

	III. CONDUCTANCES INVOLVED IN ACTION POTENTIALS

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Vertebrate olfactory receptor cells are neurons. They have an axon along which they send encoded information to the central nervous system (CNS). In this section we review some biophysical properties of the conductances that are involved in the generation of action potentials in ORNs.

As outlined in section II, the resting membrane potential appears to be determined by a very low resting K⁺ conductance, which consists of more than one type of channel: 1) an inward rectifiying conductance g_h (222, 227, 246, 343) and 2) a slowly inactivating delayed rectifier type of K⁺ conductance that has been observed in squid (217), fish (65, 246, 260), Chilean toad (69), frog (305, 343), salamander (92), rat (220, 342), and human (292). At the single-channel level, a slowly activating K⁺ current has been described (220) that could underlie the resting delayed rectifier conductance.

The reports of the voltage-gated Na⁺ conductances (g_Na) in ORNs for various species differ in some respects. 1) In the tiger salamander (92) and rat (280), it has been reported to be relatively insensitive to tetrodotoxin (TTX), whereas it is not in other species (65, 69, 246, 305). 2) It activates at about 60 mV in Xenopus laevis (305) and frog (277), 50 mV in catfish (246) and Chilean toad (69), 30 mV in squid (217) and salamander (92), and about 45 mV in rat (280, 342), human (292), salmon (260), zebrafish (65), and mudpuppy (76). This variability could be species dependent, but it could also be due to a voltage drop either along axonal compartments or across the pipette resistance. In the last case, the same voltage drop, i.e., the same right shift of I-V curves, is also seen for other conductances that are not localized to the axon.

The amplitudes of the Na⁺ currents show a considerable variability and, in some cells (292, 343), Na⁺ current could not be measured at all. The reason for this variability and occasional lack of Na⁺ currents in ORNs could be that the channels are primarily located in the axonal membrane, which is partially lost during preparation. This is consistent with the finding of Maue and Dionne (227) who were unable to observe single Na⁺ channels on the somata of mouse ORNs. Frings and Lindemann (99) found no effect of TTX when applied to the mucosal surface, whereas it blocked spike generation when applied to the interstitial surface of the mucosa. However, in the mudpuppy, Na⁺ channels were almost equally distributed on somata and dendrites (76).

Interestingly, cytosolic GTP (100 µM) shifts the inactivation curve of g_Na to the right (277). This effect is presumably not brought about by GTP itself, because the nonhydrolyzable analog guanosine 5'-O-(3-thiotriphosphate) (GTPS) exerts the same effect (277). Under perforated patch, the steady-state inactivation curve for g_Na overlaps with the inactivation curve measured in the whole cell configuration with GTPS in the pipette. These results indicate that a constitutively activated G protein maintains low half-inactivation voltages for g_Na in ORNs. These results open the possibility that g_Na might be modulated by G protein-coupled receptors.

High-voltage-ativated (HVA) Ca²⁺ currents in ORNs have been described in various species (65, 69, 92, 260, 305, 342, 343). The amplitude of this current seems to be relatively small, and in the whole cell configuration of the patch-clamp technique, the maximum current decreases with a time constant of ~3 min (305). This phenomenon, often called "washout," has been observed in many preparations and is presumably because of the diffusion between cytosol and patch pipette (14, 278). The HVA Ca²⁺ currents activate between 40 and 30 mV and have maximum amplitudes at ~0 mV; in some reports, the I-V relationship appears to be shifted to higher voltages, which may depend on high access resistances (13) or an elevated extracellular Ca²⁺ concentration (92, 305, 343). The HVA channels are thus primarily activated during the generation of action potentials, and the resulting Ca²⁺ influx presumably contributes to the repolarization by activating K⁺ channels (see sect. IIIC). Using quantitative ratiometric Ca²⁺ imaging with fluo 3 and FuraRed, Schild et al. (309) have shown that the HVA Ca²⁺ channels in Xenopus ORNs are primarily situated on the soma and the proximal dendrite. The resolution of the method does, however, not exclude that a small portion of Ca²⁺ influx could occur in other cellular compartments of ORNs.

In some species, a low-voltage-activated (LVA) Ca²⁺ current may also be involved in action potential formation. In the newt, Kawai et al. (153) have reported an LVA current being half-activated at 44 mV. Although this current is absent in some species [e.g., Xenopus (305), Chilean toad (69), and catfish (246)], its description is consistent with Trotier's finding of a TTX-insensitive inward current that could be blocked with Co²⁺ in salamander ORNs (343). This current lowers the threshold for action potential firing. It speeds up action potential generation by decreasing the inactivation of the voltage-gated g_Na during receptor potentials. The LVA Ca²⁺ current might therefore be particularly useful in relatively large ORNs that have high capacitances and long membrane time constants.

Repolarization of the action potential is achieved by the activation of various K⁺ channels: first, and above all, by a transient, 4-aminopyridine (4-AP)-sensitive K⁺ current described in squid (217), salamander (92), zebrafish (65), catfish (246), salmon (260), clawed frog (305), and rat (221). This current activates and inactivates rapidly; it activates at potentials more positive than the activation threshold of Na⁺ channels (221, 305), which precludes the traditional role of A currents in spike frequency modulation (64, 120). It rather contributes strongly to the repolarization of action potentials. However, in cultured ORNs of rat, which have a large delayed-rectifier conductance, the fast 4-AP-sensitive current as well as a Ca²⁺-dependent K⁺ current were absent (342).

Second, Ca²⁺-activated K⁺ conductances [g_K(Ca)] measured in various species (65, 69, 92, 227, 260, 305, 343) seem to contribute to the repolarization and an increase of the cell's impedance, whereby in the mouse, 130-pS channels with voltage-dependent kinetics and 80-pS channels with voltage-insensitive kinetics have been observed (227), presumably corresponding to BK and SK K⁺ channels (190, 196). In agreement with these findings, a fraction of the outward current in Xenopus ORNs was blockable by apamin (305), which blocks Ca²⁺-dependent K⁺ channels of the SK type (55). Among the various Ca²⁺ sources that could potentially activate g_K(Ca) , Ca²⁺ influx through HVA Ca²⁺ channels appears to play the major role because blocking the HVA current (69, 305, 343) or reducing the Ca²⁺ flux through HVA channels (92, 260) reduced or abolished the Ca²⁺-dependent conductances. Calcium influx-induced Ca²⁺ gradients are very steep, and the diffusion length of free cytosolic Ca²⁺ is on the order of 1 µm (143, 259). The Ca²⁺- activated conductances, or at least a major fraction of them, can therefore be supposed to be colocalized with HVA Ca²⁺ channels. This is also supported by the fact that the voltage-gated Ca²⁺-activated K⁺ current in ORNs, when activated by voltage steps under voltage clamp, activates simultaneously with the voltage steps (69, 92, 246, 305, 343).

Because most of the HVA Ca²⁺ channels seem to be situated on the soma and the proximal dendrite (309), we can conclude that g_K(Ca) is colocalized with HVA channels at the soma and the proximal dendrite. This is consistent with single-channel measurements in excised patches from olfactory neuronal soma membrane (227); it does, however, not preclude additional Ca²⁺-dependent K⁺ channels being localized elsewhere. Bacigalupo and co-workers (248, 249) have reported evidence for a Ca²⁺-dependent K⁺ conductance in the transduction compartments of the toad that can be activated by odorants (see sect. VII). Differentiation between conductances involved in action potential generation and others involved in receptor potential generation might turn out to be inappropriate because the same g_Na , g_H, and g_K(Ca) may be involved in both.

In conclusion, an action potential, initiated by a receptor potential and voltage-gated Na⁺ channels, goes through the voltage range positive to 30 mV, where HVA channels on the soma and the proximal dendrite activate. The resulting Ca²⁺ influx activates Ca²⁺-dependent K⁺ channels on soma and dendrite which, in concert with fast g_K , then contribute to the repolarization of the action potential. Maue and Dionne (227) describe two different types of g_K(Ca) , which opens the possibility that a g_K(Ca) , in addition to participating in the repolarization, may play an additional role in tuning the refractory period. The third conductance, which may play a role in both repolarization and tuning of the resting and poststimulus impedance, is the slow delayed rectifier-like K⁺ conductance (69, 92, 217, 246, 260, 292, 305, 343).

This particular combination of K⁺ conductances taking part in the repolarization seems to be very similar in all studied species, although an exact quantitative comparison is presently impossible. The general idea, however, appears to be that only very few K⁺ channels are open at rest, resulting in an extremely high resting resistance and a correspondingly high sensitivity. The K⁺ conductances that are open at rest, presumably the inward rectifier and the delayed rectifier, would not be able to repolarize the membrane back to the resting potential because the inward rectifier is closed at depolarized voltages and the delayed rectifier has too small a conductance. Repolarization seems to be done by transiently switching on the fast K⁺ conductance and the fast Ca²⁺-dependent K⁺ conductance.

	IV. ODORANT RESPONSES

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The classical recordings from single ORNs were made using either platinum-black electrodes or sharp pipettes (4, 105, 106). Although the sharp electrode recordings suffered from the leak between glass and membrane resulting in short recording durations, extracellular recordings proved useful to determine the differential responsiveness of ORNs to many stimuli (142, 325). The patch-clamp technique with gigaohm seal resistances provided several new methods of recording odorant responses: 1) tight-seal whole cell patch-clamp recordings under voltage-clamp or current-clamp conditions, 2) cell-attached patch-clamp recordings, 3) loose patch recording with a suction electrode (212), 4) perforated patch recordings using pore-forming molecules such as nystatin (76, 217) or gramicidin (361), and 5) sensory cilia attached in loose-clamp (99), tight-seal, or excised configuration (159, 164, 165). The tight-seal whole cell configuration allows voltage clamping of the membrane potential so that activation and inactivation of voltage-gated and odorant-induced conductances could be measured. However, the diffusion of molecules from and into the pipette changes the cell's milieu, resulting in loss of important factors in the cytoplasm. Washout of messengers and modulators can prevent physiological responses, especially when recording Ca²⁺ currents or odorant responses. The cell-attached mode allows the measurement of mainly capacitative currents across the patch that are associated with action potentials (219). The perforated-patch configuration prevents the diffusion of large molecules between pipette and cytosol, but the access resistance to the cell is higher than in the whole cell mode. This causes a current-dependent voltage drop across the pipette tip, and, in most patch-clamp amplifiers, the voltage-clamp control circuit is slowed considerably.

