Olfaction in Insects the Primary Processes of Odor Recognition and Coding

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Review article e-Neuroforum 2011 · 2:49–60 Silke Sachse1 · Jürgen Krieger2 DOI 10.1007/s13295-011-0020-7 1 Abt. Evolutionäre Neuroethologie, Max Planck Institut für Chemische Ökologie, Jena © Springer-Verlag 2011 2 Universität Hohenheim, Institut für Physiologie (230), Stuttgart Olfaction in insects The primary processes of odor recognition and coding

Introduction approximately 500,000 abdominal glands ent article gives a short overview of our of female chinese silkworms (Bombyx mo- current knowledge of the peripheral and We all remember spending warm sum- ri) [7]. At around the same time, detailed primary brain processes of odor percep- mer evenings outdoors and finding that research into the molecular and cellular tion. In particular, we discuss the molecu- one’s own skin is“irresistible” to mos- basis of the recognition and processing of lar elements and mechanisms responsible quitos. And we all know the apparently olfactory stimuli in insects was initiated. for the recognition of olfactory informa- magical attraction over-ripe fruit in the Still today, silkworms and other moths are tion in the antennae and present the cur- kitchen holds for fruit flies. The cause preferred objects of research due to their rently discussed models for the transduc- of these“animal attacks” lies in odorants highly sensitive and species-specific pher- tion of a chemical signal into an electrical emitted by our bodies or by fruit which in- omone systems, their characteristic pher- response in the olfactory sensory neurons dicate a potential blood donor or offer the omone-induced behavior, as well as the (OSNs). Furthermore, we will focus on prospect of food or a suitable place to lay often considerable dimensions of their an- the processing steps in the antennal lobe, eggs. The attractant chemical signals are tennae. The honey bee (Apis mellifera), in the equivalent to the vertebrate olfacto- detected from afar by the animals’ highly contrast, has established itself as a model ry bulb, paying particular attention to the sensitive sense of smell and trigger char- organism for the investigation of olfacto- function and interconnection of the neu- acteristic search behavior programs in the ry signal processing in the brain due to its ronal types involved and the significance brain, ultimately leading the animal to the marked ability to classify and learn odors. of the neuronal network for coding olfac- source of the odor. Moreover, over the past 2 decades the fruit tory signals. As these everyday examples illustrate, fly Drosophila melanogaster has become a olfaction plays a central role for most in- very important model organism in insect Structure of the peripheral sects in the registration of small, volatile olfactory research, which enables unique olfactory system in insects compounds in the environment. In this experimental approaches due to the sim- context, the ability to sensitively and spe- plicity of its olfactory system and, in par- In contrast to mammals, insects have a cifically recognize odors is often vital for ticular, due to its genetic manipulability. less complex olfactory system, which is their survival, since the chemical signals In addition, olfactory research is current- also made up of significantly fewer cells can provide essential information about ly focusing on insect species which repre- in the periphery. While there are sever- sources of food, predators, or conspecifics. sent severe food and plant pests or pose a al million OSNs in the mammalian nose, In terms of mate detection, and thus also threat to our health. Given the enormous only thousands to tens of thousands of ol- of survival of the species, the registration significance of olfactory information in factory receptor cells are found in the an- of intraspecies chemical signals (phero- the search for food and in host detection, tennae and maxillary palps of insects. De- mones) is of crucial importance. The exis- the insect sense of smell is under intensive spite its lower number of OSNs, the insect tence of species-specific substances emit- investigation for being a promising target olfactory system is by no means inferior to ted by female animals to attract their male for being the development of alternative the mammalian olfactory system in terms counterparts was first identified in 1879 by strategies to defend and fight against in- of its sensitivity to particular odorants, of- the Swiss natural scientist Auguste Farbre sect pests. ten even surpassing its mammalian coun- in his experiments with moths [14]. How- Over the last 20 years, ground-break- terparts. ever, it was not until the late 1950s that Al- ing advances in our understanding of in- In terms of their peripheral architec- fred Butenandt and his colleagues were sect olfaction have been made by combin- ture, the insect and mammalian olfactory able to successfully isolate 1 mg Bombykol, ing biochemical, molecular biological, and systems are surprisingly similar. In mam- the first sex pheromone identified, from cell-physiological approaches. The pres- mals, the sensory cilia of the OSNs are em-

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a b bedded in the aqueous nasal mucous cov- Ant ering the olfactory epithelium in the nasal cavity, whereas the axon of the olfactory neuron projects directly to the olfacto- Mp ry bulb in the brain. Similarly, the senso-

1 mm 0,1 mm ry dendrites of insect OSNs lie in cuticular hair-like structures filled with aqueous Fig. 1 c d lymph, the so called sensilla (. ); and also the OSN axons project directly–without any switching stations–to the Ant brain, in this case the antennal lobe (see . Fig. 4). A distinction is made between several olfactory hair types on the basis 1 mm 50 µm of their form and surface structure: slen- e der trichoid sensilla, cone-shaped basiconic sensilla, and pitted coeloconic sensilla can often be found. Cuticular pores through which odorant molecules can en- ter the interior of the sensillum, and thereby reach the sensory cell membrane, are 20 µm common to all olfactory hair types. Neu- rons responsive to“general” odorants are f found in all hair types. In contrast, phero- Odorant, mone-sensitive cells have only been found pheromone in trichoid sensilla to date. In adaptation Cuticle to highly sensitive pheromone detection, their dendrites, and thus also the corre-

