Diss

INTERNATIONAL GRADUATE SCHOOL OF BIOSCIENCES
RUHR-UNIVERSITY BOCHUM
MOLECULAR MECHANISM OF OLFACTORY SIGNAL

TRANSDUCTION IN DROSOPHILA MELANOGASTER
Doctoral Dissertation
Ying Deng
Department of Cell Physiology
First thesis advisor: Prof. Dr. Dr. Dr. Hanns Hatt

Second thesis advisor: Prof. Dr. Wolfgang Kirchner
Bochum, October. 2009
INTERNATIONALEN GRADUIERTENSCHULE BIOWISSENSCHAFTEN

RUHR-UNIVERSITT BOCHUM
DER MOLEKULAR MECHANISMUS DER OLFAKTORISCHEN

SIGNALTRANSDUKTION IN DROSOPHILA MELANOGASTER
Dissertation
Ying Deng
Lehrstuhl fr Zellphysiologie
Referent: Prof. Dr. Dr. Dr. Hanns Hatt

Korreferent: Prof. Dr. Wolfgang Kirchner
Bochum, October. 2009
Declaration
I certify herewith that the dissertation at hand was completed and written independently
and without outside assistance. The Guidelines for Good Scientific Practice (Leitlinien
guter wissenschaftlicher Praxis und Grundstze fr das Verfahren bei vermutetem
wissenschaftlichen Fehlverhaltens) according to 9, Sec. 3 were adhered to. This work
has never been submitted in this or similar form at this or any other domestic or foreign
institution of higher learning as a dissertation.
Hiermit erklre ich, dass ich die Arbeit selbstndig verfasst und bei keiner anderen
Fakultt eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel
verwandet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um fnf
in Wort und Bild vllig bereinstimmende Exemplare.
Weiterhin erklre ich, dass digitale Abbildungen nur die originalen Daten enthalten und
in keinen Fall inhaltsverndernde Bildbearbeitung vorgenommem wurde.
Ying Deng
Bochum, 15th Oct. 2009
Error! Reference source not found.
Table of contents
1
Introduction................................................................................................................. 1
1.1
The Drosophila olfactory system........................................................................ 1
1.1.1
Anatomy of the Drosophila olfactory system............................................. 1
1.1.2
The odor coding system in Drosophila....................................................... 7
1.1.3
New insights of Drosophila odorant receptor........................................... 14
1.2
Heterotrimeric G-proteins................................................................................. 16
1.3
Signal transduction pathway in odorant receptor neurons................................ 19
Objective ................................................................................................................... 23
Materials and Methods.............................................................................................. 24

3.1
Materials ........................................................................................................... 24
3.1.1
Fly food..................................................................................................... 24
3.1.2
Enzymes and Kits and molecularbiological supplies................................ 24
3.1.3
Immunoblotting and Immunohistochemistry............................................ 24
3.1.4
Cell culture................................................................................................ 26
3.1.5
Mammalian cell lines................................................................................ 26
3.1.6
Buffers and Solutions................................................................................ 26
3.1.7
Odorants.................................................................................................... 27
3.1.8
Miscellaneous ........................................................................................... 28
3.2
Methods............................................................................................................. 28
3.2.1
Fly rearing and fly stocks.......................................................................... 28
3.2.2
Molecular cloning and plasmid construction............................................ 29
3.2.3
Cell culture and transfection ..................................................................... 30
3.2.4
Ratiometric calcium imaging in HEK293 cells ........................................ 30
3.2.5
RNA isolation, reverse transcription, RT-PCR and Real-time quantitative
PCR
................................................................................................................... 31
3.2.6
[35S] GTPS binding assay ...................................................................... 32
3.2.7
Larval chemotaxis assay ........................................................................... 33
3.2.8
Electroantennogram (EAG) ...................................................................... 34
ii
3.2.9
Antennal single sensillum recording......................................................... 35
3.2.10
Labellar single sensillum tip recording..................................................... 36
3.2.11
Odorant application system in EAG and single sensillum recording ....... 37
3.2.12
Immunohistochemistry ............................................................................. 37
3.2.13
Whole mount fluorescent antibody staining of Drosophila larvae........... 38
3.2.14
SDS gel electrophoresis and Western-blotting ......................................... 38
3.2.15
cAMP assay .............................................................................................. 38
Results....................................................................................................................... 39
4.1
Different G subunits are expressed in Drosophila antenna. ........................... 39
4.1.1
RT-PCR analysis of G subunits in Drosophila antenna ......................... 39
4.1.2
Western-blot analysis showed that different classes of G are expressed in
the antenna ................................................................................................................ 40

