REVIEWS Olfactory signalling in vertebrates and insects: differences and commonalities U. Benjamin Kaupp Abstract | Vertebrates and insects have evolved complex repertoires of chemosensory receptors to detect and distinguish odours. With a few exceptions, vertebrate chemosensory receptors belong to the family of G protein-coupled receptors that initiate a cascade of cellu la r signa Hing events and thereby electrically excite the neuron. Insect receptors, which are structurally and genetically unrelated to vertebrate receptors, area complex of two distinct molecules that serves both as a receptor for the odorant and as an ion channel that is gated by binding of the odorant. Metabotropic signalling invertebrates provides a rich panoply of positive and negative regulation, whereas ionotropic signalling in insects enhances processing speed. Odorant A chemical compound that stimulates the sense of smell. For terrestrial animals, odorants are small, volatile molecules; for aquatic animals, odorants are water soluble. Pheromone A chemical substance that is used for communication between members of the same species ('conspecifics'). It is released by an individual and detected by a conspecific. G protein-coupled receptor G PCR). A member of a large family of membrane receptors that initiates a cellular response through G proteins. It threads through the cell membrane seven times, and the transmembrane segments adopt an a-helical secondary structure. Therefore, GPCRs are often referred to as heptahelical or 7-TM receptors. Center of Advanced European Studies and Research, Ludwig-Erhard-Allee 2, 531 75 Bonn, Germany, e-mail: u.b. kau p p ccčYxxx|xpcQcccc< Ptdlns(4,5)P2 DAG GDP O lns(l,4,5)P3 Na* Ca2*#y Pheromone Na* Ca2*0 TRPC2, \J CCCCCCCCCCQCCCCO OOOOOCOOOOOCOOOO •• ?---------------------► "OOOOO OOOOO A Na* Ca2*( In vertebrates, different anatom ical subsystems were thought tobe dedicated to the detection and processing of odorants and pheromones. However, in recent years this functional separation of subsystems has become blurred90: common odorants can be detected by the vomeronasal organ (VNO)146147 andpheromone-like molecules can be detected by the main olfactory epithelium (MOE)6'8148149. Furthermore, pheromone receptors are expressed in the MOE150, and signalling components of the MOE including olfactory receptors (ORs) are expressed in the VNO151152. Finally, human pheromone receptors, when heterologously expressed in a cell line that hosts components of the classical cyclic AMP signalling pathway, mediate responses to several volatile odorants153. The VNO hosts three kinds of pheromone receptors that belong to the G protein-coupled receptor (GPCR) family: two distinct vomeronasal receptors (VIR and V2R)9'10'11'154 and formyl peptide receptors (FPRs)12. VIR and V2R are expressed in non-overlapping apical and basal zones, respectively. The spatial organization of VI Rand V2R matches the expression pattern of the G proteins Ga and Ga (REFS 155,1 56), and axons from the apical VIR- and Gaj2-expressing neurons project to the anterior part of the accessory olfactory bulb (AOB), whereas the basal V2R-and Ga -expressing neurons project to the basal part of the AOB. Many substances excite VNO neurons at picomolarto nanomolar concentrations157. Small, volatile molecules activate VIR-positive neurons140. By contrast, V2R-positive neurons are activated by small peptides87141158-161. Recently, sulphated steroids, another class of non-volatile chemicals, have been shown to potently activate the vast majority of VNO neurons162. The binding of the pheromone to a VIR receptor successively activates G, a G protein that is often involved in inhibitory signaltransduction pathways; phospholipase Cß2 (PLCß2). which produces inositol-l,4,5-trisphoshate (lns(l,4,5)P3) and diacylglycerol (DAG) from phosphatidylinositol-4,5-bisphoshate (Ptdlns(4,5)P2); and finally the transient receptor potential cation channel C2 (TRPC2: see the figure, part a). Activation of TRPC2 mediates Na+ and Ca2+ influx, leading to a depolarization. Recovery and adaptation may involve binding of Ca2+-calmodulin (CaM)toTRPC2.The binding of pheromones to V2R receptors activates G , atrimeric G protein involved in diverse signaltransduction pathways. In some V2R-expressing neurons, TRPC2 maybe involved in generating depolarizing currents (see the figure, part b). However, because the signalling of major histocompatibility complex peptides in V2R-expressing neurons is intact in Trpc2~'~ m ice, other signalling mechanisms may exist. The gene family of FPRs has seven members in mice12. Similar to OR or VIR genes, FPR genes