Lymph Binding sponding pheromone hairs, are often ex- protein tremely long (. Fig. 1), greatly enlarg- ing the antenna’s total receptive surface. Furthermore, sensory hairs involved in Supporting cell the detection of special odorants (pher- Neuron Receptor omones, host odorants) are often very abundant and generally contain between one and three OSNs. The number of reactive neurons in sensory hairs for“general” Fig. 1 8 Organization of the peripheral olfactory system in insects. ADro- odorants, however, can go up to 30 and sophila melanogaster (fruit fly). B Head of the fruit fly with olfactory append- more cells. Irrespective of their number, ages: antennae (Ant) and maxillary palps (Mp). The olfactory organs are visualized by expression of a green fluorescent protein (GFP) in the olfactory the cell bodies of the OSNs are surround- cells. C Head of the moth Heliothis virescens with segmented antennae (Ant). ed by three supporting cells, whereby the D Scanning electron microscope image of two segments of a male H. vire- innermost, thecogen supporting cell cov- scens antenna. The surface is covered with hundreds of both long and short ers the neuron, much like a glial cell. This sensilla hairs. Pheromone-reactive neurons are found predominantly in the is followed by a tormogen and a tricho- long trichoid sensilla (reproduced with kind permission from [20]). E Im- munohistochemical visualization of HR13 expression, the receptor for the gen supporting cell, both of which are in- main component of the female sex pheromone blend of H. virescens in sen- volved in maintaining the composition of sory neurons of the long trichoid sensilla. Fluorescence-labeling on a tissue the sensilla lymph. section of the male antenna. F Structure of a sensilla hair. Olfactory sensory neurons (OSNs) are surrounded by supporting cells. The OSN dendrites proj- Odorant- and pheromone- ect into the aqueous sensillum lymph. Airborne odorant molecules (odor- binding proteins ant, pheromone) are dissolved by binding proteins in the lymph and trans- ported to the dendritic membrane. [Images A and B courtesy of Veit Grabe (MPI, Jena)] In insects, odorant detection begins when signal molecules penetrate the cuticular pores in the sensilla hair. Since odorants, and in particular pheromones, are usually highly hydrophobic molecules, their solu- bility in the aqueous sensilla lymph is ex-

50 | e-Neuroforum 3 · 2011 Summary · Zusammenfassung tremely low and their transfer via hydro- sophila mutants provide evidence that an e-Neuroforum 2011 · 2:49–60 philic fluid to specific receptor proteins in interplay between the pheromone recep- DOI 10.1007/s13295-011-0020-7 © Springer-Verlag 2011 the OSN membrane is not straightforward. tor Or67d and the PBP Lush enables rec- Nature has apparently solved this prob- ognition of the pheromone 11-cis-vaccenyl Silke Sachse · Jürgen Krieger lem by coming up with odorant-binding acetate [23, 62]. Olfaction in insects. The proteins (OBPs) and special pheromone- Comparative investigations into the primary processes of odor binding proteins (PBPs). These small structure of pheromone-free and phero- recognition and coding globular proteins (12–16 kDa) are synthe- mone-laden PBPs also indicate that bind- sized by the tormogen and trichogen sup- ing of the“correct” ligand induces a specif- Summary Odorants provide insects with crucial infor- porting cells and secreted in the sensilla ic conformational change [36, 38]. How- mation about their environment and trigger lymph, where they are found at extreme- ever, it remains unclear whether the com- various insect behaviors. A remarkably sen- ly high concentration (10–20 mM). Struc- plex formed by the ligand and the binding sitive and selective sense of smell allows the turally, binding proteins are characterized protein then directly activates the recep- animals to detect extremely low amounts of by six highly conserved cysteines in their tor, or whether the pheromone needs to relevant odorants and thereby recognize, e.g., amino acid sequence. These form three be released from the PBP first via a further food, conspecifics, and predators. In recent years, significant progress has been made to- disulphide bridges, thereby stabilizing a conformational change in order to then wards understanding the molecular elements predominantly α-helical protein structure activate the receptor alone. There are cur- and cellular mechanisms of odorant detec- [48]. Binding studies as well as nuclear rently supporting findings for both possi- tion in the antenna and the principles under- magnetic resonance (NMR) and crystal- ble mechanisms: while in Drosophila the lying the primary processing of olfactory sig- lographic structure analyses suggest that PBP Lush can activate the corresponding nals in the brain. These findings show that olfactory hairs on the antenna are specifically OBPs and PBPs are able to transport lipo- sensory neuron in the pheromone-bound equipped with chemosensory detector units. philic olfactory molecules after rendering conformation [36], a release of the pher- They contain several binding proteins, which them practically water-soluble by nestling omone preceding receptor activation is transfer odorants to specific receptors resid- them in hydrophobic binding pockets. In supported by studies showing a pH val- ing in the dendritic membrane of olfacto- this way, they likely protect the olfactory ue-dependent conformational change of ry sensory neurons (OSN). Binding of odor- molecules from decomposition or modi- moth PBPs. The latter may be effected ant to the receptor initiates ionotropic and/ or metabotropic mechanisms, translating fication by degrading or transforming en- by a low pH value near the plasma mem- the chemical signal into potential changes, zymes in the sensilla lymph. brane [61]. Binding of the free pheromone which alter the spontaneous action poten- Biochemical analyses of the sensilla to the receptor is also supported by func- tial frequency in the axon of the sensory neu- lymph as well as investigations of the ge- tional studies of pheromone and odorant rons. The odor-dependent action potentials nome and the genes expressed in the an- receptors expressed in cell cultures and in propagate from the antennae along the axon tenna have shown a multitude of OBP oocytes of the clawed frog Xenopus laevis to the brain leading to an input signal within the antennal lobe. In the antennal lobe, the types and often several PBP types in all where receptor activation even in the ab- first relay station for olfactory information, species investigated to date [53, 64]. In sence of a binding protein could be seen the input signals are extensively processed by addition, different binding protein types [21, 37, 40]. a complex network of local interneurons be- were found at the same time in the lymph fore being relayed by projection neurons to of a single sensilla hair. This suggests that Sensory neuron membrane higher brain centers, where olfactory perception takes place. various binding proteins transport a va- proteins (SNMPs) riety of odorants, thereby contributing Keywords to the specificity of the olfactory system. The precise mechanism of the interac- Odorant receptors · Binding proteins · Indeed, binding studies on various OBP tion between pheromones and phero- Signal transduction · Antennal lobe · types have detected distinct yet partially mone/PBP complexes and OSNs has not Olfactory coding overlapping ligand spectra [43]. Moreover, been definitively elucidated. Recent stud- investigations into the moth and Drosoph- ies revealed additional and compelling ev- ila pheromone systems have shown that idence that the well-known sensory neu- PBPs are more strongly“tuned” to spe- ron membrane proteins (SNMPs) may al- cial ligands than OBPs, and that they in- so play an important role in pheromone teract in a specific way only with partic- recognition. The first SNMP was discov- ular pheromone components. Using re- ered back in the mid 1990s in the dendritic ceptor-expressing cell lines and purified membrane of pheromone-sensitive neu- binding proteins, it was demonstrated rons of the Antheraea polyphemus moth that there is an interplay between a par- [44]. SNMPs belong to the diverse CD36 ticular PBP and a distinct pheromone re- family, the members of which are all char- ceptor in the recognition of a species-spe- acterized by two transmembrane domains cific pheromone component [17, 22]. Cor- and a large extracellular binding domain. respondingly, functional studies on Dro- A common feature in the hitherto func-