4.1.3
Immunohistochemical analysis of the expression patterns of different G
proteins in Drosophila antenna................................................................................. 41

4.1.4
Real-time quantitative PCR analysis of Gs and Gq expression in
Drosophila antenna and head.................................................................................... 43

4.2
Functional study of G subunit in Drosophila olfactory signal transduction
pathway ......................................................................................................................... 44
4.2.1
Overexpression study of different G subunits and G-protein specific
protein toxins in Drosophila ORNs .......................................................................... 44

4.2.2
Dose-response curves of EAG responses from CTX and control flies..... 45
4.2.3
CTX blocks the olfactory response of ORNs in Drosophila .................... 47
4.2.4
CTX does not act as a non-specific toxin on cells .................................... 49
4.2.5
Functional study of OR and G protein interactivity in the recombinant
HEK293 expression system ...................................................................................... 49

4.2.6
Overexpression of a GTPase deficient Gs mutant in Drosophila antenna
leads to a change in response dynamics.................................................................... 52

4.2.7
Odorant exposure caused Gs redistribution in antenna ........................... 53
4.2.8
Generation of a Gs knockout or knockdown fly ..................................... 54
4.3
Secondary messengers and other components in the ORNs signal transduction
cascade .......................................................................................................................... 56

4.3.1
The cAMP pathway is involved in odorant signaling in fly olfactory
neurons ................................................................................................................... 56
Expression of activated Gq in ORNs leads to resensitization deficiency. ..
4.3.2
................................................................................................................... 59
4.3.3
Effect of MIA on Drosophila odorant receptors....................................... 61
4.3.4
Functional study of Ih channels in Drosophila odorant receptor neurons 63
4.4
Larva study........................................................................................................ 65
4.4.1
Gs and Gq are expressed in larva olfactory receptor neurons ............... 65
4.4.2
CTX larvae have normal chemotaxis behavior......................................... 66
4.4.3
Expression of AcGq3 in larval olfactory receptor neurons causes olfactory
aberration .................................................................................................................. 66
5
Discussion ................................................................................................................. 67
5.1
The role of Gs in the olfactory signal transduction pathway .......................... 67
5.2
Larval olfactory system..................................................................................... 73
5.3
Other signaling components in the olfactory signal transduction pathway ...... 74
Conclusion ................................................................................................................ 78
6.1
Summary ........................................................................................................... 78
6.2
Zusammenfassung............................................................................................. 80
References......................................................................................................................... 82
Abbreviations.................................................................................................................. 100
List of Figures and Tables............................................................................................... 102
Curriculum Vitae ............................................................................................................ 104
Acknowledgements......................................................................................................... 107
Introduction
1 Introduction
Insects, as the most diverse group of animals, represent more than half of all known
living organisms and potentially represent over 90% of the differing life forms on the
planet. They have an enormous impact on global public health as disease vectors, which
carry pathogens that cause human diseases such as malaria, dengue and yellow fever, and
as agricultural enablers as well as pests (Beaty and Marquardt, 1996). Olfaction acts as an
important sensory input to their behavior, such as processing chemical signals from the
environment, leading to the detection of food, reproductive partners, oviposition sites,
hosts, prey or predators (Rutzler and Zwiebel, 2005). Therefore, it is of great value to
understand the signal transduction pathway in insect olfactory system, in addition to
nurturing a better understanding of insect neurobiology, may ultimately help in devising
novel intervention strategies to reduce crop damage and disease transmission.
The fruit fly, Drosophila melanogaster, has been used as a model orgainism for over 100
years. Today, thousands of scientists use Drosophila to study almost every aspect of
eukaryotic biology from gene organization to developmental biology to behavior and
everything in between. Advantages of choosing Drosophila for these studies include the
completely sequenced genome (Adams et al., 2000), a short live cycle and all kinds of
well-established genetic tools.
1.1 The Drosophila olfactory system