display monogenic transcription and are not co-expressed with other vomeronasal chemoreceptors. FPRs respond to structurally unrelated peptides or proteins associated with inflammation or disease and are broadly tuned; thus, these chemosensory receptors may be involved in the identification of unhealthy conspecifics12. sensitivity of sensory neurons is adjusted in a process called adaptation. Mechanisms that allow cells to recover from stimulation may also be involved in sensitivity regulation, making it difficult to experimentally dissect one from the other. ORNs display short- and long-term adaptation to brief or sustained odorant stimulation, respectively. Both modes of adaptation seem to be controlled by changes in [Ca2+]. (REE 64). Considering the central role of [Ca2+]. in feedforward and feedback mechanisms (FIG. 3), it is surprising that the precise site of Ca2+ action during adaptation remains to be identified. Response termination may occur at all stages of the OR signalling pathway (FIG. 3b). Proposed recovery mechanisms include receptor phosphorylation by protein kinase A (PKA) or G protein receptor kinase and subsequent 'capping' of the phosphorylated receptor by ß-arrestin6567, inhibition of ACIII activity by Ca2+-calmodulin (CaM)-dependent kinase IP8 and regulator of G protein signalling 2 (RGS2)69, hydrolysis of c AMP by phosphodiesterase activity, desensitization of the CNG channel by Ca2+-CaM-dependent processes70, and removal of Ca2+ by a Na+-Ca2+ exchanger71. The relative contribution of any one mechanism to recovery and adaptation is unknown. The lifetime of the ligand-receptor complex may be too short (< 1 ms72) for the complex to be phosphorylated by a receptor kinase and capped by ß-arrestin under standard conditions. However, such mechanisms may contribute to long-term desensitization during chronic stimulation67. ORNs express two phosphodiesterase isoforms: phosphodiesterase IC (PDE1C), which is selectively localized to the ciliary lumen and is stimulated by Ca2+-CaM73, and PDE4A, which is distributed throughout the cell but absent from the cilia74. Unexpectedly, the response recovery of mouse ORNs in which the Pdelc gene has been disrupted is unaltered75. Termination of the response is significantly delayed only in mice deficient in both PDE1C and PDE4A. It is therefore likely that, in the absence of degradation in the cilia, cAMP rapidly diffuses into the dendritic knob, where it is degraded by PDE4A. Results obtained by bypassing PDE activity using caged cAMP analogues or pharmacological tools suggest that PDE activity does not contribute to short-term adaptation76. Similarly, adaptation in Pdelc1' Pde4a~'~ double-knockout mice is intact75. In Pdelc~'~mice, however, adaptation is impaired. Unexpectedly, odorant sensitivity was also reduced in these mice, a paradoxical phenotype given that PDE negatively regulates transduction by removing cAMP. Perhaps other components of the signalling pathways, and thereby the balance between activating and inactivating signalling steps, are disturbed in these mice. The rate-limiting steps in response termination are the closing of the CNG channels and CaCCs. CNG channels are desensitized by Ca2+-CaM-mediated feedback inhibition, which lowers the cAMP sensitivity70. Although all three olfactory CNG channel subunits77 have CaM-binding sites7880, only a so-called TQ motif in the Bib subunit renders the channel sensitive to CaM80,81. CaM 192 | MARCH 2010 | VOLUME 11 www. natu re.com/reviews/neu ro © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS is pre-associated with the channel, allowing for rapid negative feedback80. However, adaptation was not impaired in ORNs expressing a CNG channel that lacks Ca2+-CaM regulation but is otherwise intact81. Molecule Topology OR Out PM N occcoaxox; Oligomeric state ococ ccoc Monomer IJxžpcco Out _>-------» g;dp ^--------' ctp Trimer ACIII CNGC Pseudodimer CaCC -C cNMP-binding site 'cAMP ^TI Tetramer cccocoxxxaxxxcc iiiifi hi CCCÖCOCÖCOCQCCOCQ NCX or NCKX Oligomer? PDE1C Ca2*-CaM binding domain Catalytic domain Dimer Ca2* Figure 2 I Molecules involved in mammalian olfactory signal transduction. The topology and oligomeric state of molecules involved in mammalian olfactory signal transduction are shown. These include olfactory receptors (ORs), the trimeric G protein Golf (composed of subunitsa, ß and y), adenylyl cyclase type III (ACIII), t he olfactory cyclic nucleotide-gated channel (CNGC; composed of one Bib, one A4 and two A2su bun its), a Ca2+- activated Cl~ channel (CaCC), Na+-Ca2+ exchangers (NCX); Na+-Ca2+-K+ exchangers (NCKX) and phosphodiesterase IC (PDE1C). CaM, calmodulin; cNMR cyclic nucleotide monophosphate; PM, plasma membrane. Finally, Ca2+ extrusion returns [Ca2+]. to the resting state and closes CaCCs71. As 90% of the receptor current is carried by CaCCs, this is probably the most important recovery mechanism. Ion exchangers use the inwardly directed electrochemical gradient of other ions to export Ca2+ from the cell. The NCX exchanger uses only the Na+ gradient, whereas the NCKX molecule uses both a Na+ and a K+ gradient for Ca2+ extrusion. At least three different NCX and three different NCKX molecules seem to be expressed in ORNs59,82, but electrophysiological recording from dendritic knobs provides no evidence for NCKX-mediated Ca2+ extrusion60. The olfactory marker protein (OME) may also control Ca2+ extrusion. Omp~'~ mice display significantly delayed Ca2+ clearance83 that could be due to the absence of a protein complex that consists of OMP, CaM and a Bex protein84. However, another study concluded that Ca2+ removal in cilia is not impaired by the absence of OMP85. Ca2+ extrusion by the (Ca2+)ATPases may be less important, because the pump efficiency of (Ca2+) ATPases is generally lower than that of NCX or NCKX exchangers84,86. Thus, vertebrate OR signalling is both positively and negatively regulated by a rich network of intricate mechanisms. TAARs TAARs were originally discovered in a search for the receptors of trace amines (such astyramine, ß-phenylethyl amine and octopamine) in the brain. Recently, TAARs were identified as chemosensory receptors that respond to amines8. Like ORs, TAARs are sparsely expressed in subregions of the MOE. Furthermore, TAAR-expressing neurons follow the one cell-one receptor rule and lack ORs. TAARs can increase cAMP levels in heterologous cells when stimulated with amine ligands, and TAAR-expressing neurons also express Gaolf.Therefore, TAARs probably use a c AMP-signalling pathway. Mouse TAARs specifically detect volatile amines found in urine — a rich source of social cues87,88 that control reproductive behaviour and fertility, as well as other physiological responses. The TAARs that have been functionally tested each respond to a unique set of amine ligands. TAARs are evolutionarily conserved from lower vertebrates to humans89, and they fall into three classes that are substantially expanded in fish89. Of these, class III TAARs do not have an aminergic ligand motif and probably respond to ligands other than amines. The olfactory system of insects Olfaction in insects also happens in olfactory subsystems90,91 (FIG. lb). The repertoire of chemosensory receptors in flies is smaller than that in mammals. Three different kinds of chemosensory receptors have been identified in D. melanogaster. ORs (for which there are 60 OR genes) that are unrelated to vertebrate ORs, gustatory receptors (GRs, for which there are 73 GR genes) and ionotropic 'glutamate' receptors (IRs, for which there are 61 IR genes) (FIG. 1; TABLE 1). With one exception, all ORs are localized to the basiconic and trichoid sensilla. The GRs are expressed in taste organs throughout the body, NATURE REVIEWS | NEUROSCIENCE VOLUME 11 | MARCH 2010 | 193 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS GTP | ATp GDP cAMP# A J Ja* Ca2*«-^ NCXorNCKX^ ©Ca^Na* K4 CNGCi .__. CaCC cAMP AMP Figure 3 | Signal transduction in mammalian olfactory receptor neurons, a | The odour-induced signal transduction pathway. The binding of an odorant to the olfactory receptor (OR) successively activates the trimeric, olfaction-specific G protein (Golf), adenylyl cyclase type III (AGII), the olfactory cyclic nucleotide-gated channel (CNGC; composed of one Bib, one A4 and two A2subunits) and a Ca2+-activated CI" channel (CaCC). Activation of bot h channel types finally leads to depolarization, b | Recovery and adaptation involves several Ca2+-dependent and Ca2+-independent pathways. Ca2+ controlsthe activity of t he CNGC, ACIII and phosphodiesterase ÍC(PDEIC). Moreover, export of Ca2+ by Na+-Ca2+exchange terminates signalling. OR activity seems to be terminated by several phosphorylation reactions and by the binding of ß-arrestintothe phosphorylated OR. Asterisks indicate the activated form of t he molecule. cN MP, cyclic nucleotide monophosphate; CaM, calmodulin; CaMKII, Ca2+-calmodulin-dependent kinase type II; GRK, G protein-coupled receptor kinase; NCX, Na+-Ca2+exchanger; NCKX, Na+-Ca2+-K+ exchanger; PM, plasma membrane; PKA, protein kinase A; RGS2, regulator of G protein signalling 2. and Gr21a and Gr63a are also expressed in C02-sensing basiconic sensilla. The IRs are primarily expressed in coe-loconic sensilla3. Insect