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tionally characterized CD36 proteins is breaking work in 1991, for which they later family in some insect species (e.g., the fire that they recognize, bind, and transport received a Nobel prize in 2004, Linda Buck ant) has experienced specific expansion is hydrophobic molecules such as cholester- and Richard Axel found a giant family of presently unclear. It is assumed, however, in and fatty acids, as well as lipid–protein (meanwhile) more than a thousand iden- that the variety of OR types in some spe- complexes [51]. It is therefore assumed tified genes in the rodent genome, which cies reflect evolutionary adaption to cer- that SNMPs may represent docking sites encode G-protein coupled receptors (GP- tain ecological and physiological demands for PBP–pheromone complexes near CRs) with typically seven transmembrane in the search for food or the major impor- pheromone receptors (PRs), where they domain (7TMD) proteins, each of which tance of odorants in social communica- function as co-receptors that“catch” the is expressed in subpopulations of OSNs tion between insects living in colonies. pheromone and“pass it on” to the neigh- [6]. Subsequently, very large GPCR gene Functional analyses have meanwhile boring PR [53]. Indeed, recent studies on families that corresponded functionally to shown for most of the 62 Drosophila Drosophila mutants show that SNMP1 is those found by Buck and Axel were iden- ORs and many Anopheles receptors that crucial for the pheromone response of a tified in the nematode Caenorhabditis ele- these are indispensable for the response sensory neuron, and that the protein lies gans; in addition, GPCRs encoding puta- of OSNs to odorants and they represent within the membrane in direct proximi- tive pheromone receptors in the vomero- the molecular basis for the specificity of ty to the pheromone receptor (Or67d) [3]. nasal organ of rodents were described. the various OSN types populating the an- In addition, SNMP1 was also found in the Identifying insect ORs proved to be tenna, as detected in electrophysiological supporting cells surrounding the neuron very challenging. All early approaches studies [8, 25, 30, 57]. in Drosophila. Hence, it is assumed that based on the homology of OR sequences In terms of the bandwidth of their the protein in the supporting cell mem- in vertebrates and nematodes were unsuc- odorant spectra, insect and vertebrate brane fulfils a different function, possibly cessful. A breakthrough came, however, in ORs are similar. The protein sequences of playing a role in the elimination of hydro- 1999 following the availability of the Dro- insect and vertebrate ORs also share the phobic pheromones or pheromone–PBP sophila melanogaster genome sequence 7TMD typical to GPCRs. However, the complexes from the sensilla lymph. and the application of special search pro- commonalities end here. OR proteins in In the Heliothis virescens moth, in con- gram for proteins with 7TMD combined both animal groups display no sequence trast, exclusive expression in OSNs and with the extensive sequencing of differen- similarities and are not phylogenetically co-expression with the sex pheromone tially expressed antennal cDNAs [9, 54]. related. Furthermore, in vitro and in vi- receptor HR13 was found for the SNMP1 This led to the discovery of a diverse fam- vo structural analyses demonstrated a sur- type. Interestingly, however, a further ily of 7TMD receptors, whose members prising inverse membrane topology in in- SNMP2 subtype, which is only expressed are specifically expressed in OSNs in the sect receptor proteins: in contrast to ver- in the non-neuronal supporting cells, was antennae and maxillary palps, where they tebrate ORs, the N-terminal end in in- identified in H. virescens [16]. This cell are concentrated in the sensory dendrites sects is located intracellularly, the C-ter- type-specific expression of the two Helio- of the cells. minal end extracellularly [2]. In addition this SNMP subtypes suggests that the two Compared with the more than thou- to these fundamental differences in their proteins serve different functions and sand functional OR genes found in the ORs, insect OSNs differ from their verte- could indicate an advanced specialization genome of some vertebrates, Drosophila brate counterparts in a further significant in the moth pheromone system: while for with its 62 ORs has a much lower num- point: while vertebrate OSNs display on- SNMP1 in the dendritic membrane of the ber. Meanwhile, the genomes of other in- ly one OR subtype, current studies indi- OSN an interaction with pheromone/PBP sects have been sequenced and investi- cate that, in the majority of insect OSNs, complexes or pheromones is possible, gated for the presence of OR genes using a specific ligand-binding OR type forms a SNMP2 in the membrane of supporting bioinformatic methods. Relatively few OR heteromer with a second common co-re- cells could be involved in sensilla lymph genes were also found in the malaria mos- ceptor from the OR gene family [2]. Ac- clearance. quito Anopheles gambiae (79 ORs) and in cording to the most recent nomenclature, the silkworm Bombyx mori (48 ORs). In this protein is referred to as Orco (for- Odorant receptors contrast, however, significantly more have merly Or83b in Drosophila, OR2, or OR7 been identified in other insects: 163 OR in other insects). The Orco protein is un- Receptor proteins in the dendritic mem- candidates in the honeybee genome (Apis usual in many ways: Firstly, it is the only brane of olfactory cells are key elements in melifera), 225 ORs in the jewel wasp (Na- member of the OR family which is high- the molecular recognition and discrimi- sonia vitripennis), 265 ORs in the red flour ly conserved between insect species and nation of odorants. They interact in a spe- beetle (Tribolium castaneum), and recent- it possesses a remarkably large intracel- cific manner with the relevant odorants or ly more than 400 possible ORs in the fire lular domain. Secondly, it does not bind pheromones, and initiate the transduction ant (Solenopsis invicta). Thus, extremely any odorants itself and is necessary for the of the chemical signal into an electrical re- large numbers of different ORs are clear- transfer of specific OR types to the den- sponse of the OSN. ly not exclusive to the vertebrate olfactory dritic membrane. Finally, the Orco pro- Odorant receptors (ORs) were first dis- system, but rather can also be found in the tein forms a non-selective cation channel covered in vertebrates. In their ground- insect olfactory system. Why the OR gene which, according to recent studies, is in-