1.1.1
Anatomy of the Drosophila olfactory system
Drosophila has two pairs of olfactory organs, the antennae and the maxillary palps
(Figure 1.1.1 A). They are cuticle-covered appendages, which contain approximately
1200 and 120 olfactory receptor neurons (ORNs) each (Hildebrand and Shepherd,
1997;Riesgo-Escovar et al., 1997;Shanbhag et al., 1999;Shanbhag et al., 2000). ORNs are
compartmentalized into sensory hairs called sensilla, which can be subdivided into three
morphological types: basiconic sensilla (BS) resemble a club, trichoids have sharp tips,
and coeloconics are dome-shaped and have a grooved surface (Figure 1.1.1 B, C, D, E
and F). Each sensillum contains the dendrites of one to four ORNs (Shanbhag et al.,
1999). At the base of sensillum, specialized auxiliary cells surround the cell bodies of the
Introduction
ORNs. (Figure 1.1.1 G, H). The sensory dendrites of the ORNs are bathed in the
sensillum lymph that secreted from the auxiliary cells (Shanbhag et al., 2000). Odorantbinding proteins are also secreted by the auxiliary cells and present in this extracellular
sensillum lymph (Kaissling, 1996;Park et al., 2000).
The antennae contain all three types of olfactory sensilla, whereas the maxillary palps
contain only basiconic sensilla. The respective contributions of the antenna and maxillary
palps to chemosensory-mediated behavior are not yet clear (Shanbhag et al., 1999).
Sensilla of different morphological types respond to different types of chemical stimuli.
Basiconic sensilla are sensitive to many food odorants with a variety of chemical groups
(Park et al., 2002). Trichoid sensilla do not have a strong response to food odors but to fly
odors (van der Goes van Naters and Carlson, 2007). It is also known that the volatile fly
pheromone 11-cis-vaccenyl acetate (cVA) can be detected by the trichoid sensilla (Jin et
al., 2008). One type of coeloconic sensilla (ac3) respond to a remarkably high fraction of
the tested food odors, the rest of the coeloconic sensilla are sensitive to humidity or
ammonia (Benton et al., 2009;Spletter and Luo, 2009;Yao et al., 2005).
Introduction
Figure 1.1.1 The Drosophila olfactory organ