ORs There were early hints that insect and vertebrate ORs are distinct from one another. Although vertebrate chemo-sensory receptors share some sequence similarity with other GPCRs, insect receptors do not. Unsurprisingly extensive cloning efforts based on sequence similarity failed to identify the elusive insect ORs. However, a bio-informatics approach that scanned the D. melanogaster genome for candidates with multiple transmembrane segments unveiled receptors with seven-transmembrane regions that were specifically expressed in olfactory organs92-94. The fly OR repertoire is considerably smaller than that of mammals, consisting of 62 ORs95. It quickly became clear that insect ORs are different from mammalian GPCRs. The insect receptors adopt a membrane topology that is the reverse of GPCRs9698. Moreover, most fly olfactory neurons express two distinct receptors: a universal co-receptor, Or83b, and one of the common ORs99. Co-expression of common insect ORs with Or83b or its orthologues in mammalian cell lines or Xenopus laevis oocytes greatly enhanced the cellular response to ligands compared with the expression of common ORs alone, suggesting that the two form a functional unit1,100. Indeed, oligomerization of receptors to form a functional pair may be a common theme in insects. For example, GR21 and GR63 form a C02 sensor (without Or83b). Given that OR or GR pairs form a single receptor, the one receptor-one neuron hypothesis also applies to insects, although there are notable exceptions101,102. Two recent papers showed that insect ORs are ionotropic receptors that are directly gated by odor-ants1,2. Although both studies agreed that fly ORs form heteromeric ligand-gated ion channels, the experimental findings and conclusions of the two studies were very different (FIG. 4). One study1 reported only a fast ionotropic response and found no evidence for the involvement of G proteins or intracellular messengers such as cAMP, cGMP or inositol-1,4,5-trisphoshate. By contrast, the other study2 suggested that common insect ORs activate the synthesis of cAMP through a G protein, and that this in turn activates Or83b, which serves as a cAMP-gated ion channel. The second paper concluded that the G protein-mediated pathway provides the amplification needed for low odorant concentrations, whereas at high concentrations the direct ionotropic pathway is activated. Controversial issues in this field are discussed below. Hotnotneric versus heteromeric expression. The Or83b receptor is the most conserved insect OR and is expressed in all but one type of sensory neuron. Or83b is not directly involved in odour recognition103. Rather, it associates with the common 'tuning' ORs in the early endomembrane sorting pathway and escorts the OR-Or83b complex to the cilia. Consistent with this function, in mutants that lack Or83b, dendritic localization of common insect ORs is abolished, along with cellular responses to many odorants99. Thus, Or83b may serve both as a chaperone that assists in receptor trafficking and targeting and as a cognate co-receptor of the tuning OR. However, some in vitro studies in heterologous cells104106 reported odorant-stimulated responses when a common insect OR was expressed alone. Similarly, an odorant-induce d rise of cAMP was detected in heterologous cells expressing Or22a, and cAMP-evoked currents were recorded only in cells expressing only Or83b2. It was therefore concluded that Or22a serves as a G protein-coupled odorant receptor and Or83b as a cAMP-gated ion channel. Notably, co-expression of Or22a and Or83b did not significantly enhance cAMP production or odorant-induced currents, suggesting that the respective function of each receptor is preserved in the homomer and — in principle — does not require a heteromeric complex. At present, it is unclear how these in vitro studies can be reconciled with the requirement for both a common OR and Or83b for OR signalling in insects99 and other heterologous expression systems1,100. 