52 | e-Neuroforum 3 · 2011 volved in signal transduction (see below) a Basiconic sensilla, trichoid sensilla Odorants [2, 29, 31, 35]. ORx Orco C C Pheromone receptors PM Genes for special pheromone receptors N N (PR) in insects were first identified in the tobacco budworm (Heliothis virescens) [32]. As in many moth species, the female b Trichoid sensilla Pheromones insect releases a complex mixture of pheromones made up of a main component Orco PR and several secondary components in or- x C C der to attract males. This suggests that pheromone receptors are predominant- PM ly expressed on male antennae. By com- bining genome analyses using known OR N N sequences with“screening” of an antennal cDNA library, it was possible to identify sequences for several 7TMD receptors in c Coeloconic sensilla Amines, alcohols, ammonia Heliothis that are exclusively expressed in male antennae and which occur in sensory neurons of the pheromone-sensitive N olfactory hair. In functional studies em- N ploying receptor-expressing cell lines, it was demonstrated that the HR13 receptor IR S2 S1 S2 S1 type is activated by the main component x IRY of the female sex pheromone (Z)-11-hexa- decenal [22]. Subsequently, the bombykol receptor in the Chinese silkworm Bom- byx mori was successfully identified [47, PM 33]. Since then, PR sequences for a number of other moth species have been eluci- C C dated. Pheromone-receptors are similar in structure to odorant receptors for gener- Fig. 2 8 Structure, appearance, and ligands of various olfactory receptor al odorants (. Fig. 2) and are also found types in insects. A OSNs of basiconic sensilla and trichoid sensilla, which re- together with the Orco protein in olfacto- spond to general odorants each express a distinct odorant receptor (ORx) ry cells. Remarkably, PRs form a separate and the general olfactory co-receptor (Orco). Recent studies show that ORx and Orco form heteromers, whereby the OR subunit binds the ligand while group of more conserved proteins with- x Orco functions as an ion channel. B Pheromone-sensitive OSNs have hither- in the otherwise widely diverse moth OR to been found only in trichoid sensilla. They possess a specific pheromone family [32, 58]. This conservation of PR receptor type (PRx) and also express the Orco protein. ORs, PRs, and Orcos amino acid sequences in various species each have seven transmembrane domains. The N-terminal end of the poly- may be explained by the chemical similar- peptide chain is located intracellularly, the C-terminal end extracellularly. C Two types of ionotropic olfactory receptors (IR and IR ) are expressed in ity of their pheromone ligands or, alterna- x y OSNs of coeloconic sensilla, and likely form heteromers. Their basic structure tively, reflect the high negative evolution- resembles that of ionotropic glutamate receptors. Most IRs (like IRx), howev- ary selection pressure which is exerted on er, have a shorter N-terminal. Three transmembrane domains, a pore region, the highly specific pheromone recogni- and venus fly-like extracellular S1 and S2 ligand-binding domains are char- tion system, prohibiting certain mutations acteristic of all IRs. A–C Possible receptor ligand-binding sites are marked with a yellow circle. PM plasma membrane in the receptor protein for the purposes of successful reproduction.

