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signaling in insect ORNs remains unclear and the role of G proteins in olfactory signaling is still enigmatic and is of the major focus of this thesis. Drosophila melanogaster is used as a model insect in this study due to the advantages like: accomplished genetic transformation techniques (e.g. UAS-GAL4 system), availability of complete genomic sequence, relatively simple and stereotypic organization of the olfactory system.
Olfactory organs and olfactory receptor neurons of Drosophila
Drosophila detects odors through two pairs of olfactory sensory organs on the head: antennae (3rd segment) and maxillary palp (Fig.1.1). Each antenna and maxillary palp contains about 1200 and 120 ORNs housed in a total of 410 and 60 olfactory sensilla, respectively (Shanbhag et al., 1999; Shanbhag et al., 2000; Stocker, 1994). The sensilla are of three morphological types: basiconic, coeloconic and trichoid sensilla. The most numerous are basiconic sensilla and are subdivided into large and small basiconic sensilla (distinguishable by light microscope)(Venkatesh and Singh, 1984). The sensilla house the dendrites of up to four ORNs. Basiconic, coeloconic and trichoid sensilla express ORs and gustatory receptors (GRs; detect also CO2), ORs and IRs, and ORs respectively. While ORs detect many volatile compounds, food odorants and pheromones (11-cis-vaccenyl acetate - cVA), IRs detect volatile amines, carboxylic acids and few food odorants (Su et al., 2009).
Drosophila genome encodes for 62 ORs (Clyne et al., 1999; Gao and Chess, 1999;
Hallem and Carlson, 2004; Vosshall et al., 1999; Vosshall et al., 2000). Each ORN expresses in general only one type of OR (together the co-expression of co-receptor – Orco; highly conserved across insect species). However, few ORNs express more than one OR, up to three ORs maximum. All ORNs, which express the same kind of OR, project to a distinct glomerulus (functional units of the antennal lobe (AL); ~43 glomeruli) in the AL (first neuropil in the brain that processes olfactory information, homologous to the olfactory bulb in vertebrates; Fig 1.2), suggesting that ORNs with different odor specificities show different spatial pattern or distributions (Couto et al., 2005; Fishilevich and Vosshall, 2005). The AL is the interaction site of ORNs, projection neurons (PNs; dendrites of PNs; about 150-200 PNs described (Stocker et al., 1997)) and local neurons (LNs; mostly inhibitory, intermingles with ORNs and PNs) (Fig 1.2).
ORN activity is not directly relayed on PNs, but modified by the interaction with the LN network (Shang et al., 2007; Wilson and Laurent, 2005; Wilson et al., 2004). Around 100 LNs branch across AL glomeruli, most of which innervate all or most of the glomeruli.
In contrast most PNs are uniglomerular (Marin et al., 2002; Wong et al., 2002). The PNs send their axons to the mushroom body (MBs) and lateral horn (LH) of the protocerebrum (higher centers for odor processing; Fig 1.2). Binding of an odorant to
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an OR (in the periphery) leads to a unique temporal and spatial pattern of activity in the brain (AL and MBs), which in turn leads to a behavioral response in flies.
Figure 1.1. Cartoon of Drosophila melanogaster head.
Dorsal view of a cutaway fly head showing the main elements of the olfactory pathway. Odors are sensed by olfactory receptor neurons in the antennae and maxillary palps. These neurons project axons along the antennal nerve to the antennal lobe glomeruli, where they are sorted according to chemosensitivity. From here the information is relayed by projection neurons in the inner and medial antennocerebral tract (iACT and mACT) to the mushroom body and to the lateral horn. Gustatory stimuli are sensed by gustatory receptor neurons in the labellum on the tip of the proboscis, the elongated fly mouthpiece.Adapted from (Keene and Waddell, 2007).
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Figure 1.2. Schematic of the adult fly olfactory system circuit.
Olfactory sensory neurons (OSNs) expressing the same receptor (same color) project their axons to the same glomerulus in the antennal lobe. There they probably synapse with at least three classes of interneuron: uniglomerular projection neurons (PNs, same color as directly pre-synaptic OSNs), multiglomerular inhibitory local neurons (iLNs, red) and multiglomerular excitatory local neurons (eLNs, green). PNs form synapses with Kenyon cells in the mushroom body calyx en route to terminating in the lateral horn. Some PNs bypass the mushroom body calyx and project only to the lateral horn. Adapted from (Keene and Waddell, 2007).