194 | MARCH 2010 | VOLUME 11 www. natu re.com/reviews/neu ro © 2010 Macmillan Publishers Limited. All rights reserved 05 REVIEWS cAMPC Slow, prolonged Figure 4 | Models of odorant signalling pathways in insects, a | One model1 suggeststhat the odorant receptor forms an ion channelthat isopened directly in response to the binding of odorants. The receptor consists of a common receptor (OrX) and a co-receptor (Or83b).This model does not specifythe location of the channel pore. The simplicity of the model, however, does not implythat there are no other feedback or modulatory mechanisms: they are just not known, b | An alternative model2 suggests that there are two pathways by which odour-induced depolarization can be generated. Upon odorant binding, activity is transferred to the Or83b subunit either by a direct (fast and short) or indirect (slowand prolonged) pathway. In the direct pathway, odorant binding directly opens a channel formed by t he Or83b subunit, generating a fast and short depolarization; in the indirect pathway, activation of a G protein (G )and an adenylyl cyclase (AC) leadsto cyclic AMP production. Upon binding of cAMPto Or83b, the channel opens and generates slow and prolonged depolarizing currents.The asterisks indicatethe active form of the molecule. PM, plasma membrane. Kinetics and waveform of the current response. When stimulated with brief puffs of odorants, insect ORs exhibited transient current responses with a simple waveform characterized by a short delay (<30 ms), a rapid rise and a slower decay to baseline1. The short delay together with the current fluctuations in excised inside-out patches suggested that insect ORs form a ligand-gated channel complex. By contrast, the odorant-induced current responses reported in a different study2 consisted of a small, rapid and transient response followed by a prolonged, larger component. The rapid and slow components were attributed to a direct ionotropic and a GPCR-based metabotropic mechanism, respectively. Odorants also evoked Ca2+ responses, suggesting that the ionotropic receptors are Ca2+ permeant. In both reports, the decline of currents evoked by brief odorant puffs is unexpectedly slow (up to 10 s): ligand-gated channels usually close instantaneously once the ligand has been removed (even the metabotropic response of vertebrate ORNs completely recovers in 1-2 s7lS). Perhaps the ionotropic mechanism of insect ORs is distinct from that of classical ionotropic receptors at neuronal synapses; that is, perhaps insect ORs stay active even after the ligand has been removed. Is Or83b a cAMP-gated ion channel? As described above, these two recent studies reached different conclusions, on the basis of different results, concerning the mode of action of insect ORs. Further work is required to resolve this issue. However, I would argue that, for several reasons, the proposal that Or83b is a CNG channel is less compelling. First, the odorant-induced rise of cAMP was detected by co-expression of either hyperpolarization-activated cation (HCN) or olfactory CNG channels that served as cAMP sensors. Under physiological conditions, CNG channel currents are highly outwardly rectifying, owing to Ca2+ blockage of more permeant Na+ ions63. By contrast, the odorant-induced CNGA2-mediated currents recorded in this study under presumably physiological conditions were not outwardly rectifying2. Second, the membrane-permeant analogues 8-Br-cAMP and 8-Br-cGMP stimulated currents in Or83b-expressing cells at extracellular concentrations of >10 nM. By contrast, classical mammalian CNG channels require at least 0.1-1 mM extracellular concentrations of these analogues for activation, and the most c AMP-sensitive mammalian CNG channel opens at micromolar concentrations of cAMP in excised-patch recordings63. Thus, the ligand sensitivity of the presumed Or83b channel must be much higher than that of classical CNG channels — probably in the picomolar range. In fact, novel CNG channels with 25-100 nM ligand sensitivity have recently been described107,108. Nevertheless, in my opinion, unequivocal demonstration of CNG channel activity requires cAMP-gated currents to be recorded in excised inside-out membrane patches, or the use of caged cAMP in the whole-cell configuration. Third, Or83b lacks known motifs for a pore region or a cyclic nucleotide-binding domain, although mutations in a putative pore motif in S6 changed the ion selectivity2. Moreover, high signal amplification by a second messenger is not required for sufficient sensitivity72-108 (BOX 1). Finally, dual activation of OrX-Or83b complexes by ligand and cAMP poses a host of conceptual difficulties. Activation would require both high-affinity ligand-binding sites that stimulate cAMP synthesis at low concentrations of odorant and low-affinity sites that activate the channel directly at high concentrations of odorant. Alternatively, odorants may initially act as partial agonists (see Supplementary information S2 (box)), and cAMP may fully open the channel. The physiological importance of a slow and sustained cAMP odour response in a rapidly moving fly is also unclear. On stimulation, D. melanogaster receptor neurons increase their action potential frequency within a few hundred milliseconds, and most