Ionotropic receptors [10]. These proteins belong to a subfam- structure. However, they lack certain ami- ily of so called ionotropic receptors (IRs) no acids which interact with glutamate in A completely new class of putative olfac- and differ fundamentally in structure iGluRs. In addition, the corresponding li- tory receptors was recently discovered in from ORs and PRs (. Fig. 2). IRs are re- gand binding region of iGluRs in IRs, in Drosophila melanogaster antennae [4], lated to ionotropic glutamate receptors line with a possible function as a bind- as well as in a number of other insects (iGluRs) in terms of their sequence and ing site for various odorants, is extremely

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a b

OBP OBP Ca2+ Odorant Ca2+ Odorant Na+ K+ Na+ K +

Extracellular

OR Orco Plasma membrane ORx Orco x

AC Gs Intracellular Indirect Direct

ATP cAMP

c d PBP PBP Pheromone Pheromone Ca2+ Na+ K + Ca2+ Ca2+ Ca2+ Na+ K + Na+ K +

Extracellular PIP2 SNMP SNMP Plasma membrane CC CaC PR x Orco PR x Orco PLC β Gq Intracellular

Ca2+ DAG IP3

Fig. 3 8 Models of olfactory signal transduction in insects. A A specific odorant receptor (ORx) and the common co-receptor (Orco) form a ligand-activated receptor–ion channel complex in OSNs which respond to general odorants. An odorant molecule is transferred by an odorant-binding protein and binds to the ORx subunit, thereby causing the non-selective cation channel to open [49]. B According to Wicher et al., the variable ORx subunit is a G-protein coupled receptor, while Orco is a direct and cyclic nucleotide-activated ion channel [59]. In the presence of high concentrations of a general odorant, a direct ORx/Orco interaction causes the Orco channel to open. In the case of low odorant concentrations, the model proposes an indirect opening of the channel. Firstly, the receptor activates a G-protein (Gs), which leads to cAMP production via an adenylyl cyclase (AC). This second messenger in turn leads to the opening of the Orco channel. C Findings in pheromone-sensitive OSNs suggest the involvement of a sensory neuron membrane protein (SNMP) in signal transduction. The SNMP may serve as a docking site for the ligand-laden pheromone-binding protein and/or be involved in the release of the pheromone. The Orco channel opens following binding of the pheromone to the PRx subunit. Alternatively, it has been proposed that binding of the PBP–pheromone complex to SNMP reverses an inhibition of the pheromone receptor (PRx)–Orco complex, thus triggering an influx of cations into the cell [24]. D Several lines of evidence indicate a G-protein-mediated pathway in pheromone recognition [52]. Receptor activation could thus activate a phospholipase C (PLCβ) via a Gq-protein, which converts phosphoinositol- (4,5)-biphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 then opens a calcium-selective ion channel (CaC); the intracellular increase in calcium concentration subsequently activates other non-selective cation channels (CC)

variable. Current studies demonstrate the ants could be demonstrated. Interesting- Olfactory signal transduction expression of IRs particularly in OSNs of ly, between two and five IR types are ex- coeloconic sensilla which do not express pressed in a single OSN. As with the Or- Odorant-specific ORs, PRs, and IRs are other 7TMD-ORs, as well as a concentra- co protein in OR cells, there are also espe- the receptive proteins in the dendritic tion of the IR protein in the dendritic pro- cially conserved IR types which heterodi- membrane of OSNs. But how, after bind- cesses of the sensory neurons. By means merize with variable IR types [1]. ing of a suitable ligand, is a chemical odor- of“miss”-expression of IRs in a different ant signal transduced into an electrical cell OSN type, which usually does not possess response of the OSN that is first of all a these receptors, the direct involvement of change in the membrane potential? IRs in the recognition of distinct odor-