(A) Drosophila melanogaster head with the olfactory appendages, the antenna and the maxillary palps. S:
Scapus, P: Pedicellus, F: Funiculus. Figure A was adapted from Jrgen Berger, Max Planck Institute for
developmental Biology, Tbingen, Germany. (B, C) Low magnification scanning electron micrographs of
antenna surface. C: coeloconic sensillum; I: intermediate sensillum; LB, SB, TB large, small, thin basiconic
sensillum, respectively; Sp spinule; T s. trichoid sensillum. Scale bar in B and C = 10 m. (D) High
resolution scanning electron micrographs of thin basiconic sensillum (TB), large basiconic sensillum (LB),
coeloconic sensillum (C) and trichoid sensillum (T). Different types of sensilly have different surface
structure and arrangement of wall pores. Scale bars in TB and LB = 1 m, Scale bars in T and C = 0.5
m.(E) Structure of basiconic sensillum, trichoid sensillum and coeloconic sensillum (Stocker, 1994). (F)
Cross-sections through various types of sensillum. Scale bars = 0.5 m. (G, H) Longitudinal section (G)
and schematic drawing (H) of a small basiconic sensillum with two ORNs. D1 and D2 are the dendrites of
two odorant receptor neurons (R). The three types of auxiliary cells are thecogen Th, trichogen Tr, and
tormogen cells To. Epidermal cell E. The large sensillum-lymph cavity (S1) is bordered by the microvilli
and microlamellae of trichogen and tormogen cells. Scale bars = 1 m. B, C, D, F, G were adapted from
Shanbhag, S.R. et al. 1999. H was adapted from Park, S.K. et al 2002 (Park et al., 2002).
ORNs send axons to the antennal lobe (AL), which has a functional organization
remarkably similar to that of the olfactory bulb in vertebrates (Hildebrand and Shepherd,
1997). In the AL, ORNs form synapses onto second order neurons called projection
Introduction
neurons (PNs) (Stocker, 1994) (Figure 1.1.2 A). The AL can be subdivided into about 43
spherical units called glomeruli (Laissue et al., 1999). Each ORNs send axons to only one
or a few glomeruli (Stocker, 1994), and individual PNs typically innervate only a single
glomerulus (Jefferis et al., 2001;Marin et al., 2002;Wong et al., 2002). The glomeruli also
contain the processes of local interneurons that branch in multiple glomeruli (Stocker et
al., 1990;Stocker, 1994), providing a means for information transfer between glomeruli.
The axons of PNs project to the mushroom body (MB) and lateral horn of the brain
(Figure 1.1.2 A).
Larvae of Drosophila also exhibit a dorsal organ mediated olfactory response (Ayyub et
al., 1990;Cobb et al., 1992;Heimbeck et al., 1999;Monte et al., 1989;Oppliger et al.,
2000). The larva cephalic chemosensory apparatus consists of 3 external sensory organs,
dorsal organ (DO), terminal organ (TO), and ventral organ (VO), plus 3 pharyngeal
organ, as the dorsal, ventral, and posterior pharyngeal sense organs (DPS, VPS, PPS)
(Figure 1.1.2 B) (Gerber and Stocker, 2007;Python and Stocker, 2002). Each of them
includes several sensilla, a sensillum comprising one to several sensory neurons and 3
accessory cells, all housed below a common cuticular structure or terminal pore.
The dorsal organ houses the dendrites of 21 olfactory receptor neurons in the central
dome and six peripheral sensilla. An olfactory function of the dorsal organ is proved by
electrophysiological recordings (Kreher et al., 2005;Oppliger et al., 2000) and by
combined toxin expression and behavioral studies (Fishilevich et al., 2005;Heimbeck et
al., 1999;Larsson et al., 2004). The experiment of genetically ablation the OR83b
receptor and expressing diphtheria toxin in OR83b neurons demonstrate that these 21
sensory neurons, which express OR83b, are the sole larval ORNs.
The DO, TO, and VO all correspond with their internal ganglion. The ganglion of the DO
(DOG) contains 36 to 37 sensory neurons (Python and Stocker, 2002). The 21 ORNs
among them extend their dendrites as seven triplets into the dome. The dendrites of three
of the rest neurons project towards the dorsolateral sensilla of the TO (Heimbeck et al.,
1999;Python and Stocker, 2002), whereas the remaining neruons innervate the 6
peripheral sensilla of the DO. The TO and VO ganglia include 32 and 7 sensory neurons,
respectively (Python and Stocker, 2002). Each of the three pharyngeal sense organs
consists of several sensilla, comprising one to nine sensory neurons. The presence of
Introduction
pores and bristles suggest the gustatory and mechanosensory function of these pharyngeal
sense organs. The dorsal and ventral pharyngeal sense organs, both of these sensory
organs are located behind the mouth hooks, contain 17 and 16 neurons, respectively;
while the posterior pharyngeal sense organ contains two sensilla with three neurons each
(Figure 1.1.2 B).
In contrast to adults, all olfactory projections remain ipsilateral in larva. Both olfactory
and gustatory neurons from the DO ganglion connect to the brain by the antennal nerve
(Python and Stocker, 2002;Tissot et al., 1997). The supraesophageal labral nerve receives
information from the dorsal pharyngeal organ and from the posterior pharyngeal organ,
whereas the subesophageal maxillary and labial nerves comprise the afferents from the
TO and VO ganglia and from the ventral pharyngeal organ, respectively (Gendre et al.,
2004;Python and Stocker, 2002) (Figure 1.1.2 B).
Introduction
Figure 1.1.2 Organization of the Drosophila olfactory system.