responses NATURE REVIEWS | NEUROSCIENCE VOLUME 11 | MARCH 2010 | 195 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Odorant-binding protein A member of a diverse family of proteins that have been proposed to serve either as odorant scavengers or carriers that deliver the odorant or pheromone to the receptor cease within a few seconds109. Therefore, perhaps the native receptor and channel properties in vivo are different from the properties of the heterologously expressed complex. Open questions. The two studies raise interesting questions as to the mechanism of channel activation. Is the channel formed by one or by both subunits? Does Or83b co-determine the ligand affinity and selectivity of OrX? Initial experiments suggest that the ion selectivity, rectification and pharmacology of the channel depend on the subunit composition and that the respective members of the Or83b family from D. melanogaster, the silk moth Bombyx mori and the malaria mosquito Anopheles gambiae have different electrical properties1. What is the stoichio-metry between OrX and Or83b? How is the electrical response terminated? The receptor heteromers form non-selective channels that also pass a Ca2+ current. Does the Ca2+ influx control sensitivity and response termination as in vertebrate ORNs? Does the receptor, similar to many neuronal ionotropic receptors, desensitize in the presence of the ligand? Chemosensory properties of insect ORs The relatively small number of chemosensory receptors in insects compared with the number in vertebrates has allowed researchers to systematically study the chemical receptive range of individual sensilla and ORNs, providing some important insights. For example, ORNs are spontaneously active and can be either further activated or inhibited by ligands110,111. Similar to GPCRs, insect ORs may be partially active when no ligand is bound, and some odorants may act as inverse agonists (see Supplementary information S2 (box)) to suppress baseline activity; however, the antagonistic action of a specific ligand in the presence of other ligands has also been observed, suggesting that ligands can act as antagonists or as inverse agonists110,112. Whether a ligand activates or inhibits an ORN is dictated by the OR expressed113. This high baseline activity, which can be enhanced or suppressed, is recapitulated in heterologously expressed OrX and Or83b receptors1,2 and provides an additional dimension with which to encode odorants. There is no compelling evidence for a functional distinction of generalist versus specialist receptors in insects. At high ligand concentrations, the response profiles of most ORNs are smooth and broad. At low concentrations, the profiles sharpen considerably, and a given receptor responds to only one or two ligands112. Thus, the concentrations used may determine whether a receptor is classified as generalist or specialist in a continuum of specificity. Odorants can also elicit different temporal response patterns109,113, depending on both the odorant molecule and the receptor type. For example, the kinetics of response termination for a receptor varies between odorants. This may be due to the rate of dissociation of the odorant from the receptor102. Temporal coding of a stimulus also enhances the ability to recognize and discriminate odours114. Ionotropic glutamate receptors Recently, 61 members of a novel family of chemosensory receptors that are expressed in the dendrites of ORNs innervating coeloconic sensilla have been identified3. The receptors, designated IRs, are related to ionotropic glutamate receptors, although the two receptor families are divergent115 and IRs lackthe residues that are important for glutamate binding. Although the functional properties of IRs have not been studied in heterologous expression systems, their localization and structural features suggest that they are chemosensory receptors that may function as ligand-gated ion channels. The discovery of IRs strengthens the concept that insect and vertebrate olfaction are fundamentally different, in that insect odorant receptors function primarily as ionotropic receptors. Although insect ORs and IRs may both be ionotropic, their oligomeric structures are probably different. Up to five IRs and only two ORs can be co-expressed in an ORN, each probably forming a functional receptor. Two IRs (IR8a and IR25a) are ubiquitously expressed in coeloconic ORNs — a situation that is reminiscent of the co-receptor function of Or83b. If IRs represent channel subunits, their assembly into tetrameric or pentameric complexes would create an enormous combinatorial diversity of