In addition to electronic amplifiers, charge-coupled device (CCD) and laser scanning microscope imaging techniques (296, 306) became available and were used to measure odorant responses, whereby the increase of intracellular Ca²⁺ was monitored as an indirect measure of excitation.

The first manuscripts to present studies of ORNs using the patch-clamp technique were published in 1985 (7) and 1986 (343). Trotier (343) described voltage-gated currents, single channels including the inward rectifier and currents induced by addition of odorants (7 µM isoamylacetate and 10 µM butanol).¹ Although the description of the odorant-induced current was short, it contained some important points that were confirmed later: the reversal potential of the odorant-induced current was ~0 mV, the I-V relationship was almost linear, and there was a considerable delay in the response (in this study ~2 s). Trotier (343) cautiously suggested as a working hypothesis that the odor-activated channels were nonselective cation channels modulated by a diffusible cytosolic metabolite. The equilibrium potential for both Cl and cations was ~0 mV in his study.

The history of the discovery of odor-activated currents in vertebrates took three main routes: 1) independently the laboratories of Firestein, Kurahashi, and Gold followed Trotier's working hypothesis and consequently carried out detailed analyses of the odor-activated currents. Kleene and Gesteland (165) discovered that what was believed to be an odor-gated "cation" current was actually a mixture of a Ca²⁺-permeable cation current and a Ca²⁺-activated Cl current. These experiments led to the present view of the cAMP-mediated second messenger cascade, which is described in section V. 2) Evidence regarding odorant-induced Ca²⁺ and cation currents that were different from the cAMP-gated current did not fit in the model of a cAMP-mediated second messenger cascade. Work from the laboratories of Restrepo, Ache, and Schild as well as from studies in insects (327, 328, 363, 373) indicated that there might be at least one more second messenger pathway mediated by inositol phosphates, Ca²⁺, or guanosine 3',5'-cyclic monophosphate (cGMP). This work is described in sections VI and VII. 3) Several studies reporting odorant-mediated currents did not primarily aim at analyzing elements of the cAMP or inositol trisphosphate (InsP₃) pathway. They rather pointed at the kinetic and biophysical characterization of odorant responses, which are reviewed in the rest of this section.

After Trotier's 1986 paper (343), one study² reported the block of the odorant-induced current by amiloride in frog ORNs (98), another reported an odorant-stimulated depolarization that reversed at 2.7 mV (8), and studies from two laboratories presented a detailed characterization of odor-activated currents in ORNs (91, 175, 179, 182). Kurahashi (175) characterized an odorant-gated cation conductance in newt ORNs using n-amyl acetate (10 mM) as odorant stimulus (Fig. 2). This current had the following properties: 1) it reversed at ~0 mV, the reversal potential shifting by ~57 mV upon reducing Na⁺ concentration by a factor of 10; 2) the current was maximum when the odorant was applied at the apical dendrite; 3) the I-V curve was nonlinear, showing a block of inward current at potentials more negative than 30 mV; 4) its permeabilities for alkali ions were P_Li/P_Na/P_K/P_Rb/P_Cs = 1.25:1:0.98:0.84:0.8; 5) removal of Ca²⁺ increased the odorant-gated current and prolonged its duration, but did not cause a change in reversal potential; and 6) removal of Cl did not change the reversal potential. The size of the odor-induced current was maximal when the odor plume was directed to the apical side of the cell. The pipette solutions in these studies contained 5 mM ethylene glycolbis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) so that [Ca²⁺]_i was heavily buffered. In addition, in a separate manuscript, Kurahashi and Shibuya (183) suggested that the inactivation of the odorant-induced current was mediated by Ca²⁺ influx through the odor-regulated conductance.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   A: odorant-induced currents recorded at various holding voltages (Vh; indicated to left of each current trace) from a newt (Cynops pyrrhogaster) olfactory receptor neuron. Current traces were arbitrarily shifted. Bottom trace indicates timing of n-amyl acetate (10 mM) application from a glass pipette positioned <20 µm away from apical dendrite. B: voltage dependence of odorant-induced current; replotted from A and additional records from same cell. [Modified from Kurahashi (175).]

Firestein and Werblin (91) stimulated terrestrial phase salamander ORNs using a mixture of acetophenone, n-amyl acetate, cineole, phenylethylamine, and triethylamine (each at a concentration between 0.1 and 1 mM). The reversal potential for cations (u_cat) and u_Cl were both at ~0 mV in this study. These authors confirmed and extended Trotier's finding of a purported cation current with respect to the following points: 1) again the I-V curve of the odor-activated currents was linear with a reversal potential of ~0 mV, 2) the delay of the odorant response was assessed more accurately (140-570 ms), 3) a dose-response curve extending from ~10⁵ to 10⁴ M for a mixture of stimuli was given, and 4) the odor response was shown to be cooperative with a Hill coefficient of 2.7. Any of the steps between binding of the odorant and flow of ions through odorant-activated channels could thus be cooperative, leading to a sigmoidal input-output (i.e., concentration-current) relationship (see also Ref. 87). In a later study, Firestein et al. (90) confirmed that the odorant sensitivity is restricted to the cilia and possibly the distal dendrite. They also found that the latency of the response was relatively independent of the stimulus concentration (10⁵ and 10⁶ M).

The spatial distribution of odorant sensitivity and of the odorant-induced current was carefully examined in a series of experiments in salamander ORNs by Lowe and Gold (212). Using an odorant mixture containing 2-hexylpyridine, isoamyl acetate, acetophenone, and cineole, these investigators determined that the odorant response increased approximately linearly as a function of the fraction of the ciliary bundle that was exposed to the odorant. No further increase in responsivity was found upon stimulation of the entire ciliary bundle and a fraction of the dendrite. This indicated that olfactory receptor sites are uniformly distributed along the length of the cilium and are excluded from the dendrite. In addition, the odorant-induced current was found to be localized to the cilia, and the resting K⁺ conductance was found to be much lower in the ciliary membrane than in the dendritic membrane. The pipette contained a pseudointracellular solution with 0.1 mM EGTA, pH 7.2, providing little buffering of intracellular Ca²⁺.

Obviously, in a sensory system that can possibly detect single molecules (215, 238), concentrations in the range between 10⁵ and 10⁶ M are high. However, the physical chemistry of lipophilic odorants in water is by no means trivial, and it has to be kept in mind that in the experiments described, the odorants were applied in the absence of mucus or odor-binding proteins, i.e., the perireceptor microenvironment (107, 139, 224, 235, 271) was far from physiological. The experiments by Frings and Lindemann (99), who recorded from single cilia of ORNs in an at least partly intact epithelium, are interesting in this respect because they recorded an increase in action potential rate upon application of cineole (dissolved in ethanol), whereby 9, 5, and 3 of 22 responding cells responded to odorant concentrations of 1, 10, and 100 pM, respectively (99).

Optical recordings of odorant-induced changes in [Ca²⁺]_i with CCD cameras or laser scanning microscopes have the advantage of spatial resolution, and they are minimally invasive, although the endogenous Ca²⁺ buffering capacity of the cells (158) is increased by adding the reporter dye (118). Optical recording of [Ca²⁺]_i was first used by Restrepo and co-workers (288, 290) to monitor odorant responsiveness of catfish ORNs. These and subsequent studies determined that odorants elicit increases in intracellular Ca²⁺ (16, 145, 192, 257, 288, 291, 292, 302, 337) and that removal of extracellular Ca²⁺ abolished the odorant-induced increases in [Ca²⁺]_i in ORNs, suggesting that the increase was due to influx of Ca²⁺ through the plasma membrane (16, 288, 291, 292, 302, 337). However, some olfactory neurons from bullfrog (Rana catesbeiana) were found to respond even in the absence of extracellular Ca²⁺ (302). Imaging experiments determined that shortly after stimulation, and in some ORNs even after prolonged stimulation, the increases in [Ca²⁺]_i took place at the apical end of the cell, suggesting that the source of Ca²⁺ was at the apical compartments of the cell (16, 257, 291, 302, 337). Interestingly, recent measurements with confocal microscopy in salamander (A. tigrinum) ORNs has demonstrated odor-induced changes in [Ca²⁺]_i in individual olfactory cilia (192). The increase in [Ca²⁺]_i in the cilia is rapid and transient and contrasts with a much slower and prolonged increase in [Ca²⁺]_i in the cell body.

Imaging of [Ca²⁺]_i has also been used to study channels and transporters involved in [Ca²⁺]_i regulation in ORNs and the regulation of these channels and transporters by second messengers. Thus it has been possible to localize the HVA Ca²⁺ current in ORNs (309), the Na⁺/Ca²⁺ antiport (136), and InsP₃-activated, Ca²⁺-permeable channels of the plasma membrane of ORNs (145, 311).

Studies of odorant responsiveness utilizing [Ca²⁺]_i imaging techniques have provided information on the odor specificity of neighboring ORNs. Takebayashi and co-workers (122, 303) imaged a partly dissociated preparation of mouse ORNs in which the cells retained their original spatial relationships. With the use of three different odorants in one study (122) and a series of aliphatic odorants in another study (303), the responsiveness of ORNs was estimated from the odor-induced increase in [Ca²⁺]_i . These authors were able to demonstrate that cells with a similar odor responsivity were located next to cells with a different odor responsivity. The distribution patterns were, however, not consistent with a completely random distribution.

Finally, optical recording with voltage-sensitive dyes (VSDs) has been used to determine neuronal activity in the olfactory system (60, 149). This technique has the advantage that the average activity in a volume of tissue can be determined upon stimulation with many odorants (150). Staining of the olfactory epithelium with VSDs could be used to study signal transduction in ORNs, which would be particularly useful in resolving questions about signaling between neighboring ORNs. However, the use of VSDs in the olfactory epithelium at high magnification has not been reported in refereed publications. Voltage-sensitive dyes have been used to record distributed activity within the olfactory epithelium and the olfactory bulb at low magnification (cf. Refs. 59, 61, 62, 155, 156, 356). These experiments have provided an important contribution of our understanding of patterns of responsivity to odors in the olfactory epithelium and form a basis for understanding olfactory quality coding. Studies of distributed patterns of responsivity with VSDs are not reviewed in detail here, and the reader is referred to recent reviews (60, 149).