Chemosensory receptors
Like other gene families, OR families have expanded over time by gene duplication and divergence. Invertebrate olfaction is mediated by receptor families that are evolutionarily distinct from vertebrate receptors. The genome of the nematode worm Caenorhabditis elegans encodes more than 1,500 predicted GPCRs (Robertson and Thomas, 2006) and most of them are expressed in chemosensory neurons, primarily involved in olfaction and gustation (Troemel et al., 1995). C. elegans also uses non-GPCRs for chemosensation, as receptor-like guanylate cyclases may also function as nematode chemoreceptors (Gray et al., 2004; Yu et al., 1997). OR gene family is the largest gene family in the mammalian genome and is larger than any other gene family in any other species and comprises 1% of genes (Hoover, 2010). Mammalian genome encodes ~ 205-1200 functional OR genes (Niimura and Nei, 2007) and four types of chemosensory GPCRs are described : 1) odorant receptors, ORs (Buck and Axel, 1991); 2) trace amine associated receptor family, TAAR (Liberles and Buck, 2006); 3) type I vomeronasal receptors, V1Rs (Dulac and Axel, 1995); and 4) type II vomeronasal receptors, V2Rs (Matsunami and Buck, 1997). The number of chemosensory receptor genes in most insects are smaller than in most mammals and more closely approaches the number found in fish (~100 OR genes). Genomic analysis has identified 62 or 79 or 170 ORs and 68 or 72 or 13 gustatory receptors (GRs) in Drosophila melanogaster, Anopheles gambiae and Apis mellifera respectively. Several types of chemosensory receptors are described in insects as well: 1) odorant receptors, ORs (detects food odors and pheremones) including the highly conserved receptor across insect species co-receptor Orco (formerly termed as Or83b in Drosophila) (Clyne et al., 1999; Gao and Chess, 1999; Vosshall et al., 1999); 2) gustatory receptors (GRs; detects taste molecules and CO2); and 3) ionotropic receptors, IRs (detect ammonia, amines, water vapor and alcohols). ORs and GRs were predicted to have seven transmembrane domains like classical GPCRs (Benton et al., 2006; Clyne et al., 2000; Clyne et al., 1999; Lundin et al., 2007; Zhang et al., 2011), whereas IRs are related to ionotropic glutamate receptors and predicted to contain three transmembrane domains and a pore loop (Benton et al., 2009).
Chapter 1. General Introduction
8 Olfactory signaling
In both vertebrates and nematodes, odor molecules bind with the dedicated GPCRs (ORs), present on the dendritic membrane of the olfactory receptor neuron (ORNs), and activates a signaling pathway that produces intracellular messenger (through a specific G protein) called the metabotropic pathway (canonical signaling pathway). G proteins are important signal transducing molecules in a cell and are heterotrimeric and are composed of three subunits called α, β, γ. When a GPCR activates a G protein, it leads to the disassociation of the heterotrimer into Gα-GTP and Gβγ (heterodimer). Though the specificity in GPCR coupling and effectors activation is provided by the Gα subunits, both Gα-GTP and Gβγ activate different transduction pathways in the cell depending on their particular effectors (Gilman, 1984). The signaling pathways initiated by insect ORs are poorly understood. Though insect ORs were predicted to have seven transmembrane topology reminiscent of classical GPCRs, their topology is inverted (N terminal end of the protein is intracellular and the C terminal end is extracellular; Fig 1.3) and low sequence homology to all known GPCRs (Benton et al., 2006; Clyne et al., 1999; Gao and Chess, 1999; Lundin et al., 2007; Vosshall et al., 1999).
Moreover, insect ORs form heteromeric complexes with a conserved ortholog protein called Orco (Fig 1.3) (Benton et al., 2006; Larsson et al., 2004). It remains unclear whether and if so how insect ORs depend on G proteins for olfactory signaling. Two different hypotheses have been proposed for insect olfactory signal transduction: either insect ORs may act as ligand gated ion channels (ionotropic or noncanonical signaling pathway) or combine an ionotropic and a G protein-dependent pathway (metabotropic pathway) for olfactory signaling (Sato et al., 2008; Smart et al., 2008; Wicher et al., 2008).
However, the involvements of different G proteins in insect olfactory signaling are still enigmatic.
Figure 1.3. Insect olfactory receptors.
In the olfactory receptor neuron of insects, together the ORX (ligand binding OR) a conserved ortholog protein called – Orco (co-receptor) is also expressed. Both ORX and Orco were predicted to have seven transmembrane domains like classical GPCRs but it has an inverted topology; N terminus of the protein is intracellular and the C terminus of the protein is extracellular. Adapted from (Kaupp, 2010).