54 | e-Neuroforum 3 · 2011 a To date, functional studies on special OSNs of the coeloconic sensilla equipped with IRs have shown that IRs themselves form odorant-activated ion channels from combinations of between two and three different IR types. In addition to an IR type which determines ligand specificity, the heteromer contains one or two (often the same) IR types, which function as co- receptors [1]. In OSNs with IRs, it is assumed that the binding of odorants to the b receptor/ion channel complex, analogous to the IR-related ionotropic glutamate receptors, results in a cation influx into the MGC sensory neuron and depolarization of the Fig. 4 Organization of the antennal lobe in He- 7 Ordinary cell. liothis virescens. A Frontal view of the head with an- glomeruli However, the molecular processes in- tennae connected to the head capsule above the volved in olfactory signal transduction compound eyes. The white box denotes the area in which the left antennal lobe lies within the head. in those OSNs with 7TMD odorant or B Glomerular structures in the antennal lobe of a pheromone receptors, which are preva- male moth. These round neuropil structures are lent on the antennae, are largely unclear formed from the endings of OSNs and the projec- and the literature reports partially contro- tions of projection neurons and local neurons. In versial findings. More recent functional addition to the macroglomerular complex (MGC) (comprising four glomeruli) where incoming pher- c studies by two research groups who car- omone signals are processed, many“ordinary ried out electrophysiological investiga- glomeruli,” where“general” odorants are processed, tions on mammalian cells heterologous- are visible. To visualize glomerular structures, ly expressing combinations of a ligand- the antennal lobe has been fluorescence stained specific Drosophila OR and the Orco pro- with an antibody against a synaptic protein and an“optical section” was imaged with a confocal la- tein unanimously point to an odorant-ac- ser scanning microscope. C Three-dimensional re- tivated heteromeric receptor–ion chan- construction of the antennal lobe of a male moth, nel complex made up of one variable OR generated by using optical section series of the an- subunit and the conserved Orco protein tennal lobe seen in B. The MGC is marked in gray, [49, 59]. However, there is disagreement while the various“ordinary glomeruli” are marked in different colors. [Images courtesy of P.H. Olsen as to whether ORs are only subunits of li- (A), B.B. Løfaldli (B and C) and H. Mustaparta (all gand-activated OR/Orco channels which from the NTNU, Norway)] open upon odorant binding and directly cause OSN depolarization (ionotropic signal pathway). Alternatively, insect ORs are studies by Wicher et al. [59] demonstrate of odorants and would represent an inter- also G-protein-coupled receptors which, that odorant binding to the OR subunit al- esting analogy to the vertebrate olfacto- following their activation, trigger down- so activates a Gs protein and that, addi- ry system, where the binding of odorants stream intracellular reaction cascades and tionally, Orco is an ion channel which can to ligand-specific ORs, also via G-protein lead to the formation of second messen- be opened by cAMP, a classic second mes- activation (Golf) and an adenylyl cyclase, gers, which in turn indirectly open the Or- senger. This group’s model thus propos- leads to cAMP synthesis, which opens a co ion channel (metabotropic signal path- es the existence of dual (combined) iono- cAMP-gated cation channel. way). tropic and metabotropic signal transduc- With regard to the transduction of dis- The results obtained by Sato et al. gave tion (. Fig. 3B), according to which a di- tinct pheromone signals, investigations no support for any involvement of G-pro- rect OR–Orco interaction occurs in the on Drosophila mutants have shown that tein-activated reaction cascades, point- presence of high odorant concentrations, a specific pheromone receptor, the Or- ing to a purely“ionotropic signal path- hence a rapid opening of the Orco chan- co protein, and SNMP1 are essential for way” (. Fig. 3A). These findings were nel, while in the presence of low odor- the olfactory cell’s electrophysiological re- consistent with the view that insect ORs ant concentrations, the ligand-specific sponse. On the basis of the most recent could not be GPCRs, since they have no OR triggers a G-protein cascade, leading Drosophila data, an exclusively“ionotropic sequence similarities with vertebrate and to the formation of cAMP, which in turn signalling pathway” has been suggest- nematode ORs, or other classic GPCRs, opens the Orco channel. This slower G- ed, which includes involvement of the and are furthermore characterized by an protein-mediated amplification may be SNMP1 protein but rejects G-protein ac- inverse membrane topology. However, crucial for the highly sensitive recognition tivation. In contrast, biochemical, elec-

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Ethyl-3-hydroxybutyrate Ethyl benzoate

PN tion neurons Projec

AL s y neuron Sensor OSN

Fig. 5 8 Odorant representation in the input and output neurons of the Drosophila antennal lobe (AL). The calcium-sensitive protein G-CaMP was selectively expressed in olfactory sensory neurons (OSN, bottom) or projection neurons (PN, top). Cal- cium signals for two distinct odorants were superimposed onto morphological images of the antennal lobe. Red indicates a strong and yellow a medium increase in the intracellular calcium concentration (weaker calcium signals were cropped). Both odorants activate a specific combination of glomeruli. The patterns of the right and left antennal lobe are bilaterally symmetric. A comparison of the activity patterns between the two processing levels shows similar yet non-identical patterns

trophysiological, and genetic investiga- ant signals, but may be relevant for setting tory glomeruli (. Fig. 4), within which tions carried out in the last 20 years also and modulating the spontaneous activity OSN axons are synaptically interconnect- suggest the existence of a G-protein-medi- in OSNs [52]. ed to projection neurons and a network ated metabotropic mechanism in phero- Taken together, findings to date show of local interneurons. It has already been mone signal transduction: in this context, an inhomogeneous and complex picture shown in many insect species that the ax- studies on various moth species support of olfactory signal transduction in insect ons of OSNs expressing the same odorant a pheromone-activated IP3 signal cascade olfactory sensory cells. Further experi- receptor types converge in one glomeru- in pheromone-sensitive OSNs [39, 52]. ments in the future are needed to clarify to lus and that, in most cases, each glomeru- A corresponding model which attempts what extent ionotropic and metabotropic lus receives only the afferent input of one to combine experimental findings ob- mechanisms, either alone or in combina- receptor type [55]. It could also be dem- tained to date (. Fig. 3D) proposes that tion, mediate the transduction of odorant onstrated for some species that the num- the binding of a pheromone molecule to and pheromone stimuli. ber of glomeruli corresponds approxi- a specific pheromone receptor mediates mately 1:1 to the number of different odor- the activation of a G-protein and, sub- The olfactory pathway ant receptor types. In this way, the activi- sequently, a phospholipase C (PLC); this ty of an OSN population which responds enzyme catalyzes the hydrolysis of phos- As mentioned above, the interaction be- to certain odorants is concentrated in phatidyl-4,5-bisphosphate to yield inosi- tween an odorant molecule and a spe- one glomerulus. Thus, olfactory glomer- tol trisphosphate (IP3) and diacylglycerol cific odorant receptor is transduced into uli represent not only structural but also 2+ (DAG). IP3 then opens a calcium (Ca )- neuronal excitation of the OSN. This se- functional units of olfactory coding. The selective ion channel, which in turn“gates” quence of action potentials is transmit- number of glomeruli is genetically deter- further non-selective cation channels via ted via the antennal nerve to the first re- mined and specific to each species: the the calcium influx [52]. How the expres- lay station in the brain, the antennal lobe. fruit fly has approximately 50 glomeru- sion of the cAMP-activatable Orco chan- The antennal lobe represents the prima- li, the moth around 60, and the honeybee nel in pheromone-responsive OSNs is ry olfactory neuropil for the integration approximately 160. More than 400 glom- reconcilable with this signal transduction and coding of olfactory information and eruli have been found in some ant species. cascade remains as yet unclear. However, is structurally similar to the primary ol- Not only is the number of glomeruli ge- it has been suggested that Orco in pher- factory center in vertebrates, the olfacto- netically determined, but also their size omone-responsive neurons is not direct- ry bulb [26]. Both brain centers consist of and position in the antennal lobe, mak- ly involved in the transduction of odor- discrete round structures known as olfac- ing it possible to compile a digital, 3D atlas