4. Are ORNs modulated by the autonomic nervous system?

A further interesting point within the context of diversity in transduction mechanisms is the possible influence of the autonomic nervous system on olfactory transduction pathways. Some neurotransmitter receptor antagonists such as propranolol, a -adrenergic antagonist and a serotonin (5-HT₁) agonist, and the muscarinic antagonist atropine can block odorant responses in isolated ORNs via an unknown mechanism (

It has proven difficult to culture functionally responsive ORNs. The only report of odorant-induced changes in electrical activity is from cultured ORNs of rat bearing multiple long processes that showed odorant-induced depolarizations in conjunction with an increase in conductance. Interestingly, these cells also displayed a second type of odorant-activated current that resembled very much synaptic currents (275). Others have reported odorant-induced changes in second messenger formation and/or odorant-induced changes in [Ca²⁺]_i in cultured olfactory neuroblasts. Long-term cell cultures of neuroblasts from human fetal olfactory epithelium and olfactory neuronal cell cultures from rat embryo responded to stimulation by odorants with biochemically measured increases in cAMP or InsP₃ formation (297, 347), and human olfactory neuroblastoma cells (110) and neuronal cells cultured from adult human olfactory epithelium (354) have been shown to respond to odors with increases in intracellular Ca²⁺.

Cell cultures of olfactory neuroblasts have also been used in the study of olfactory mechanisms in invertebrates. Cultured ORNs of lobster responded to odorants even before they had developed processes (83), suggesting that these cells insert all transduction components in the soma membrane during their development. Cultured ORNs of the silkmoth Manduca sexta responded to a pheromone (synthetic bombycal) with a sequence of inward currents. The early component of the response to the pheromone was a Ca²⁺ current that has been suggested to be regulated by InsP₃ (328). This current was inhibited by Ni²⁺ and did not appear in the absence of Ca²⁺ or Ba²⁺ in the bath. It was followed by a slower inwardly rectifying current component with a reversal potential near 0 mV. The nonspecific current had the characteristics of a Ca²⁺-dependent nonspecific cation current described in insect ORNs (327). Finally, a third sustained component reversed near 0 mV and was proposed to be a protein kinase C-dependent cation current (327, 328).

Responses of single vomeronasal ORNs to odorants or pheromones have as yet not been reported. However, Trotier et al. (345) and Liman and Corey (200) have measured voltage-gated currents in microvillous vomeronasal neurons using the patch-clamp technique. Interestingly, Liman and Corey (200) report a lack of a response upon dialysis into the cytosol of vomeronasal receptor neurons with cAMP (200), whereas Taniguchi et al. (335, 336) report current responses upon dialysis of vomeronasal receptor cells of the turtle with cAMP, cGMP, or InsP₃ . Furthermore, Okamoto et al. (265) have shown a forskolin-induced increase of adenylyl cyclase in turtle vomeronasal receptor cells, but odorants that increase the activity of this enzyme in ORNs of other species had no effect. Hence, there appear to be some dissimilarities between the receptor cells in the main olfactory epithelium and those in the vomeronasal organ (197). This has been confirmed by Dulac and Axel's (77) finding that a family of putative receptor genes of the rat vomeronasal organ is unrelated to the receptors expressed in the rat main olfactory epithelium, and by studies from Berghard et al. (24) indicating that, unlike ORNs, vomeronasal neurons do not express mRNA for the G protein G_olf , for subunit 1 of the olfactory cAMP-gated channel (g_cn1) and for adenylyl cyclase III.³ Vomeronasal neurons do express mRNA encoding for the second subunit of the olfactory cAMP-gated channel (g_cn2) (24). In the absence of g_cn1 , g_cn2 does not open in response to increases in the concentration of cyclic nucleotides, but does open upon exposure to nitric oxide (NO) (44). A channel with characteristics consistent with those of g_cn2 opens in response to NO in patches excised from rat vomeronasal neurons (44) (see Ref. 197 for review).

	V. ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE-MEDIATED SIGNALING IN OLFACTORY CELLS

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It is now generally believed that most odorants bind to seven transmembrane (TM) receptors that couple to G proteins resulting in second messenger formation (40, 46, 51, 52, 181, 189, 232, 262, 268, 274, 281, 283, 322, 324) (Fig. 3). A G protein called G_olf , which was first cloned on the basis of enriched expression in the olfactory system, is localized to the olfactory cilia (132, 234). Virtually identical G proteins have also been found in the striatum (119) and tissues outside the CNS such as the pancreas (365). Although no functional data are available directly implicating G_olf in olfactory transduction, it is most likely that G_olf couples certain olfactory receptors to cAMP formation by mediating stimulation of type III adenylyl cyclase (17). Accordingly, biochemical studies of adenylyl cyclase activity and measurements of cAMP formation in isolated olfactory cilia implicated a G protein-mediated increase in cAMP in olfactory transduction (267, 326).⁴ However, some potent odor stimuli failed to activate adenylyl cyclase (see sect. VI) (49, 326).

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Schematic diagram of cAMP-mediated transduction cascade of an olfactory neuron. Olfactory receptor neuron consists of at least 5 compartments (left): axon, soma, dendrite, dendritic knob, and cilia. Elements of transduction cascade reside in cilia membrane. A simplified model of cAMP-mediated signal pathway is shown on right. R, receptor; G, G protein; AC, adenylyl cyclase; gcn , cyclic nucleotide-activated conductance; gCl(Ca) , intracellular Ca2+-activated Cl conductance. For simplicity, intracellular Ca2+-dependent phosphodiesterase that transforms cAMP to AMP (29, 30), protein kinase A (26), and receptor kinase responsible for receptor desensitization (68, 312) are left out.

These findings were obtained with conventional biochemical methods and were confirmed and extended in experiments in the subsecond time range utilizing stopped flow methods that demonstrated rapid transient stimulation of either cAMP or InsP₃ formation in isolated olfactory cilia from rat and catfish (28, 37, 38, 287). It must be emphasized that a strict comparison of time courses between these biochemical measurements and measurements of odor-activated currents in intact olfactory neurons is not meaningful because the biochemical measurements were performed on detergent-treated fragments of olfactory cilia where the reactants and products are free to diffuse, whereas in the intact cell, diffusion within the cilia is restricted to a small volume. However, the rapid stimulation of second messenger formation by odorants in isolated olfactory cilia, with a time to peak ranging from 25 to 50 ms, indicates that the biochemical reactions can be fast enough to mediate olfactory responses, which have latencies of 50-500 ms in intact cells (cf. Refs. 90, 175, 213, 246). In addition, biochemical measurements showed that stimulation of both second messenger pathways by odorants was dependent on guanine nucleotides (28, 208, 210, 287) and that the cAMP and InsP₃ pathways were differentially sensitive to bacterial toxins (28), implicating different G proteins in mediating the cAMP and InsP₃ responses.

A cAMP-gated channel (g_cn), analogous to the cGMP-gated channel of vertebrate photoreceptors (5), was first detected in excised patches from olfactory cilia of toad (Bufo marinus) ORN by Nakamura and Gold (255). These authors reported activation of nonspecific cation channels by cAMP and cGMP in excised patches from olfactory cilia membrane. The cAMP-gated channels were found to be permeable for Ca²⁺, and the authors suggested that voltage-dependent block by extracellular Ca²⁺ and Mg²⁺ elicited outward rectification in the I-V relationship. These observations were confirmed and extended by subsequent work from various laboratories (cf. Refs. 35, 49, 72, 86, 93, 97, 100, 101, 111, 133, 164, 170, 176-178, 181, 183, 199, 213, 218, 223, 245, 273, 332, 370, 371, 374, 376). Similar channels have been found in various other tissues including retina, aorta, and testis (151, 152, 353, 357). Some odorants appear to directly suppress the cAMP-gated conductance g_cn via an unknown mechanism (180).

The evidence in support of the mediatory role of cAMP-gated channels in olfactory transduction is conclusive. Lowe and Gold (213) have shown that odorant-stimulated currents are, except for a difference in latency, identical to currents induced by photorelease of cAMP (a mixture of odorants was used in this study including cineole, isoamylacetate, and 2-hexylpyridine) (213) (Fig. 4). Importantly, however, a large fraction of the cAMP-induced current is carried by Cl (see sect. VC). In addition, on-cell patch measurements at the olfactory knob from Zufall and co-workers (93) indicate that the channels opening in response to odorant stimulation are identical to cAMP-gated channels measured in excised patches in the inside-out configuration (the odorant mixture used contained amylacetate, acetophenone, and cineole) (93). Blockers of cAMP-gated channels including amiloride, cadmium, and L-cis-diltiazem inhibit odorant responses (86, 98, 99, 273, 291, 292), and odorant responses and responses to increased cytosolic cAMP levels display the same ionic dependence (86, 97, 175, 176). In addition, a cAMP-gated channel-deficient transgenic mouse is anosmic to all odorants tested as assessed by electroolfactogram (EOG) measurements (50), and a mutation in a cyclic nucleotide-gated channel subunit in C. elegans disrupts responsivity to selected odorants (63, 172).

View larger version (17K): [in this window] [in a new window]   FIG. 4.   A: whole cell current responses to photolysis of caged cAMP and odorant stimulation in a tiger salamander (Ambystoma tigrinum) olfactory receptor neuron. Odorant stimulus (O; duration, 30 ms; pressure, 100 kPa) and flash (F; duration, 40 ms; log attenuation, 0.54) both began at zero on time axis. Notice that response to flash is immediate, whereas response to odorant has a significant delay. B: summation of odorant and photolysis responses. Odorant stimulus (O) was followed by a flash (F) that was limited to cilia and timed to coincide approximately with peak of odorant response. Intensity and duration of flash were fixed (log attenuation, 0.54; duration, 10 ms), whereas amplitude of odorant response was varied by adjusting ejection pressure applied to stimulus micropipette (7-175 kPa; duration, 20 ms). Notice that response to flash is not simply additive, but increases and subsequently decreases in magnitude as odorant concentration is increased. This behavior is expected if both stimuli activate cAMP-gated channel at level of increases in cAMP concentration. Nonlinear behavior of flash response is predicted from Hill equation, which describes cAMP concentration dependence of gcn (see Ref. 213 for details). [From Lowe and Gold (213).]