(A) Overview of the Drosophila adult olfactory system organization, with mammalian counterparts in
parentheses (Komiyama et al., 2003). Axons of ORNs expressing the same receptor (represented by the
same color) project to the same glomeruli in the AL. PNs send dendrites to glomeruli and axons to the
mushroom body and the lateral horn, two higher olfactory centers approximately analogous to vertebrate
primary olfactory cortex. (B) Overview of larval chemosensory system (Gerber and Stocker, 2007). From
the three external chemosensory organs, the DO is a mixed structure composed of the central olfactory
dome (grey) and a few putative taste sensilla (small circles). The cell bodies of the sensory neurons are
collected in ganglia (DOG, TOG, VOG) below each sense organ (DO, TO, GO). Some of the neurons
innervating the dorsolateral sensilla of the terminal organ are situated in the ganglion of the DO. ORNs
(blue) send their axons via the antennal nerve (AN) into the larval antennal lobe (LAL). Local interneurons
(LN) interconnect the glomeruli of the larval antennal lobe. PNs (PN; green) travel in the inner antennocerebral tract (iACT) to link the larval antennal lobe with the mushroom body calyx and the lateral horn
(LH). An intrinsic mushroom body Kenyon cell (KC) extending its process via the pedunculus (PD) into
the mushroom body lobes (not indicated) is shown in red. Axons from putative taste receptor neurons
(brown) extend via the antennal nerve, the labral nerve (LN), the maxillary nerve (MN) and the labial nerve
(LBN) to the suboesophageal target region (SOG).
Introduction
1.1.2
The odor coding system in Drosophila
1.1.2.1 Identification of Drosophila odorant receptor genes