56 | e-Neuroforum 3 · 2011 a b of the antennal lobe using morphological Projection neurons data [34, 5]. Regarding the processing of female sex pheromone signals, the males of some insect species, in particular many moth species such as the moth Heliothis eLN iLN virescens, exhibit an interesting feature: a comparison of male and female antennal lobes demonstrates specific glomeruli in the male which are located separately at the entrance of the antennal nerve and are significantly larger than other glomeruli (. Fig. 4B,C). This macroglomerular complex (MGC) receives and encodes the activity of pheromone-sensi- Sensory neurons tive OSNs. Various components of the sex pheromone mixture have been identi- Types of synapses fied and their coding could be attributed to distinct MGC glomeruli in many moth Chemical inhibitory species. In the fruit fly Drosophila, how- Chemical excitatory ever, only one substance, 11-cis-Vaccenyl- acetate, could be identified as a sex phero- Electrical mone to date. Moreover, the antennal lobe in the latter shows no MGC comparable Fig. 6 8 Connectivity model of the antennal lobe. A Morphological images of the three neuron types with that seen in any moth species. How- in the Drosophila antennal lobe, in each of which sensory neurons (bottom), inhibitory local interneu- ever, there is consensus that other phero- rons (middle), or projection neurons (top) are marked in green; the remaining neuropil is marked in red. mone components exist also for Drosoph- B A model of the hitherto identified synaptic connections in the antennal lobe between sensory neu- ila. Thus, pheromone detection is not nec- rons (green), excitatory and inhibitory local interneurons (blue; iLN, eLN), and projection neurons (red). essarily linked to the presence of an MGC. Sensory neurons form chemical excitatory synapses with projection neurons and both types of local interneurons. Inhibitory local interneurons inhibit projection neurons, sensory neurons (presynaptic The antennal lobe receives input from inhibition), as well as other glomeruli, via chemical inhibitory synapses. Excitatory local interneurons sensory neurons and accommodates a excite other glomeruli with chemical excitatory synapses. They also possess electrical synapses with complex neuronal network in which a which they are able to modulate projection neurons and inhibitory local interneurons vast receptor-neuron input is transferred to comparatively few output neurons, the projection neurons. The OSN termi- centers, such as the lateral protocerebrum neuronal activity at different sites in the nals and projection neuron terminals in and the mushroom bodies, where odor brain to be measured simultaneously. In each individual glomerulus are connect- perception takes place. this way, the spatial and temporal activity ed with a further neuron type in the an- of neurons involved in encoding an olfac- tennal lobe, the local interneurons. These The representation of tory impression can be visualized. Stud- limit their branches to the antennal lobe, odorants in the brain ies on honeybees have been able to show forming a multitude of connections be- that odorant stimulation evokes a repro- tween the various glomeruli. Both inhib- How are odorants represented and pro- ducible spatio-temporal activity pattern in itory (GABAergic) and excitatory (cho- cessed at the various processing levels in the antennal lobe [46]. All odorants acti- linergic) local interneurons have been de- the antennal lobe? Functional calcium vate a specific combination of glomeruli, scribed in Drosophila. To date, only inhib- imaging studies in honey bees have made whereby each glomerulus can be activat- itory local interneurons have been iden- an important contribution to our under- ed by several odorants. Thus, the olfacto- tified in other insect species, such as the standing of olfactory coding in the brain ry system has developed a combination- moth and honeybee. Local interneurons [18, 19, 28, 46]. This method is based on al strategy with which the enormous va- process and transform incoming olfactory staining the brain with a calcium-sensi- riety of odorants can be represented with information from the antennal OSNs and tive dye which changes its fluorescence in only a few coding units, the glomeruli. strongly modulate the outgoing signals a calcium-dependent manner. Calcium is These odorant-specific patterns are bilat- from individual glomeruli. This integrat- a very good indicator of neuronal activi- erally symmetric and conserved between ed olfactory information is received by ty since it is involved in a series of signal different individuals of the same species uni- and multiglomerular projection neu- cascades as a second messenger substance [19], i.e., odorant patterns are genetically rons, which innervate only one or several and also plays an important role in syn- determined. glomeruli. The projection neurons convey aptic transmission. By using a highly sen- In recent years, functional imaging the olfactory information to higher brain sitive camera, functional imaging enables has also been applied in the olfactory sys-