The cAMP-gated channels have been shown to be localized mainly to olfactory cilia where they can reach a density of 205-2,400 channels/µm² (166, 177, 178, 371), although channels are found at lower densities (0.2-25 channels/µm²) on the soma of the receptor neurons (169, 177, 178, 332) (some of these values may be overestimates due to the assumption that the patch area is equal to the area of the tip of the pipette, see Refs. 166, 301). Addition of low concentrations of cAMP to excised patches of ORN soma membrane in the nominal absence of divalent cations in the pipette elicits current fluctuations typical of single-channel openings. The unitary conductance has been reported to be in the range from 12 to 55 pS depending on the species (Table 2), and in some species a subconductance level is present (111, 178). The apparent binding constant for cGMP is slightly smaller than the binding constant for cAMP, except in the salamander where the apparent binding constant for cGMP is five times smaller than the binding constant for cAMP (compare native channels in Table 2). The apparent binding constants for cAMP and cGMP decrease slightly when the holding potential is shifted from negative to positive values (35, 101, 169, 199). Interestingly, the dose-response relationship for cAMP-gated channels from all species studied, except for the carp, has a Hill coefficient larger than unity, indicating that multiple cyclic nucleotide molecules interact with a single cAMP-gated channel in a cooperative manner (Table 2).

Two subunits of the olfactory cAMP-gated channel sharing a considerable degree of homology have been cloned to date (22, 35, 63, 72, 111, 172, 199, 218). The first subunit (g_cn1) (see footnote 3) can form functional cAMP-gated channels when expressed in a heterologous cell system (72, 111, 218), whereas the expression of the second subunit (g_cn2) alone does not result in expression of functional channels (35, 199). When g_cn1 is expressed alone, the affinity for cAMP is two orders of magnitude lower than the affinity of the native channel (Table 2). However, when g_cn1 and g_cn2 are coexpressed, the affinity for cAMP increases to within the same order of magnitude of the affinity for the native cAMP-gated channel. On the basis of the similarity in affinity for cyclic nucleotides, and because of similarities in single-channel properties, it has been suggested that the native cAMP-gated channel is a heterooligomeric g_cn1/g_cn2 protein complex (35, 199). It is important to state, however, that the native channel has not been definitively shown to be a g_cn1/g_cn2 heterooligomer.

The cAMP-gated channels are permeable for monovalent alkali cations including Na⁺ and K⁺ and for divalent cations including Ca²⁺ (22, 72, 101, 170, 176, 179). The permeabilities of organic cations through these channels have been investigated by Balasubramanian et al. (19). The cAMP-gated channels have been reported to be inhibited by amiloride (101, 162, 273, 332) and dichlorobenzamil (160, 169, 170), the Ca²⁺ channel blockers cadmium (193), nifedipine (370), L-cis-diltiazem (101, 160, 170), D-cis-diltiazem (101), and D-600 (101); the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, calmidazolium, and trifluoperazine (162); the guanylate cyclase inhibitor LY-83583 (195); and Rp-cAMPS, a structural analog of cAMP (42). The use of these blockers to classify odor responsivity must be exercised with care because of known effects of the drugs on other conductances or enzymes and because of the fact that the action of these drugs has only been reported for certain species.

The conductance of cAMP-gated channels is modulated by both [Ca²⁺]_i and extracellular Ca²⁺ concentration ([Ca²⁺]_o) (18, 56, 102, 161, 163, 173, 175, 206, 223, 255, 370, 376). Increases in [Ca²⁺]_i inhibit the cAMP-gated channel. This was first described by Zufall et al. (376) and interpreted as a direct allosteric effect of Ca²⁺ on the g_cn channels (376). The inhibitory effects of [Ca²⁺]_i and intracellular Mg²⁺ concentration on g_cn channels were then analyzed in more detail (161, 223) and are now believed to be mediated by calmodulin (56, 206) and at least another unidentified endogenous factor (18). By stimulating newt ORNs with caged cAMP and odorants, Kurahashi and Menini (181) have shown that short-term adaptation occurs downstream of the olfactory receptors and provide a calculation showing that that the kinetics of adaptation are consistent with mediation by modulation of the cAMP-gated channel by [Ca²⁺]_i/calmodulin (181).

The effect of [Ca²⁺]_o on g_cn is a permeation block; Ca²⁺ reduces the permeabilities for other ions while passing through the pore (102) so that, under physiological conditions, i.e., with [Ca²⁺]_o in the low millimolar range, the current through these channels is almost exclusively carried by Ca²⁺. They are thus Ca²⁺ channels. As all Ca²⁺ channels, they have an increasing permeability for other cations when external Ca²⁺ is removed. Under resting conditions, the Ca²⁺ influx through these channels can lead to a standing Ca²⁺ gradient in ORNs (204), but the current amplitude is presumably in the range of a few picoamperes (102). With a membrane resistance of ~10 G, this current could cause sufficient membrane depolarization to induce a small tonic increase in the rate of firing of action potentials but would be unlikely to result in the characteristic strong phasic increase in action potential firing rate that odorants elicit in olfactory neurons (106, 108). Hence, the major effect of the activation of g_cn is to increase [Ca²⁺]_i , which then acts as a third messenger.

Kleene and Gesteland (165) first described a ciliary Ca²⁺-activated Cl conductance (Fig. 5). They postulated that the increase in [Ca²⁺]_i resulting from the opening of cAMP-gated channels activates ciliary Ca²⁺-activated Cl channels, thereby amplifying the generator current (Fig. 3), a suggestion that has been confirmed by experiments from various laboratories (89, 160, 167, 184, 214, 361). These Cl channels display a steep dependence on [Ca²⁺]_i (Hill coefficient of 2) with a K_1/2 of 4.8 µM (165). Calcium concentrations in this range can be reached easily because of the small volume of olfactory cilia (106, 284) and the restricted diffusion for Ca²⁺ in the cytosol (144, 259). In a ciliary compartment of 1 fl (0.2 µm in diameter and ~30 µm in length), 1 pA Ca²⁺ influx corresponds to ~10⁴ Ca²⁺/10 ms. Adding this number of Ca²⁺ to a volume of 1 fl corresponds to an increase of total Ca²⁺ concentration of 100 µM. If 99% of the Ca²⁺ entering the cilium is buffered, as in other systems (118, 259), the increase of free [Ca²⁺]_i would be 1 µM/10 ms. This does not, of course, exclude much higher concentrations on a shorter time scale.

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Effect of cytoplasmic Ca2+ on ciliary membrane conductance of olfactory receptor neurons of northern grass frog, Rana pipiens. A: current-voltage relationship of membrane of 1 cilium was measured in each of 8 baths. Each bath consisted of a standard pseudointracellular solution plus free Ca2+ from 0 to 300 µM as indicated. Recording pipette contained standard extracellular solution. B: slope of current-voltage plot, measured in linear range between 50 and 0 mV, is plotted against concentration of free Ca2+ in bath. Each point is mean of determinations in 7 cilia. Conductance in 300 µM Ca2+ was arbitrarily defined as 100, and other values were normalized to this. Actual conductance in 300 µM Ca2+ was 4.1 ± 0.8 nS (n = 7; range, 1.8-8.3 nS). No controls have been subtracted. Curve is best-fitting Hill equation, which has a K1/2 of 4.8 µM and a Hill coefficient of 2.0. [Modified from Kleene and Gesteland (165).]

Calcium-activated Cl current resulting from this increase in [Ca²⁺]_i is depolarizing because the reversal potential for Cl u_Cl is considerably more positive than the resting potential in these neurons. Kurahashi and Yau (184) measured Cl inward currents using voltage clamp at a holding potential of 50 mV, indicating that u_Cl >50 mV (Fig. 6). Dubin and Dionne (76) obtained a u_Cl value of 45 mV and Zhainazarov and Ache (361) determined a u_Cl value of 2.3 mV using the perforated patch technique (125) and the Cl-impermeable pore gramicidin. As a result, the opening of the Ca²⁺-activated Cl channels results not only in amplification of the odor-induced current, but also in enhanced stability of the transduction current in a wide variety of extracellular ionic environments (167, 184).

View larger version (15K): [in this window] [in a new window]   FIG. 6.   Perforated-patch recording from a dissociated newt (C. pyrrhogaster) olfactory receptor neuron. Patch pipette contained no Cl to avoid loading cell with Cl. Holding potential was 50 mV. Chloride channel blocker 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) blocks a substantial part of odor-induced current (top trace). Time course of odorant stimulation is shown below current trace. A mixture of n-amylacetate, isoamylacetate, and limonene was used as stimulus. [From Kurahashi and Yau (184).]

Presumably, u_Cl is not constant during activation of the Ca²⁺-activated Cl channels because of the small volume of the cilia: intracellular Cl concentration = 100 mM means ~60 × 10⁶ Cl/cilium (V_cilium = 1 fl). On the other hand, 60 × 10⁶ is the number of ions that cross the membrane if a current of 10 pA over 1 s is assumed. A depletion of Cl within the cilia and a corresponding shift of u_Cl to more negative values can be expected during activation. This Goldman-Hodgkin-Katz-like mechanism would be a negative feedback: I_Cl = g_Cl(Ca)(u  u_Cl), where I_Cl is Cl current, would decrease because of a shift of u_Cl based on I_Cl itself. Future studies are necessary to better understand the Cl concentrations in ORNs and the mucus (see Table 4) as well as the mechanism(s) responsible for Cl ion homeostasis in ORNs.

Although the exact mechanisms of how bicarbonate permeation (31, 187), Cl exchangers (289), and pumps affect u_Cl remain to be elucidated, it appears that the major component of the cAMP-stimulated transduction current in ORNs is a Ca²⁺-activated Cl current. In retrospect, the initial failure of many laboratories to record odorant-induced currents in isolated receptor neurons may have resulted from choosing a physiological [Ca²⁺]_o and setting a low u_Cl , whereas successful recordings were made using unphysiologically low [Ca²⁺]_o and an unusual, but physiologically correct, u_Cl . However, under the latter conditions, the cation current through g_cn contributes significantly to the overall odor-gated current and was therefore initially assumed to be identical to it.