The discovery of the first ORs was elusive for many years due to the nature of the
receptors themselves. The presence of a large number of ORs, their sequence divergence
and the low expression level made them difficult to detect until, in 1991, the first
mammalian ORs were cloned from the rat olfactory epithelium (Buck and Axel, 1991).
These proteins showed aspects that were consistent with their classification as ORs: they
are expressed specifically in the olfactory epithelium, they are members of the
superfamily of G-protein-coupled receptors (GPCRs) with seven membrane-spanning
domains as hypothesized by previous studies (Jones and Reed, 1989) and their sequences
are related. The proteins physiological function was confirmed a few years later
(Wellerdieck et al., 1997;Zhao et al., 1998), and genes with similar properties were soon
described in other organisms (Freitag et al., 1999;Nef et al., 1996;Selbie et al.,
1992;Sengupta et al., 1996). Insect ORs were first identified in Drosophila melanogaster
by three independent groups in 1999 using different approaches (Clyne et al., 1999;Gao
and Chess, 1999;Vosshall et al., 1999). One successful approach to their isolation began
with the assumption that odorant receptors in flies, like those in mammals and
Caenorhabditis elegans, were GPCRs. A computer algorithm was then devised that
recognized proteins on the basis of structure and was trained to examine DNA databases
for proteins with structures like those of GPCRs. This algorithm identified members of
the OR gene family from the genome of Drosophila.
The Drosophila OR gene family contains 60 members that are distributed throughout the
genome, often in small clusters (Clyne et al., 1999;Robertson et al., 2003). Two of these
genes are alternatively spliced, resulting 62 odorant receptor proteins in total (Robertson
et al., 2003). 39 ORs are expressed in the adult (no including OR83b) (Vosshall et al.,
2000), and 25 ORs are expressed in the larva (Fishilevich et al., 2005) (Table 1). By
comparison, humans are believed to have about 350 functional OR genes (Malnic et al.,
2004), mice have about 1000 OR genes (Godfrey et al., 2004;Zhang and Firestein, 2002)
and mosquitoes have about 80 OR genes (Hill et al., 2002). In general, Drosophila
odorant receptor proteins are highly diverse, in many cases showing only 20% identity to
Introduction
each other and no similarity to mammalian odorant receptors. This diversity among
odorant receptor proteins is apparent throughout the length of the protein, albeit
conserved residues shared by many of the genes have been identified (Clyne et al.,
1999;Vosshall et al., 1999). However, closely linked genes often show a higher degree of
similarity, the two most similar receptors, OR19a and OR19b, differ by only three amino
acids, suggesting that OR gene clusters are likely to have arisen through recent genome
duplication (Robertson et al., 2003). Each ORN expresses only one or a small number of
OR genes, resulting in molecular diversity among ORNs (Clyne et al., 1999;Vosshall et
al., 1999).
Most Drosophila odorant receptor neurons co-express two different types of odorant
receptors: OR83b, a broadly expressed receptor, and one of the 61 ligand specific ORs.
OR83b is highly conserved among insect species whereas the ligand-specific receptors
are highly divergent. Electrophysiological and behavioral experiments in OR83b
knockout flies revealed that OR83b is essential for the normal function of other ORs
(Larsson et al., 2004;Neuhaus et al., 2005). Benton and colleagues later demonstrated that
not only is OR83b served as a chaperone protein that transports the ligand-binding ORs
from the cell body to the dendrite where ORs can detect odorants, but also acted as a
functional part of the receptor-complex (Benton et al., 2006). However, it still remains to
be elucidated whether OR83b is involved at all in the odorant binding.
Expression patterns of the complete repertoire of Drosophila odorant receptors
Antenna
maxillary palp
larva
not detected
Or47a (47F1)
Or47b (47F6)
Or7a (7D14)
Or85a (85A3)
Or85f (85D15)
Or19a (19B3-19C)
Or13a (13F16-18)
Or22a (22A5)
Or56a (56E1)
Or82a (82A3-4)
Or2a (2E1)
Or23a (23A3)
Or65a (65A7-11)
Or22b (22A5)
Or33b (33B10)
Or33a (33B10)
Or67c (67D2)
Or43b (43F5)
Or69b (69E8-F1)
Or69a (69E8-F1)
Or67a (67B2)
Or43a (43A1)
Or35a (35D1)
Or10a (10B15)
Or9a (9E1)
Or88a (88B1)
Or49b (49D1)
Or98a (98B5)
Or85b (85A9)
Or83c (83D5)
Or42b (42A2)
Or59b (59E1)
Or1a (1A8)
Or33c (33B10)
Or46a (46E7-8)
Or59c (59E1)
Or71a (71B1)
Or85e (85B2)
Or85d (85A11)
Or1a (1A8)
Or2a (2E1)
Or7a (7D14)
Or13a (13F16-18)
Or22c (22C1)
Or24a (24E4)
Or30a (30A3)
Or33b (33B10)
Or35a (35D1)
Or42a (42A2)
Or42b (42A2)
Or45a (45F1)
Or45b (45F1)
Or47a (47F1)
Or49a (49A5)
Or59a (59E1)
Or63a (63B1)
Or67b (67B2)
Or74a (74A6)
Or82a (82A3-4)
Or83a (83A6)
Or85c (85A9)
Or94a (94D9)
Or94b (94D9)
Or92a (92E8)
Or46b (46E7-8)
Or98b (98D4)
Table 1 Expression patterns of the complete repertoire of Drosophila odorant receptors.