receptors. Furthermore, if all subunits in a complex bind to a ligand and are able to gate the channel pore, cooperativity among subunits may tune channel activity to a narrow range of odorant concentrations. Insect pheromone receptors The most well-understood D. melanogaster pheromone is cis-vaccenyl acetate (cVA). Insect pheromone receptors — unlike those of the mammalian nose — belong to the superfamily of ORs. A single OR (Or67d) is responsible for sensing cVA116,117. However, Or67d requires both Or83b and another membrane protein, sensory neuron membrane protein (SNMP), for proper function118,119 (see Supplementary information S3 (figure)). Although this receptor complex is activated at high cVA concentrations in vifro116,118,119, an odorant-binding protein (OBP) facilitates activation in vivo. One such OBP, LUSH, is formed in the lymph of a subset of triochoid ORNs, including cVA-sensitive ORNs120. Mutants that lack this OBP do not respond to cVA121. cVA is deeply buried inside LUSH, and it is the cVA-occupied LUSH that activates neurons122. LUSH is an inactive ligand, perhaps a weak partial agonist121 that is converted to a full agonist on cVA binding. Commonalities and differences: a summary As described above, vertebrates and insects use similar strategies to recognize and discriminate odours TABLE 2). Both have several large families of receptors to detect odorants, although the mammalian OR repertoire is considerably larger than that of insects. Moreover, the tuning of ORs (including some that are more broadly tuned and others that are more specific) and the action of odorants as agonists, antagonists or inverse agonists are also processes that are shared by mammals and insects. However, the high baseline 196 | MARCH 2010 | VOLUME 11 www. natu re.com/reviews/neu ro © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Table 2 | Commonalities and differences of olfactory receptors in vertebrates and insects Characteristic Vertebrates Insects Class GPCR Non-GPCR Repertoire Large, variable Smaller, constant Topology Heptahelical Inverse heptahelical Activation Metabotropic lonotropic Pseudogene fraction High None to low Stoichiometry Monomers Heteromers One receptor-one neuron rule Yes Yes* Gene selection Stochastic Deterministic Expression pattern Zonal and rand Dm Zonal and random Instructive role Yes Unknown Ectopic expression Yes Unknown Inhibitory action of odorants Rare Common Convergence of axonsto glomeruli Yes Yes Glomeruli perreceptortype Variable, <2 up to 20 ~1 GPCR, G protein-coupled receptor. *Thereare notable exceptions to this rule, which have been excluded from this table for clarity. receptor activity and inhibitory action of ligands seems to be a general feature of insect ORs, whereas it is an exception for mammalian ORs. In vertebrates, each neuron expresses only a single receptor gene. Insect ORNs express between two (ORs) and four (IRs) different receptors. However, if we assume that many insect ORs assemble into a unique receptor complex13, the one receptor-one neuron rule is also valid for insects in a functional sense. This rule forms the logical basis of the combinatorial strategy of odorant recognition. The expression of vertebrate ORs is organized in several overlapping zones that are continuously arranged along the dorsoventral axis of the MOE123125. The distribution of ORs in their respective zone has been described as random or stochastic. Similarly, in the D. melanogaster antenna, insect ORs segregate in different zones along the proximal-distal and dorsal-ventral axes94. Again, functionally identical sensilla are randomly distributed in each zone. The functional importance of the zonal organization is not precisely known. There is also overwhelming evidence that vertebrate ORNs expressing a given OR send their axons to one or two glomeruli in the medial and lateral halves of the olfactory bulb (OB)17,126. Stimulation results in a glomerular pattern of activity that is unique for each odorant, referred to as an odour map127. The equivalent of the OB in insects is the antennal lobe. The OB and the antennal lobe are organized in a surprisingly similar way128, underscoring the common principles that govern odour recognition in vertebrates and insects. Despite these commonalities, there are several differences (TABLE 2). Mammalian and insect ORs differ greatly in their sequences, share no common ancestors and adopt a different membrane topology. Moreover, the signalling mechanisms are entirely different: mammalian ORs are GPCRs, whereas insect ORs are ligand-gated ion channels. The ionotropic signalling mechanism is well suited to the tracking