The cAMP-mediated olfactory transduction cascade (Fig. 3) consists of seven TM receptors that couple to G proteins (G_olf). An adenylyl cyclase (type III) is stimulated, and the resulting increase in cAMP directly activates cyclic nucleotide-gated channels (g_cn). These channels are blocked by both [Ca²⁺]_o and [Ca²⁺]_i . Under physiological conditions, the g_cn channels are highly permeable for Ca²⁺, which enters the cell and activates a Ca²⁺-dependent Cl conductance (K_1/2 = 4.8 mM), the current through which is depolarizing because u_Cl is considerable less negative than the resting potential.

	VI. INOSITOL TRISPHOSPHATE-MEDIATED SIGNALING IN OLFACTORY CELLS

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The involvement of a second messenger other than cAMP in mediating olfactory transduction was first suggested by biochemical studies with isolated olfactory cilia showing that certain odorants fail to elicit substantial increases in cAMP formation (49, 326). For some of these odorants, the weak responsiveness in biochemical experiments might be explained by the possibility that the odorants elicit increases in cAMP in a small fraction of olfactory neurons as indicated by the small magnitude of their EOG responses (216). However, odorants like lyral, isovaleric acid, and isobutyric acid in frog (216), trimethylamine, isovaleric acid, and lilial in mouse (50) and L-cysteine in catfish (49) elicit relatively strong EOG responses but fail to elicit a substantial increase in cAMP formation (37, 49, 326).

Huque and Bruch (127) first determined that certain amino acids known to be potent odorants in catfish (53, 334) elicit increases in the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP₂) to diacylglycerol (DAG) and InsP₃ in a GTP-dependent manner. These experiments, performed with isolated catfish olfactory cilia, showed that the elevation in InsP₃ formation was absolutely dependent on GTP, implicating a G protein in this response. These and other studies with conventional biochemical methods using membrane preparations (208, 210) or olfactory neuronal cultures (297) have been confirmed and extended in experiments in the subsecond time range utilizing stopped flow methods that demonstrated rapid transient stimulation of cAMP and/or InsP₃ formation in a variety of species (27, 28, 37, 38, 287, 297, 308). In addition, stopped flow measurements showed that stimulation of both second messenger pathways by odorants was dependent on guanine nucleotides (28, 208, 210, 287) and that the cAMP and InsP₃ pathways were differentially sensitive to bacterial toxins (28), implicating different G proteins in mediating the cAMP and InsP₃ responses. Finally, Raming et al. (281) have shown in a preliminary study that Sf9 insect cells transfected with a putative olfactory receptor responded to odorants with a dose-dependent increase in InsP₃ formation.

The identity of the G protein(s) that couples odorant receptors to InsP₃ formation is not firmly established. In other cell systems, phospholipase C-catalyzed InsP₃ formation is activated by G protein -subunits of the G_q and G_o types, by the ,-subunits released after the interaction of G protein heterotrimer with ligand-bound receptor, or by tyrosine kinase receptors (79). In stopped flow studies, Breer and co-workers (28) found that the odorant-stimulated InsP₃ response was inhibited by pertussis toxin, suggesting that the G protein mediating InsP₃ formation in the olfactory system is pertussis toxin sensitive (28). A number of laboratories have localized immunoreactivity to G_q , a G protein known to be linked to InsP₃ formation in other cell systems, to the olfactory cilia (1, 71, 237). In addition, in cultured lobster olfactory neurons, antibodies raised against G_q and G_o inhibit InsP₃-mediated whole cell responses (82). This indicates that G_q and G_o could be G proteins linking olfactory receptors to InsP₃ formation. However, in contradiction to the studies of Breer and co-workers (28), G_q is not a pertussis toxin-sensitive G protein. Therefore, it remains unclear whether G_q or a pertussis toxin-sensitive G_q-like G protein is involved in olfactory transduction.

Inositol trisphosphate receptors have been characterized biochemically in isolated olfactory cilia. Isolated olfactory cilia from catfish and rat have been shown to be enriched with an InsP₃-binding protein that displayed stereospecificity and affinity consistent with olfactory InsP₃-gated conductances studied in bilayers (see sect. VIB2) but different from that of the InsP₃ receptor found in the endoplasmic reticulum (ER) of CNS neurons (141, 294). Antibodies raised against rat brain InsP₃ receptors labeled the olfactory cilia layer (67).

View larger version (12K): [in this window] [in a new window]   FIG. 7.   Simultaneous current and charge-coupled device intracellular Ca2+ concentration ([Ca2+]i) recording in an olfactory receptor neuron of clawed frog, Xenopus laevis, upon dialysis of 100 µM inositol trisphosphate (InsP3) through patch pipette. A: current recording at a holding potential of 80 mV. First response was completely blocked by Ni2+ (Ni), whereas second response was only partly blocked by Ni2+. B: simultaneous with current measurement shown in A, [Ca2+]i was imaged and evaluated in dendritic knob (dotted line) and soma (continuous line). Time axes in A and B are identical. [Modified from Schild et al. (311).]

The most direct evidence for odor responses mediated by opening of InsP₃-gated channels is found in invertebrates (spiny lobster P. argus ORNs). Fadool and Ache (80) showed that an inward current induced by a mixture of odorants (tetramine) in cultured lobster ORNs is enhanced fourfold by addition of an antibody raised against the COOH terminus of the mammalian InsP₃ receptor. The same antibody increased the open probability of InsP₃-gated ion channels recorded in excised patches or lobster ORN plasma membrane by a factor of 4. The I-V relationship and pharmacology of the odorant-induced inward current in lobster are currently unknown.

In vertebrates, the only published report of the ionic basis of responses to odorants known to elicit InsP₃ formation in biochemical experiments in the same species is from catfish ORNs (130, 246). Extracellular recordings from single units in the channel catfish I. punctatus indicate that amino acids elicit both excitatory and suppressive responses in individual olfactory neurons (142). Biochemical experiments with isolated olfactory cilia from catfish indicate that amino acid odorants stimulate formation of InsP₃ (48, 287), although formation of cAMP was also stimulated at high concentration and after long exposure to the odorant (49). A single stimulus could be excitatory in one cell by eliciting an increase in the frequency of action potential firing and cause inhibition of action potential firing in another cell, and a single cell could be excited by one amino acid and inhibited by another. These experiments suggest at least two pathways for olfactory signal transduction in catfish. In experiments with freshly isolated catfish ORN under whole cell patch clamp, some cells responded to amino acids with inward currents that reversed near 0 mV, consistent with either nonspecific cation channels or a Cl conductance (130, 246). In some of these cells, the current was presumably carried by a cAMP-gated nonspecific cation current as evidenced by the marked outward rectification in the presence of extracellular divalent cations, and the sensitivity of the response to pretreatment with forskolin (246). However, in other cells, the response reversed at positive potentials (23 mV), and the response was augmented, and the reversal potential became more positive upon addition of 1 mM extracellular Ca²⁺ (246). This later response cannot be mediated by nonspecific cation or Cl conductances alone and is consistent with mediation by a Ca²⁺- or Na⁺-permeable or an inwardly rectifying cation conductance. It is at present unclear whether this odor-activated conductance is regulated by InsP₃ or another second messenger.

Two groups have reported responses to odorants known to stimulate InsP₃ formation in biochemical experiments with rat olfactory cilia in frog ORNs. Working with Rana ridibunda ORNs, Kashiwayanagi (145) found that a mixture of lilial, lyral, and ethyl vanillin elicited a substantial (41 pA on the average) increase in inward current at a holding potential of 70 mV with a parallel increase in [Ca²⁺]_i . These odorants elicited a response even in ORNs exposed to 1 mM cAMP by dialysis through the patch pipette. In contrast, in Chilean toad (Caudiverbera caudiverbera), a mixture of isovaleric acid, pyrazine, and triethylamine elicited a small (5 pA) inward current in the presence of 10 mM extracellular BaCl₂ . In the presence of amphibian Ringer solution in the bath, these investigators were unable to detect an odorant-induced inward current but detected an odorant-induced increase in [Ca²⁺]_i in the dendritic knob (16). The increase in [Ca²⁺]_i elicited opening of Ca²⁺-activated K⁺ channels in this preparation (discussed in sect. VII). The nature of the second messenger mediating the odorant response in C. caudiverbera is unknown.

	VII. CALCIUM AS A THIRD MESSENGER IN OLFACTORY TRANSDUCTION

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As discussed above, odorants induce increases in cAMP and/or InsP₃ , which activate Ca²⁺-permeable conductances and increase Ca²⁺ concentration. Odorants thus cause an increase in [Ca²⁺]_i in olfactory neurons (16, 145, 290-292, 302, 337) (see also sect. IVE). Even after prolonged stimulation, the odor-induced increases in [Ca²⁺]_i are spatially inhomogeneous, with the largest changes occurring in the apical portion of the cells (16, 290, 302, 337). Studies of the effects of the cAMP and/or InsP₃-induced elevation in [Ca²⁺]_i have made it increasingly clear that Ca²⁺ plays the role of a third messenger by eliciting opening of Ca²⁺-regulated conductances, thereby greatly amplifying or even changing the polarity of the generator potential (Fig. 8).

View larger version (22K): [in this window] [in a new window]   FIG. 8.   Schematic diagram showing parallels between cAMP-mediated pathway and presumed InsP3/Ca2+-mediated pathway. In either case, odorants act via olfactory receptors (OR), G proteins (G), and an enzyme that forms a second messenger, cAMP or InsP3 . Second messenger activates a Ca2+-permeable conductance. Resulting increase in [Ca2+]i then activates a second conductance, e.g., a Cl, a cation, or a K+ conductance, thereby leading to generation of receptor potential. PLC, phospholipase C; DAG, diacylglycerol; PKC, protein kinase C; gcn , nucleotide-activated conductance; gCa,cat , Ca2+ conductance or Ca2+-permeable cation conductance; gCl(Ca) , Ca2+-dependent Cl conductance; gcan, Ca2+-dependent cation conductance; gK(Ca) , Ca2+-dependent K+ conductance.