(Fishilevich et al., 2005;Vosshall et al., 2000)
Introduction
Another fact should be noticed is that except for the odorant receptors, different odorant
binding proteins are expressed in the adult and larval olfactory organs. Compared to the
thorough understanding of the odorant receptor map of the odorant receptor neurons,
research on Drosophila odorant binding proteins or pheromone binding proteins are
largely shrinked in the last decade. Except the well described functional requirement of
OBP76a/lush in the trichoide sensillum (Jin et al., 2008;Smith, 2007), general food odor
are thought to activate odorant receptors via direct interactions without the involvement
of OBPs. This idea is supported by the fact that a lot of odorant receptors were
deorphanized in a heterologous system (Nakagawa et al., 2005;Sakurai et al.,
2004;Wetzel et al., 2001), a large number of odorant receptors expressed in the halo
empty neuron system have the same odorant response profile as in the native sensillum
(Hallem et al., 2004;Kreher et al., 2005) and the dissociated olfactory receptor neurons
from the moth Manduca sexta responds to the conponent of the female moths sexpheromene blend (Stengl et al., 1992b). 35 OBP genes have been identified in the
genome of Drosophila by the use of reporter genes. The expression pattern study
revealed that 9 genes are expressed exclusively in the adult olfactory organ, several genes
that are not found in the adult olfactory organs are expressed in both larval dorsal organ
and adult gustatory organ or peripheral gustatory sensillum, for example OBP19c,
OBP56b and OBP56g (Galindo and Smith, 2001).
1.1.2.2 Characterization of Drosophila odorant receptors
The first Drosophila odorant receptor that has been functionally characterized is OR43a,
which was initially characterized physiologically following antennal overexpression
(Stortkuhl and Kettler, 2001) and heterologous expression in Xenopus oocytes (Wetzel et
al., 2001). Both studies identified that cyclohexanone, cyclohexanol, benzaldehyde and
benzyl alcohol are ligands for OR43a.
Odorants that pass through pores on the sensillum bind to ORs expressed on the dendrite
of ORNs and induce an action potential, which can be monitored using the single
sensillum recording (SSR) technique (Bestmann et al., 1996;Stensmyr et al.,
2003;Wojtasek et al., 1998). By performing the single sensillum recording, a recording
electrode is placed in the desired sensillum and captures voltage changes due to the firing
Introduction
of the ORNs (Figure 3.2.11). Because the sensillum contains more than one ORN, the
recording trace represents the summed activity of all the neurons housed within the
sensillum (Figure 3.2.11). In most of the sensilla, it is possible to distinguish the spikes
from different ORNs by their distinct spikes amplitude. Electrophysiological recordings
of antennal basiconic sensilla have revealed that ORNs are classified into distinct
functional classes, each with a unique odorant response spectrum (deBruyne et al., 2001).
A fundamental step forward was achieved when John Carlsons group established a
mutant fly strain with a deletion in the locus of the receptor OR22a/b, thereby abolishing
odor-evoked responses in the ORN where the receptor is expressed without eliminating
the ORN itself, the so-called empty neuron (Hallem et al., 2004). With this system,
based on a combination of the SSR technique and the GAL4-UAS system (Hallem et al.,
2004), it is possible to express virtually any OR, study its properties in vivo and use it as a
medium-throughput tool for Drosophila OR deorphanization, i.e. a simple way to assign
ligands to each OR (Hallem and Carlson, 2004;Kurtovic et al., 2007) (Figure 1.1.3).
Based on this approach, it was shown that the OR is not only responsible for the odorant
response spectrum in ORNs, but also for its spontaneous activity and response dynamics
(Hallem et al., 2004).
10
Introduction
Figure 1.1.3. Analysis of odor response spectra of individual odorant receptors