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Olfactory signaling pathways in vertebrates and nematodes
In vertebrate and nematode ORNs the binding of odorants activates a G protein mediated signaling pathway. Once activated, Golf in vertebrates (also called as Gs) and Gi in nematodes increase the levels of cyclic nucleotides, cAMP and cGMP respectively.
These cyclic nucleotides directly act on the cyclic nucleotides-gated (CNG) channels expressed on the dendritic membranes of the ORNs. Upon activation CNG channels allow the cations to enter into the neurons and which eventually leads to depolarization and generation of the action potential (Kaupp, 2010; Pellegrino and Nakagawa, 2009).
Signal transduction pathway in mammalian ORNs is shown in Fig 1.4.
Figure 1.4. Signal transduction in mammalian olfactory receptor neurons.
The binding of an odorant to the OR activates the trimeric, olfaction-specific G protein (Golf). Activated G protein disassociates to Gα-GTP and Gβγ subunits. The Gα subunit then activates the enzyme; adenylyl cyclase type III (ACIII), which, in turn, catalyzes the conversion of ATP into cyclic adenosine monophosphate (cAMP). Increase in the concentration of the second messenger cAMP leads to the activation of the olfactory cyclic nucleotide-gated channel (CNGC) and a Ca2+ activated chloride channel (CaCC). Activation of both channel types finally leads to depolarization. Adapted from (Kaupp, 2010).
Binding of pheromones to V1Rs and V2Rs also activates a G protein mediated signaling pathway (via the Go/i subgroup of G proteins; Fig 1.5). Once activated V1Rs activates Gi2 and stimulates Gβγ mediated calcium signaling, whereas V2Rs activates Go and also stimulates calcium signaling (Berghard and Buck, 1996; Berghard et al., 1996;
Kaupp, 2010; Lucas et al., 2003).
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Figure 1.5. Pheromone signaling in vertebrates.
(A) The binding of a pheromone to a V1R receptor activates Gi, a G protein that is often involved in inhibitory signal transduction pathways. Activated Gβγ subunit then activates a specific enzyme;
phospholipase Cβ2 (PLCβ2), which in turn cleaves the membrane phospholipid - phosphatidylinositol-4,5-bisphoshate (PIP2) into inositol-1,4,5-trisphoshate (IP3) and diacylglycerol (DAG). DAG then activates the transient receptor potential cation channel C2 (TRPC2), which mediates the influx of Na+ and Ca2+ ions and thus leads to the depolarization. (B) The binding of a pheromone to V2R receptor activates Go, a trimeric G protein involved in diverse signal transduction pathways. The activated Gα subunit is thought to play a role in signaling and activates TRPC2 channel and leads to the depolarization. But the mechanism by which the Gα subunit activates the channel is unknown. Adapted from (Kaupp, 2010).
Olfactory signal transduction in insects
In order to understand the signaling mechanisms of insect ORs, several researchers turned towards the expression of insect ORs in heterologous cells systems (in vitro) while others investigated these mechanisms in the native tissue (in vivo). Results of these experiments proposed contradicting models for olfactory signaling – the ionotropic or metabotropic or combined pathways.
Ionotropic signaling pathway
Some studies support the idea that insect ORs acts as ligand gated ion channels.