Three ciliary Ca²⁺-activated conductances thought to be regulated by odorant-induced increases in [Ca²⁺]_i have been described in ORNs: a Ca²⁺-activated Cl conductance, a Ca²⁺-activated K⁺ conductance, and a Ca²⁺-activated cation conductance (g_can). In the case of the cAMP-mediated transduction pathway, Ca²⁺ concentration activates a Cl current (160, 165, 184, 214, 361), which underlies the receptor potential (see sect. VC).

With regard to the action of an InsP₃-mediated increase of [Ca²⁺]_i , there are divergent reports. Evidence for three possibilities has been accumulated: 1) activation of a Ca²⁺-dependent nonspecific cation conductance, g_can; 2) activation of a Ca²⁺-dependent K⁺ conductance, g_K(Ca); and 3) activation of both g_can and g_K(Ca) .

1) The activation of a Ca²⁺-dependent nonspecific cation conductance g_can in vertebrate ORNs has first been demonstrated in Xenopus olfactory neurons (307). This conductance is situated in the transduction compartments of Xenopus ORNs as shown by simultaneous patch-clamp recordings and [Ca²⁺]_i imaging (310). Its ionic properties as well as the modulation by [Ca²⁺]_i and [Ca²⁺]_o differ markedly from the corresponding properties of the cAMP-gated cation conductance g_cn (Table 3) so that g_can and g_cn appear to be different conductances. Its properties further suggest that g_can is identical to the nonspecific cation conductance stimulated by dialysis of InsP₃ into the cytoplasm of Xenopus olfactory neurons (g_cat,IP3) (see Table 3) (311), although the molecular identity of g_can has not been demonstrated directly. It remains to be established whether the action of InsP₃ on g_can is direct. In any case, the resulting current is depolarizing at resting membrane potential.

2) In Chilean toad C. caudiverbera, there is evidence implicating odorants known to stimulate InsP₃ formation in isolated olfactory cilia from rat in an inhibitory (hyperpolarizing) response (248, 249). In ORNs from this species, odorants such as isovaleric acid, pyrazine, and triethylamine elicit a hyperpolarization that is mediated by opening of charybdotoxin-sensitive K⁺ channels, presumably Ca²⁺-activated K⁺ channels of the BK type (133, 248). Morales and co-workers (16) have shown that these odorants elicit an apically localized increase in [Ca²⁺]_i due to influx of Ca²⁺ from the outside. The Ca²⁺ conductance is sensitive to nifedipine, an inhibitor of L-type Ca²⁺ channels as well as of cAMP-gated channels (370). The identity of this odorant-activated Ca²⁺ conductance in toad is unknown, and it remains to be determined whether its activation is mediated by second messengers (cAMP or InsP₃).

The net result of the opening of the K⁺ channel would be to turn a response that would otherwise be depolarizing into a hyperpolarizing (suppressing) response. This is consistent with the fact that odorants that stimulate this K⁺ conductance elicit suppression of the basal rate of firing of action potentials in Chilean frogs. It has, however, to be kept in mind that mucosal K⁺ concentration can be much higher than the basolateral K⁺ concentration (see sect. IX), which would result in a less negative reversal potential for K⁺ and turn the activation of K⁺ channels localized to the mucosal side into a depolarization.

3) In catfish olfactory receptor cells, a charybdotoxin-sensitive Ca²⁺-activated K⁺ conductance was activated by dialysis of InsP₃ into the cytoplasm, but this current developed more slowly than the InsP₃-gated nonspecific cation conductance (245). Consequently, it was postulated that in catfish this current participated in repolarization of the plasma membrane after prolonged stimulation. Similarly, in rat olfactory neurons, dialysis of InsP₃ into the cytosol elicited opening of a Ca²⁺-activated K⁺ conductance, but the overall current (the sum of the K⁺ and nonspecific cation component) reversed at 35 mV, a potential more positive than the resting potential for these cells (264). Indeed, under whole cell current clamp, catfish and rat olfactory neurons responded to increased cytosolic InsP₃ with membrane depolarization, not hyperpolarization (245, 264).

In summary, Ca²⁺ seems to play the role of a third messenger in olfactory neurons. In the cAMP-mediated transduction pathway, it is increased by Ca²⁺ entering the cell through cyclic nucleotide-gated channels, whereas in the InsP₃-mediated transduction pathway, it may either be released from Ca²⁺ stores or enter the cells through cation channels. There are four different actions of Ca²⁺ reported in ORNs: 1) it activates a Ca²⁺-dependent Cl conductance, 2) it activates a Ca²⁺-dependent nonspecific cation conductance, 3) it activates a Ca²⁺-dependent K⁺ conductance, or 4) it activates, in the same cell, both a Ca²⁺-dependent nonspecific cation conductance and a Ca²⁺-dependent K⁺ conductance, the overall effect being a depolarization.

	VIII. GASEOUS MESSENGERS IN OLFACTORY NEURONS (CARBON MONOXIDE AND NITRIC OXIDE)

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Nitric oxide and carbon monoxide (CO) are recognized as gaseous second messengers that can act as local neurotransmitters because of their ability to diffuse across cell membranes and stimulate the soluble form of guanylate cyclase (36, 103, 350). A role for gaseous second messengers in signal transduction in the peripheral olfactory system was first proposed by Breer and Shepherd (41) based on experiments with isolated rat olfactory cilia, demonstrating that odorants stimulate slow increases in cGMP formation that are abolished by addition of inhibitors of NO synthase, the enzyme that catalyzes the formation of NO from L-arginine (39). These authors speculated that the increase in cGMP could activate the cyclic nucleotide-gated channel, which is slightly more sensitive to cGMP than cAMP (Table 2). Subsequent experiments by Lischka and Schild (205) in Xenopus ORNs showed that NO induced an inward current at negative holding potentials with the ionic characteristics of the cAMP-gated conductance. The development of this current was dependent on the presence of GTP in the pipette, suggesting that the inward current induced by NO was mediated through an elevation in cGMP concentration. In addition, a direct stimulatory effect of NO on cyclic nucleotide-gated channels has been shown in tiger salamander olfactory neurons (42). Nitric oxide causes a chemical modification at a cysteine residue in the region of the cAMP-gated channel linking the S6 transmembrane domain to the ligand-binding domain. This chemical modification favors the open-channel conformation. Nitric oxide has also been shown to elicit opening of the second subunit of the cAMP-gated channel (g_cn2) (44). These observations suggest that odorant-stimulated NO formation plays a role in olfactory transduction. However, immunohistochemical studies show that mature olfactory neurons do not express the neuronal form of NO synthase (70, 174, 299, 362). Possible explanations for the lack of expression of neuronal NO synthase in mature ORN may be expression of another form of NO synthase in the olfactory epithelium (70), or a role for NO in signal transduction at early stages in the development of the olfactory epithelium, with a switch to CO in later stages (193).

In contrast, heme oxigenase II, the enzyme catalyzing the degradation of heme to biliverdin and CO, has been localized to ORNs in adult rats (350). In addition, primary cultures of embryonic rat olfactory neurons produce CO, and changes in the amount of CO produced as a function of time in culture parallel changes in cGMP levels in the embryonic ORNs (129). Furthermore, addition of CO to freshly isolated tiger salamander (A. tigrinum) ORNs elicits opening of a conductance with the characteristics of the cyclic nucleotide-gated conductance (193). As in the case of NO (205), the effect of CO on this conductance is absolutely dependent on the presence of GTP in the patch pipette (193), suggesting that the effect of CO is mediated by cGMP.

Because the biochemically measured odor-induced increases in cGMP concentration in isolated olfactory cilia take place slowly, Breer and Shepherd (41) proposed a role for gaseous second messengers in olfactory adaptation. Indeed, Leinders-Zufall et al. (194) have shown that prolonged exposure to the membrane-permeable cGMP anaglog 8-bromo-cGMP elicits a decrease in the sensitivity of the response to odors in tiger salamander ORNs. The decrease in odorant sensitivity can also be elicited by prolonged exposure of the cells to CO. Furthermore, the effect of CO is absolutely dependent on the presence of extracellular Ca²⁺, suggesting that it is mediated by the changes in [Ca²⁺]_i elicited by opening of the cyclic nucleotide-gated (CNG) channels. On the basis of these experiments and the effect of inhibitors of the CO/cGMP pathway on olfactory adaptation, Zufall and Leinders-Zufall (375) suggest that CO/cGMP are involved in long-term adaptation in ORNs. Alternatively, [Ca²⁺]_i could be simply modulated by [Ca²⁺]_o . Interestingly, what on the surface appears to be an opposite effect has been suggested for exposure of ORNs to the other gaseous second messenger NO. In Xenopus ORNs, exposure to NO prevented washout of odor-induced currents measured under whole cell patch clamp (205). The apparent discrepancy in the effects of cGMP and gaseous second messengers on odor responses in salamander and Xenopus ORNs may be due to differences in the experimental protocols. A crucial parameter, which was different in the experiments made in the tiger salamander (193) and in Xenopus (205), was the GTP concentration. With 1 mM (salamander) or 3 mM (Xenopus) GTP, both NO released from sodium nitroprusside and CO activated the cyclic nucleotide-dependent conductance g_cn (193, 205). But are GTP concentrations between 1 and 3 mM physiological? Among the almost nonexistent reports on cytosolic GTP concentrations, Kleinecke et al. (168) determined the concentration of total GTP to be ~300 µM in hepatocytes. In yeast, the ratio of GTP concentration to ATP concentration is ~0.2 (266). With the assumption of ATP concentration in the range between 1 and 2 mM, the total GTP concentration would again be in the range of 300 µM. The free GTP concentration is presumably at least one order of magnitude lower. Lischka and Schild (205) applied 10 µM GTP through the patch pipette; under these approximately physiological conditions, NO did not activate g_cn . Unfortunately, it is not known whether CO activates g_cn using physiological GTP concentrations or in recordings under perforated patch conditions, nor is it established in detail whether low concentrations of cGMP (~100 nM), which do not activate g_cn , affect the activation of g_cn by cAMP. In Xenopus, low cGMP concentrations prevented washout of odorant responses (205) so that there appeared to be a silent effect of cGMP on g_cn channels, which became operative when the cell was stimulated. The Hill coefficients of the g_cn channels have usually been determined with respect to either cAMP or cGMP (Table 2), whereas the physiologically relevant case, in which cGMP and cAMP act cooperatively upon g_cn , has so far not been analyzed.