of rapid changes in odour concentration and quality by a rapidly flying insect. In insects, ORNs hosting the same OR gene target a single glomerulus, and the number of ORs is similar if not identical to the number of glomeruli. In mammals, the number of glomeruli is considerably larger (in humans there are around 400 ORs and 6,000 glomeruli129). In this respect, the mammalian system is more flexible. Both mammalian and insect ORNs must choose which OR gene to express from sizeable repertoires. The mammalian repertoires of functional ORs are large (300-1,300 ORs), whereas insect OR repertoires are much smaller (50-160 ORs). Both deterministic and stochastic models have been proposed to explain the choice of a receptor gene. In mammals, the choice of an OR is thought to follow a stochastic mechanism followed by a negative-feedback inhibition130,131. By contrast, in D. melanogaster, deterministic selection is accomplished by a molecular 'zip code' comprising three classes of regulatory elements that specify expression in the correct organ, activate OR genes in a subset of ORNs and restrict expression to a unique class of ORNs in that organ132,133. The reason why mammals and insects adopted different selection mechanisms is unclear. However, the larger OR repertoires in mammals, and consequently enhanced complexity, may have required a different selection procedure13. Although the mechanisms — adaptation to different environments and genomic drift due to gene duplication and deletion — underlying evolutionary changes in OR genes are similar in mammals and D. melanogaster", the result is different. The repertoire of OR genes in D. melanogaster, other insects and their ancestral species has been amazingly constant, whereas the repertoire of ORs varies extensively among different mammalian orders. NATURE REVIEWS | NEUROSCIENCE VOLUME 11 | MARCH 2010 | 197 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Finally, in mammals, ORs serve an instructive role that determines the projection of the ORN axon to a specific glomerulus126,134. Homophilic or heter-ophilic interactions between axons that involve ORs or OR-containing complexes cause axons to coalesce into a glomerulus135,136. Alternatively, the stimulation of cAMP synthesis by ORs may be involved in axonal sorting137. Such an instructive role of ORs has not been reported for insects. Future directions We have observed considerable advances in our understanding of how organisms register and distinguish molecules in the olfactory system. The complete set of the principal molecules in the canonical cAMP signalling pathway of vertebrates has been identified, and the cellular signalling events are known with reasonable precision. What is lacking is a complete quantitative model that takes into account the restrictions imposed by the biophysical and kinetic properties of each signalling component, particularly with regard to short- andlong-term adaptation. Moreover, we need a rigorous quantitative understanding of the molecular receptive range of receptors. Many technical issues limit our ability to generalize from and compare previous conclusions. Substantial advances will require atomic-resolution three-dimensional structures of receptors with different ligands bound. Advancing the concept of a 'conformational' space of a receptor will greatly help us to decipher the coding strategy on a more quantitative level by dissecting the contributions of each level of olfactory processing from the receptor to higher sensory areas in the brain. 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Cell 136, 23-25 (2009). Acknowledgements I thank H. Fried and A. Aho for preparation of the figures and H. Krause for preparing the manuscript. I am particularly grateful to O. Ernst, Humboldt-University, Berlin, Germany, for the GPCR structures in the Supplementary information. Because of space limitations, I was unable to cite all relevant primary literature. Competing interests statement The authors declare no competing financial interests. DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/gene Pdelc UniProtKB: http://www.uniprot.org ACIII | anoctamin 2 | ß-arrestin | CNCA2 | Cr21a | Cr63a | IR8a | IR25a | LUSH |OME| Orfild | Or83b |EDE1C | PDE4A |ELCJfi |RCS2|TRPC2| FURTHER INFORMATION U. Benjamin Kaup's homepage: http://www.caesar.de/rnolekulare-neurosensonik.ritml SUPPLEMENTARY INFORMATION See online article: SI (box) | S2 (box) | S3 (figure) ALL LINKS ARE ACTIVE IN THE ONLINE PDF 200 | MARCH 2010 | VOLUME 11 www. natu re.com/reviews/neu ro © 2010 Macmillan Publishers Limited. 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