Both ORs and Orco were predicted to contribute to the pore of the odor gated ion channel complex (Nakagawa et al., 2012; Nichols et al., 2011; Pask et al., 2011; Sato et al., 2008). Sato et al., expressed ORs from various insects (fruit fly, mosquito and silk moth) together the co-receptor Orco in heterologous cell systems (HEK293 and HeLa cells) and characterized a fast ionotropic response, mediated by several odorants. The
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fast ionotropic response persisted in the presence of second messenger inhibitors (e.g. a PLC inhibitor) or general G protein inhibitors (such as GDPβS), which led the authors to conclude that odor evoked currents mediated by insect ORs signal via a G protein independent pathway. Also they found no evidence for odor stimulated cAMP production. Further they showed that the specific subunit composition governs the biophysical properties of the OR/Orco receptor, suggesting that these proteins function as a complex to form an odor gated ion channel whose initial activation does not depend on G protein signaling (Sato et al., 2008). By expressing Drosophila OR (Or43b) together Orco in an insect cell line (Sf9 cells), Smart et al., found that inhibitors of the second messengers activated by Gαq or Gαs pathways and general a G protein inhibitor GDPβS did not block odor evoked calcium increases but changed the inactivation kinetics, which made the authors to conclude that Drosophila ORs are unlikely to be a member of GPCR superfamily proteins and in HEK cell they signal via non selective cation channels in the plasma membranes, without the involvement of G proteins (Smart et al., 2008). Wicher et al., expressed Drosophila OR (Or22a) together with Orco in HEK293 cells and found that odors evoke a fast ionotropic current independent of G proteins and a slow late response dependent on G proteins (involvement of cAMP, implicating a metabotropic pathway mediated by Gαs). Also odor evoked currents persisted in the presence of the general G protein inhibitor GDPβS, but the cells were less sensitive to odorants. These results made the authors to conclude that ligand-bound ORs couple to Gαs (although any direct interaction was not shown) which produces cAMP, which in turn activates Orco (which, as they hypothesize, has the properties of cAMP-gated nonselective cation channels). A model proposed from these experiments is that ORs function as GPCRs and Orco functions as an ion channel (Wicher et al., 2008). Yao et al., studied the involvement of G proteins in Drosophila OR signaling in vivo by loss or gain of function of G proteins (using RNAi constructs, competitive peptides and constitutively active Gα protein constructs). Through single sensillum recordings (SSRs) they found that in these mutant conditions the odor evoked spike activity was unaffected when compared to control flies (although Gαq mutants exhibited a ca. 30%-reduced response, the authors considered it to be insignificant). These results made the authors to conclude that ORs rely on ligand-gated ionotropic signaling mechanisms that require Orco (Yao and Carlson, 2010). These four independent research groups support that insect ORs act as ligand gated ion channels and do not require G proteins (except that Wicher et al.
propose the involvement of G proteins as well) for olfactory signaling.
Chapter 1. General Introduction
12 Metabotropic signaling pathway
In insects, especially in fruit flies and moths, involvement of Gαs and Gαq pathways in odor and pheromone detection was reported. Also there is an evidence for the involvement of Gαo in olfactory signaling.
Gαq mediated signaling
The evidence for the involvement of Gαq pathway is mostly provided for the pheromone transduction of moths, but also involvement in general odor detection has been shown. Pheromone transduction in moths depends on PLCβ (phospholipase C β).
PLCβ hydrolyzes phospholipids generating IP3 and DAG. A rapid transient rise of IP3 was observed in the antennal tissue of moths upon pheromone and general odorant stimulation (Boekhoff et al., 1993; Breer et al., 1990). Also perfusion of cultured ORNs from M. sexta with IP3 mimicked pheromone-dependent currents (Stengl, 1993; Stengl, 1994). Apart from IP3, DAG was also shown to play a role in insect olfaction. DAG acts as an activator of PKC (protein kinase C) or as an activator of TRP channels (transient receptor potential type ion channels). Application of DAG in in situ, in vitro tip recordings and patch clamp recordings depolarized ORNs or activated depolarizing inward currents in B. mori, A. polyphemus, and M. sexta (Maida et al., 2000; Pophof and Van der Goes van Naters, 2002). Changes in the levels of membrane-bound phospholipids have also been shown to alter the primary response to odorants in crustaceans (Zhainazarov et al., 2004). Mutations in the Drosophila dgq gene encoding the Gq subunit was shown to reduce odor responses across to different concentrations in D. melanogaster (in vivo; SSR) and the responses were further attenuated by additional mutations in plc21C, a gene encoding for a PLCβ (Kain et al., 2008). Further, knockdown of Gαq by targeted RNA interference (RNAi) resulted in behavioral deficits in Drosophila (Kalidas and Smith, 2002). The Gαq pathway was also shown to regulate the function of the co-receptor Orco. Orco phosphorylation via PKC was shown to regulate the sensitivity of OR to cAMP and therefore to odorants. The metabotropic pathway affecting the phosphorylation state of Orco is proposed to regulate OR function and thereby shapes the odor response of ORNs (Sargsyan et al., 2011). The metabotropic regulation of Orco was proposed as a key mechanistic difference between ORs and IRs (Getahun et al., 2013).
Gαs mediated signaling
Gαs activates the cAMP dependent pathway by stimulating the production of cAMP from ATP. cAMP is shown to be involved in insect OR transduction. cAMP activated currents were described in cultured olfactory receptor neurons of the moth
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Manduca sexta (Krannich and Stengl, 2008). Drosophila ORs have been shown to stimulate cAMP production, which in turn activates the co-receptor Orco (Wicher et al., 2008).