Interestingly, the soluble guanylyl cyclase, which is coexpressed with type 2 heme oxygenase in ORNs, is not only present in cilia, but also in somata and axons (129). These findings, together with a positive correlation of CO production and high endogenous cGMP concentrations in differentiating cultured ORNs of rat, suggest an involvement of CO/cGMP signaling in developmental processes of ORNs (129).

In summary, both NO and CO have been shown to play a role in ORNs. Although the neuronal form of the NO synthase could not be demonstrated in cilia from mature ORNs using immunohistochemistry, it has been shown that some odorants stimulate increases in cGMP that are abolished by the addition of NO synthase inhibitors. Furthermore, NO blocked the washout of odorant responses and directly activated g_cn channels. On the other hand, heme oxygenase II has been localized in cilia, somata, and axons of ORNs. Under certain conditions, CO elicited opening of g_cn channels and appeared to be involved in adaptation processes. It may also be involved in the signaling during developmental processes in ORNs.

	IX. IONIC CONCENTRATIONS IN MUCUS AND ION HOMEOSTASIS

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Ion concentrations in the mucus have been measured using spectrophotometry (45, 134) or ion-sensitive electrodes (58, 243, 244) (Table 4). The spectrophotometric methods yield total ion concentrations, whereas ion-sensitive electrodes measure concentrations of the free ions. This may explain why Joshi et al. (134) obtained 10.7 mM as the Ca²⁺ concentration of the mucus, whereas Minor et al. (243, 244) found values in the range between 1 and 2 mM (mean, 1.5 mM) in the same species (Rana temporaria). The ion concentrations in frog varied during the year, being highest in February (45). Quite surprising are the high values for K⁺ concentrations in the mucus ([K⁺]_muc). Whereas in frog two reports obtained 7.9 mM (243, 244) and 10.6 mM (134), Bronstein and Leont'ev (45) obtained a [K⁺]_muc of 69.6 mM. This difference might be explained by injured cells or cilia, which would easily increase [K⁺]_muc . In mammals, values as high as 88 mM have been measured (45, 243), suggesting a remarkably steep K⁺ gradient across the tight junctions. The high [K⁺]_muc implies equilibrium potentials for K⁺ that are less negative than the resting potentials. The high concentration of extracellular K⁺ is also typical for outer dendrites of insect ORNs (138).

What are the consequences for the transduction cascades? During odorant stimulation (using n-amyl acetate, geraniol, iso-eugenol, -pinene, o-xylene, and peppermint oil as stimuli, personal communication), Minor et al. (243, 244) obtained a decrease in [Ca²⁺]_muc of ~50 µM during odorant application, which is consistent with influx of Ca²⁺ as the first electrochemical step in the transduction cascade. A change in [Cl]_muc did not exceed 0.5% during stimulation (243, 244). Whereas Joshi et al. (134), using cineole as stimulus, reported an increase in [Na⁺]_muc during stimulation, Minor et al. (243, 244) recorded the time courses of an increase in [K⁺]_muc and a decrease in [Na⁺]_muc . As to the InsP₃/Ca²⁺ pathway, depolarization is brought about by the influx of cations (cf. Refs. 245, 290, 310, 311), possibly including K⁺ (see below). In this case, K⁺ would enter the cell at the mucosal side during stimulation, whereas K⁺ efflux occurs at the serosal side at rest and much more so during action potential repolarization. In addition, a Na⁺-K⁺-ATPase localized to the cilia and the dendritic knob seems to be involved in the equilibrium of K⁺ (157, 209).

As to the odorant-induced activation of Ca²⁺-dependent K⁺ channels (248, 249) that are localized in the transduction compartments and thought to be inhibitory, the flux through these channels depends directly on u_K . In the case of mammals, where the mucosal E_K clearly appears to be much less negative than the resting potential (Table 4), the activation of mucosal K⁺ channels would be excitatory. In amphibians, with [K⁺]_muc equal to 7.9 mM (243, 244) or 10.6 mM (134), u_K is approximately at the threshold of voltage-gated Na⁺ channels. Whether or not spikes are generated depends on the exact values of u_K and the threshold of the Na⁺ conductance. If, however, [K⁺]_muc equals 69.6 mM as measured by Bronstein and Leont'ev (45), then the Ca²⁺-dependent K⁺ channels would clearly depolarize the cells and induce spiking. Because of this uncertainty, this question needs further study.

Finally, high mucosal equilibrium potentials for K⁺ pose, although indirectly, another question: If u_K > u_m , and u_Cl > u_m as reported by Dubin and Dionne (76), Kurahashi and Yau (184), and Zhainazarov and Ache (361), which conductance could be responsible for odorant-mediated inhibition, given that u_Ca and u_Na are positive?

The precise ionic composition of the mucus, although still controversial, seems thus to be of crucial importance. In most patch-clamp experiments, it is completely neglected. The in vitro mucosa preparation developed by Frings and Lindemann (99) leaves the polarity of the olfactory neurons intact. It thus allows investigating the function of ORNs under nearly physiological conditions. Using this preparation, Frings et al. (97) were able to establish the modulatory effects of mucosal Ca²⁺, Mg²⁺, Na⁺, and K⁺ on forskolin-induced excitation and spiking. Whereas forskolin increased the basal spike rate, mucosal Ca²⁺ and Mg²⁺ as well as the application of L-cis-diltiazem lowered the spike rate. Interestingly, mucosal Ba²⁺ and 4-AP increased the spike rate, indicating the presence of apical K⁺ channels that render ORNs sensitive to mucosal K⁺. Lowering the spike rate by removing mucosal Na⁺ could be counteracted by adding Ba²⁺ or 4-AP. Taken together, Na⁺, K⁺, Mg²⁺, and Ca²⁺ all modulated the basal or the forskolin-induced activity of frog ORNs (97).

Little information is available regarding the ion homeostasis of Na⁺, K⁺, and Ca²⁺ in ORNs. A Ca²⁺-Mg²⁺-ATPase present in ORNs of salmon has been described by Lo et al. (211). This pump was shown to be thapsigargin-insensitive and particularly active in a cilia preparation with an unusually low dissociation constant of 20 nM, consistent with measured resting Ca²⁺ levels in fish ORNs (293). The function of this Ca²⁺-ATPase is presumably Ca²⁺ extrusion from the transduction compartments (211).

Kern et al. (157) have localized the Na⁺-K⁺-ATPase in the cilia, apical dendritic knob, and dendrite of gerbil and guinea pig ORNs, with the highest activity being localized to the cilia. In Atlantic salmon, Na⁺-K⁺-ATPase activity has also been localized to the olfactory cilia (209). At first glance, this is an unusual localization of a Na⁺-K⁺-ATPase, since the Na⁺-K⁺-ATPase in epithelial cells is typically expressed at the basolateral side (12, 202). However, typically, the apical K⁺ concentration of epithelia is low, an exception being the high K⁺ concentration of the endolymph of the inner ear, maintained by the stria vacularis (359), and the extracellular medium found in insect outer dendrite (138).

The 3Na⁺/1Ca²⁺ exchanger has been reported to be localized primarily in the dendrite of ORNs (136). It was suggested that this localization corresponds to an uncoupling of soma and transduction compartments so that action potential-related Ca²⁺ increase in the soma would not affect processes in the dendritic knob and the cilia, and vice versa, transduction-related Ca²⁺ increases in the distal compartments of the cell would not affect Ca²⁺-dependent processes in the soma (136).

Little is known about the role of sustentacular cells in the maintenance of ion homeostasis in the mucus, although from parallels with other epithelia, it is likely that these cells play an important role. Persaud et al. (272) found that basal ion transport across frog olfactory mucosa was inhibited by apically applied furosemide, but not by amiloride. They suggested that a furosemide-sensitive Na-Cl or Na-K-Cl cotransport system may exist in the apical membranes of sustentacular cells. In addition, these authors found that K⁺ pathways are available on the apical side of the mucosa. They speculated the existence of K⁺ channels on the apical membranes of the sustentacular and/or olfactory receptor cells. Further work is necessary to determine the precise role of sustentacular cells in mucosal ion homeostasis.

In summary, the ion concentrations in the mucus of the olfactory mucosa have as yet received relatively little attention, although they obviously may affect the responses to odorants crucially. The ion composition of the mucosa is similar to that of the extracellular space, the important exceptions being [Ca²⁺]_muc which seems to be slightly lower and [K⁺]_muc which, at least in some species, is markedly higher than in the extracellular fluid. The resulting elevated equilibrium potential of K⁺ would mean that Ca²⁺-dependent K⁺ currents would be depolarizing. The concentration gradients are maintained by the action of ion pumps and exchangers. A Ca²⁺-ATPase and the 3Na⁺-2K⁺-ATPase have been reported to be expressed in the transduction compartments of ORNs, whereas the 3Na⁺/1Ca²⁺ exchanger appears to be localized mainly on the dendritic compartment.

	X. MULTIPLE PATHWAYS: IMPLICATIONS FOR OLFACTORY CODING

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In view of the current controversy about the existence of multiple second messenger pathways (see sects. IVD and VIB4), it is instructive to consider the consequences of the existence of one or multiple second messenger pathways for odorant quality coding.

View larger version (36K): [in this window] [in a new window]   FIG. 9.   Working model of dual olfactory transduction pathways in olfactory receptor neurons of spiny lobster, Panulirus argus. Top: odorants activate either a cAMP-mediated cascade including receptors, G protein, and adenylyl cyclase, or an InsP3-mediated pathway, including receptors, G protein, and phospholipase C. In the former case, cAMP directly gates K+ channels leading to a hyperpolarization (bottom left), whereas InsP3 activates nonselective cation channels, thereby causing a depolarizing receptor potential (bottom right). [From Ache (2).]