e-Neuroforum 3 · 2011 | 57 Review article

tem of the fruit fly Drosophila melanogas- corded showed that projection neuron re- nor on the basis of their partially contro- ter [15]. Various molecular genetic tech- sponses were more unspecific, i.e., broad- versial results. However, recent findings niques have been established in recent er, than those of OSNs which innervate already impressively show how the anten- years in the fruit fly, which have contrib- the same glomerulus [60]. Furthermore, nal lobe network extensively transforms uted to making it a genetic model organ- recent studies on Drosophila show that the input signal from sensory neurons via ism. With the help of the GAL-UAS sys- OSNs are also presynaptically inhibited by intra- and interglomerular interactions to tem, for example, specific gene products GABA, suggesting a“gain control“ mech- ensure reliable recognition, discrimina- can be ectopically expressed in subpop- anism which modulates neural transmis- tion, and perception of odors. ulations of individual neurons. This sys- sion from OSNs to the downstream pro- tem makes it possible for a calcium-sen- jection neurons [42]. Thus, it can be seen Corresponding address sitive protein, such as the GFP derivate in Drosophila that various glomeruli are Dr. Silke Sachse G-CaMP, to be selectively expressed in modulated differently in the network ac- Abt. Evolutionäre Neuroethologie, Max Planck Institut für Chemische Ökologie OSNs or projection neurons of the Dro- cording to the odorant [50]. Hans-Knöll-Straße 8, 07745 Jena sophila olfactory system (. Fig. 5). Thus, With regard to the synaptic connec- Germany it enables the representation of odorants at tions in a single glomerulus, basic stud- [email protected] various processing levels of the antennal ies have been carried out in cockroach- lobe to be visualized. Results to date show es [11, 12, 13]. Supported by findings from Silke Sachse studied biology at the Free University Ber- lin, gaining her PhD in 2002 in Prof. Dr. C. Giovanni Gal- that odorants can also evoke a reproduc- current research on other insects, it could izia’s research group. She subsequently worked from ible and combinatorial activity pattern in be shown that all neuron types in the an- 2002 to 2005 as a post-doctoral fellow at the Rockefell- the fruit fly, both in sensory and projec- tennal lobe are synaptically interconnect- er University in New York, in Prof. Dr. Leslie B. Vosshall’s . Fig. 5 . Fig. 6 laboratory. Following her post-doctoral period, she be- tion neurons ( ), highlighting the ed ( ): OSNs are cholinergic neu- came a principal investigator at the Max-Planck Insti- fact that combinatorial odorant coding is rons and transmit olfactory information tute for Chemical Ecology, Jena, in Prof. Dr. Bill. S. Hans- a general principle in the insect olfacto- to both projection neurons and local in- son’s department. Since 2008, she is leading an inde- ry system. terneurons. Moreover, OSNs receive the pendent junior research group supported by the BMBF. above-mentioned presynaptic inhibition PD Dr. Jürgen Krieger Processing in the antennal from inhibitory, i.e., GABAergic, local Universität Hohenheim, Institut für Physiologie (230) lobe network neurons. In turn, local interneurons are Garbenstr. 30, 70599 Stuttgart connected to OSNs, other local interneu- Germany Since the antennal lobe is a complex net- rons, and projection neurons. Ultimately, work of excitatory and inhibitory neuro- projection neurons are generally choliner- Jürgen Krieger studied biology at the University of Os- nal circuits, the question arises as to how gic, i.e., excitatory, and connected to both nabrück. He gained his PhD in 1991 at the Chair for Zoophysiology at the University of Hohenheim un- odorant representation of the input, i.e., other neuronal types. The only synaptic der Prof. Dr. Heinz Breer. Since ending his post-doctor- the sensory neurons, is transformed into connection that has not been found so far al period in 1995, he has held the post of lecturer and an output representation in the projection is a connection from projection neurons senior researcher at the Institute for Physiology at the University of Hohenheim. Since 2002, he has lead the neurons, which comprise only a fraction back to OSNs. research group on“Chemoreception in insects”. He re- of the number of neurons compared with Interestingly, it could be shown recent- ceived his professorship at the University of Hohen- the vast number of OSNs. When compar- ly that excitatory local neurons in Dro- heim in 2008. ing odorant representation in the senso- sophila excite projection neurons and in- Conflict of interest. The corresponding author states ry input with representation in the projec- hibitory local neurons via electrical syn- that there are no conflicts of interest. tion neurons (. Fig. 5), we see that the apses, leading to lateral excitation be- activity patterns are similar yet not iden- tween glomeruli in the antennal lobe [27, tical. Numerous studies have addressed 63] (. Fig. 6). These new findings ex- References the question of the input–output relation- plain, firstly, why odorant representation ship in the antennal lobe, reaching par- in the projection neurons of Drosophila is 1. 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Benton R, Vannice KS, Vosshall LB (2007) An essential role for a CD36-related receptor in pheromone cium signals from sensory neurons and In conclusion, a homogeneous mod- detection in Drosophila. Nature 450:289–293 output neurons in Drosophila are identi- el of how the antennal lobe processes the 4. Benton R, Vannice KS, Gomez-Diaz C, Vosshall LB cal [41, 56]. However, a further study in afferent input of OSNs cannot be devel- (2009) Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell Drosophila in which individual projec- oped as yet on the basis of previous stud- 136:149–162 tion neurons and individual OSNs on the ies, which used a variety of techniques, antennae were electrophysiologically re- methods of analysis, and animal models,

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