Manipulating the levels of cAMP changed the kinetics of odor response in heterologously expressed Drosophila ORs (Smart et al., 2008). Expression of cholera toxin (CTX; ribosylates the Gαs subunit of heterotrimeric G proteins) or GTPase defective (constitutively active) Gαs subunit in Drosophila ORNs led to abolishment of firing frequency of the ORNs and prolonged the duration of spike activity, respectively, compared to the control flies (Deng et al., 2011). Heterologous expression of a Drosophila OR (Or43a) together the co-expression of chimeric Gαs and Orco led to a calcium increase upon odor stimulation (Deng et al., 2011). cAMP produced by the activation of other receptors in dendrites apart from ORs was also shown to be involved in odor responses. Endogenous circadian rhythms of cyclic nucleotides, generated by varying octopamine levels, contribute to the control of the intracellular Ca2+ concentrations in ORNs from Manduca sexta, and generate more sensitive pheromone responses during the night (Flecke et al., 2010; Flecke and Stengl, 2009).
Other G protein mediated signaling
The majority of the studies indicate a role of Gαs or Gαq in insect olfactory signaling. The role of other Gα subunits and of the βγ heterodimer is largely unknown.
Only a few studies focused on the role of the Go/i subgroup of G proteins in olfactory signaling. Chatterjee et al., showed that flies with reduced levels of the Gαo subunit had a reduced odor response regardless of odor identity and intensity (measured as the spike activity and EAG amplitudes) (Chatterjee et al., 2009), whereas Yoa et al., and Deng et al., showed that reduced or higher levels of Gαo or Gαi subunits in Drosophila ORNs had no effect on odor response (measured as the spike activity and or EAG amplitudes) (Deng et al., 2011; Yao and Carlson, 2010). However, co-expression of almost all Gα subunits (Gαs, Gαq, Gαo, Gαi and Gαf) encoded by the Drosophila genome together with Or43a and Orco in a heterologous cell system was shown to induce calcium increase upon odor addition (Deng et al., 2011).
Although these results are mutually contradicting, it could be possible that insects use multiple signaling pathways for odor transduction. Much remains to be done to provide detailed and consistent data for an integrated model for olfactory transduction. First the role of all G proteins encoded by the insect genome or those expressed on the dendrites of the ORNs should be known. For example very little is known about the role of Go/i subgroup of G proteins in olfactory signaling of insects.
Go/i subgroup of G proteins is shown to play a role in pheromone signaling in vertebrates. But, to date the involvement or a role of Go/i subgroup of G proteins in insect olfactory signaling is greatly unknown. Second whether all ORs activate a same G
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protein or not should be known. It may be possible that different ORs activate different G proteins and also a G protein can be activated to a varying degree. Most studies in the literature mutated the G proteins in all ORNs. But in order to address the above hypothesis mutation of G proteins should be targeted to a receptor of interest.
Objectives of this study
The major objective of this work is to study the role of G proteins in olfactory signaling of insects. Drosophila odorant receptors, Or22a and Or92a were used as the model for insect ORs and the role of G proteins in olfactory signaling was studied by combing in vivo and in vitro methods. I developed an in vitro olfactory receptor assay for insect ORs as a tool to study G proteins in olfactory signaling. This assay is explained in detail in Chapter 2. With this assay, screening of the molecules involved in signal transduction can be studied by excluding any possible indirect interactions. This assay is used to study the role of G proteins (Go/i subgroup) in heterologously expressed Drosophila odorant receptors (dOr22a and Orco). Any role of the Go/i subgroup of G proteins in insect olfaction is largely unknown. I studied a role of the Go/i proteins in olfactory signaling of Or22a neurons by using a combined behavioral, in vivo (calcium imaging of dendro-somatic compartment) and in vitro (calcium imaging of Drosophila ORs expressed heterologously) approach. My key findings were that Go and Gi contribute to
The major objective of this work is to study the role of G proteins in olfactory signaling of insects. Drosophila odorant receptors, Or22a and Or92a were used as the model for insect ORs and the role of G proteins in olfactory signaling was studied by combing in vivo and in vitro methods. I developed an in vitro olfactory receptor assay for insect ORs as a tool to study G proteins in olfactory signaling. This assay is explained in detail in Chapter 2. With this assay, screening of the molecules involved in signal transduction can be studied by excluding any possible indirect interactions. This assay is used to study the role of G proteins (Go/i subgroup) in heterologously expressed Drosophila odorant receptors (dOr22a and Orco). Any role of the Go/i subgroup of G proteins in insect olfaction is largely unknown. I studied a role of the Go/i proteins in olfactory signaling of Or22a neurons by using a combined behavioral, in vivo (calcium imaging of dendro-somatic compartment) and in vitro (calcium imaging of Drosophila ORs expressed heterologously) approach. My key findings were that Go and Gi contribute to