Information on odorant quality, which is directly related to the chemical structure of the odorants, is conveyed to the olfactory bulb by the ORNs through synaptic connections with mitral/tufted and periglomerular cells (84). In the simplest scheme, individual ORNs could express a single olfactory receptor per cell which, when occupied by an appropriate odorant, would stimulate a single second messenger pathway, thereby eliciting a generator potential that would increase the neuron's firing rate. Such a model has been suggested on the basis of in situ hybridization studies that show that each olfactory neuron expresses one or a few putative olfactory receptors that project to one or a few glomeruli (191, 258, 261, 285, 286, 348, 349) and electrophysiological and molecular biological studies that favor the role of cAMP as the sole second messenger in olfactory transduction (50, 256, 257). In this model, the second messenger system could be viewed as a black box that would simply serve the role of faithfully transducing information on receptor occupancy into an increase in the frequency of action potential firing. In this case, the only relevance of the study of the transduction mechanisms in the olfactory receptor cell for odor quality coding would be the exact determination of the function relating receptor occupancy to the output of the cell. Other details of peripheral olfactory transduction in the ORN would be irrelevant to quality coding.

In the case of multiple second messenger pathways, peripheral transduction can still be considered a black box if each olfactory neuron expresses only one pathway. In this case, the only change is that there will be a number of black box types (one for each pathway). Otherwise, the implications for olfactory coding remain the same as for a single second messenger pathway.

The situation is quite different if two olfactory receptors are expressed in a single ORN as suggested by electrophysiological experiments (27, 75, 142, 146, 245, 249, 291) and as shown elegantly by genetic manipulations in C. elegans (21, 320, 321, 341), especially if the two second messenger pathways are coexpressed in a single cell as demonstrated or suggested by various electrophysiological studies (27, 75, 142, 146, 245, 249, 291). If stimulation of second messenger formation in olfactory neurons could lead to depolarization or hyperpolarization depending on which second messenger-regulated conductances are activated in a given neuron (27, 105, 108, 109, 142, 249), or if the two pathways interact with each other, then interactions among second messenger pathways in the periphery could contribute to coding of odor quality information. Figure 9 depicts a working model of a dual olfactory transduction pathway in the lobster, where fish food led to a depolarizing response via activation of a cation conductance; the depolarizing response was, however, short-circuited by an odorant-activated K⁺ conductance (2, 240a). Figure 10 gives an example of a catfish ORN that clearly responds differentially to different stimuli (142). As suggested by Ache (2) and Dionne and Dubin (74), the ability of the primary receptor neurons in the olfactory system to respond with excitation or inhibition would enhance the ability of the olfactory system to discriminate between odorants, because a complex odor could be represented as more than simply the sum of the components. Cells that respond to one molecular determinant with suppression and to another with excitation may play a role in enhancing contrast between odorants that share partial structural homology.

View larger version (54K): [in this window] [in a new window]   FIG. 10.   Representative responses of single olfactory receptor neurons of channel catfish, Ictalurus punctatus, to 6 stimuli and to a water control. A: no significant change from spontaneous activity to water control. B: suppressive response to 104 M methionine (Met). C: suppressive response to 104 M alanine (Ala). D: excitatory response to 104 M arginine (Arg). E: excitatory response to 103 M glutamic acid (Glu). F: excitatory response to 3 × 104 M MBS (sodium salts of cholic acid, taurocholic acid, and taurolithocholic acid each at 104 M). G: excitatory response to 104 M ATP. Vertical dotted line indicates beginning of neural responses as defined by onset of simultaneously recorded electrolfactogram response. Both excitatory and suppressive responses recovered to spontaneous level of activity. [Modified from Kang and Caprio (142).]

Differential stimulation or suppression of olfactory neurons by odors could be used by the olfactory system as a mechanism for contrast enhancement. In addition, the responses resulting from simultaneous stimulation and inhibition of different neurons by one odorant could be contrasted in the olfactory bulb in such a way that low odorant concentrations could be detected at signal levels that could not be resolved from noise in a purely excitatory system.

In summary, although it has not yet been shown conclusively that peripheral olfactory transduction plays a role in quality coding in the olfactory system, it is likely that second messenger pathways do not simply play the role of a black box as far as quality coding is concerned. Multiple olfactory transduction pathways could lead to increased odorant discriminability and detectability.

	XI. SUMMARY AND PERSPECTIVES

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Our understanding of the molecular mechanisms of olfactory transduction has advanced greatly during the last decade because of the application of modern techniques of molecular biology, electrophysiology, and biophysics to the study of ORNs. First proposed to be the second messenger mediating olfactory transduction on the basis of biochemical studies, cAMP was shown to directly gate nonspecific cation channels. The evidence supporting a mediatory role for these cAMP-gated channels in olfactory transduction is conclusive. In addition, the influx of Ca²⁺ through cAMP-gated channels elicits an increase in [Ca²⁺]_i leading to opening of Ca²⁺-activated Cl channels. Although the precise nature of the regulation of intracellular Cl in olfactory neurons must be addressed in future studies, it is clear that the Ca²⁺-activated Cl channels participate in the transduction cascade by amplifying the response.

However, studies carried out throughout the last decade also indicated that cAMP is not the only second messenger involved in olfactory transduction and confirmed early reports of inhibitory odorant responses. In addition, there is a considerable amount of evidence indicating that InsP₃ plays a role in olfactory transduction. Although the precise nature of the involvement of InsP₃ in olfactory transduction remains controversial, the discovery of plasma membrane ion channels activated by InsP₃ suggests that this second messenger may mediate olfactory responses for some odorants. Future work must determine whether InsP₃ is indeed a second messenger mediating responsivity to odors, or whether it plays a modulatory role. In particular, detailed work by different laboratories using the same animal model systems must determine whether apparently discrepant observations on this alternate second messenger pathway are because of species differences or differences in experimental conditions.

The finding that different odorants can cause either excitation or suppression of action potential firing in the same olfactory neuron is exciting because it opens the possibility that peripheral olfactory transduction plays a role in odor quality coding. Future studies must seek to conclusively determine the molecular events underlying odorant-induced suppression of action potential firing. The role, if any, of peripheral olfactory transduction in odor quality coding must be qualified.

Experiments carried out in the last decade have established a role for Ca²⁺ as a "third messenger" in olfactory transduction. The influx of Ca²⁺ elicits amplification of the odor-induced current through opening of Ca²⁺-activated Cl channels and participates in adaptation during sustained stimulation. Both the cAMP-gated channel and the two InsP₃-regulated conductances are permeable for Ca²⁺, and opening of these channels causes a spatially restricted increase in intracellular Ca²⁺. This increase in [Ca²⁺]_i elicits opening of Cl or nonspecific cation channels amplifying the response to odorants and may elicit opening of Ca²⁺-activated K⁺ channels. In addition, other actions have been described for the increase in [Ca²⁺]_i , including inhibition of the activity of the cAMP-gated channel through calmodulin, modulation of InsP₃-regulated conductances, regulation of the actions of CO and NO, and modulation of enzymatic activity. Consistent with the complex action of [Ca²⁺]_i , it has been determined that [Ca²⁺]_i is tightly regulated in a compartmentalized manner within ORNs. The understanding of Ca²⁺ homeostasis and of the regulatory action of [Ca²⁺]_i is incomplete, and future experiments must address this issue to obtain a better understanding of olfactory transduction.

Studies of olfactory transduction during the last decade have concentrated on the function of isolated ORNs, but little is known about the mechanisms of olfactory transduction in ORNs in situ. Studies at the cellular level with more intact in vitro systems in which the integrity of the neuroepithelium is conserved are needed to clarify the role of proposed intercellular communication through molecules such as CO and NO and of efferent control of the function of ORNs. In addition, because ion concentrations in the mucus affect the reversal potential of odorant-regulated channels in the cilia, and the presence of odorant binding proteins in the mucus may affect the interaction of odorants with receptors, determination of the mechanisms for ion homeostasis and odorant delivery in the mucus overlaying the olfactory epithelium is crucial to the complete understanding of olfactory transduction.

In conclusion, a review of the literature on olfactory transduction in the last decade reveals unprecedented progress in the study of olfactory transduction. Previously intractable questions about signal transduction in ORNs have been answered. As a result, not only has a better understanding of olfactory transduction been obtained, but the study of olfactory transduction has provided new findings relevant for understanding the mechanisms of signal transduction in other types of neurons and sensory receptor cells. The olfactory receptor cell might even turn out to be the first neuron that can be completely characterized in terms of signal input, second messenger processing, and output generation. A survey of the literature also reveals the fact that many questions remain unanswered. It is likely that the next decade of research in olfactory transduction will be just as prolific as the last, resulting in a deeper understanding of intra- and intercellular interactions within and among ORNs.

	ACKNOWLEDGEMENTS

  We thank Drs. G. Lowe and H. P. Zippel as well as anonymous reviewers for critical reading of the manuscript and F. I. Vespolina for suggesting to include the section on ion homeostasis.

	FOOTNOTES

   This study was supported by National Institutes of Health Grants DC-00566 and DC-00214 and the Human Frontier Science Program Grant RG112 (to D. Restrepo) and by Deutsche Forschungsgemeinschaft Grants SFB236/A19 and SCHI 260/4-1 (to D. Schild).

¹   Whenever odorant-induced responses are discussed, the molecular nature of the odorant used is stated. This is important because, as discussed in section VIA, individual odorants might stimulate different second messenger pathways leading to activation of different conductances.

²   The odorants used in this study were cineole, amyl acetate, and isobutylmethoxypyrazine at a concentration of 0.5 µM.

³   Because there is no consistent nomenclature for the two subunits of the olfactory cAMP-gated channel, we have chosen to use nomenclature consistent with the rest of the review for the two subunits. We use g_cn1 for the first subunit (72, 111, 218) and g_cn2 for the second subunit (35, 199). In different manuscripts, g_cn1 is labeled either rOCNC1 or , whereas g_cn2 is refered to as rOCNC2 or  (cf. Refs. 35, 44, 199).

⁴   The term isolated olfactory cilia is somewhat misleading in the sense that this preparation contains not only cilia from olfactory receptor neurons but also microvilli from sustentacular cells and, depending of the care taken during the dissection, cilia from respiratory cells (11, 34, 57). This fact must be taken into account in evaluating biochemical and electrophysiological experiments utilizing isolated olfactory cilia. Furthermore, it has to be kept in mind that isolated cilia are usually obtained by a "Ca²⁺ shock," i.e., a sudden increase of the extracellular Ca²⁺ concentration, whose effects on olfactory transduction remain to be studied in detail.

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