(Hallem and Carlson, 2004) (A) An in vivo expression system for odorant receptors. A mutant ab3A
antennal neuron (Dab3A) lacks odor response due to the deletion of its endogenous receptor genes, OR22a
and OR22b. Odorant receptors are introduced specifically into Dab3A using the GAL4UAS system; an
OR22aGAL4 driver promotes transcription of the OR gene. The odorant response of the neuron
(Dab3A:OrX) is then assayed electrophysiologically. (B) Mapping odorant receptors to olfactory neurons.
The normal odor response of the ab3A neuron (first panel) is absent from the Dab3A neuron (second
panel). Expression of Or7a in the Dab3A neuron (Dab3A:OR7a; third panel) results in an odor response
spectrum resembling that of the wild-type ab4A neuron (fourth panel), indicating that E2-hexenal is a
ligand for OR7a, and that OR7a is the odorant receptor in ab4A. Graphs depict the response of the neuron
in spikes per second (spikes/s) to the diagnostic panel of odorants at the left. (C) Odorant receptors that
have been mapped to functional classes of neurons. The eight different functional types of basiconic
sensilla are designated ab1ab8, and the neurons are named according to the sensillum in which they are
found (for instance, the ab2 sensillum contains ab2A and ab2B neurons). Odorant receptors that have been
mapped to basiconic neurons are indicated below the corresponding neurons. (D) Odor response spectra of
antennal odorant receptors. The colored dots depict strong responses.
Electrophysiological studies in vivo have also been complemented by studies in cell

culture: Some insect ORs can be functionally expressed in human embryonic kidney 293
(HEK293) cells, HeLa cells or Xenopus laevis oocytes (Nakagawa et al., 2005;Neuhaus
et al., 2005;Sato et al., 2008;Wetzel et al., 2001;Wicher et al., 2008). The functional
characterization of insect ORs in heterologous expression systems has provided several
new insights into the molecular mechanism of insect ORs, including functional
11
Introduction
interaction between OR subunits (Neuhaus et al., 2005), novel signaling properties of
insect ORs (Sato et al., 2008;Smart et al., 2008;Wicher et al., 2008) and the role of
OR83b (Nakagawa et al., 2005;Neuhaus et al., 2005).
1.1.2.3 Odor representations in the antennal lobe and higher brain center
Several studies showed that in rodents, odorant receptor neurons expressing the same OR
converge to single glomeruli in the olfactory bulb (Mombaerts et al., 1996;Ressler et al.,
1994;Vassar et al., 1994). Studies using OR promoters to drive expression of receptors
have revealed that in Drosophila, axons of ORNs expressing the same OR also converge
onto only one or a few glomeruli (Gao et al., 2000;Vosshall et al., 2000). Recent studies
essentially completed the OR-to-glomerulus map (Couto et al., 2005;Fishilevich and
Vosshall, 2005) (Figure 1.1.4). The ORNs axon projection map is highly rigid in a sense
that the expression of ORs in ORNs does not influence the targeting of glomeruli in the
antenna lobe (Dobritsa et al., 2003;Wang et al., 2003).
Different odorants activate distinct but overlapping subset of glomeruli and the number of
activated glomeruli increases with increasing odorant concentration, as revealed by
optical imaging (Ng et al., 2002;Wang et al., 2003) and metabolic labeling studies
(Rodrigues, 1988). Therefore, odor coding in the antennal lobe appears to involve a
spatial map of odorant receptor activation. Similar electrophysiological analysis revealed
that different odorants activate different populations of projection neurons, and PN
responses were found to differ in breadth of tuning, signaling mode and response
dynamics (Wilson et al., 2004).
Another interesting question raised from these studies is to compare the odorant receptive
field of antenna and the antennal lobe. Based on the optical imaging study, the pre- and
post-synaptic odor-evoked glomerular activity was compared (Ng et al., 2002;Wang et
al., 2003), and it was found that a given odor evokes essentially the same activation
pattern regardless of whether the reporter is driven pre- or post-synaptically, suggesting
that activation of a PN simply reflects activation of its pre-synaptic ORNs.
Another group using electrophysiological recordings obtained different conclusions.
Evidence was provided that PNs are more broadly turned than ORNs, suggesting that
12
Introduction
PNs output is not only modified by the input of ORNs, but also by lateral connections
within the antennal lobe.
Studies have shown that the odor-evoked activity in the mushroom body also has a spatial
pattern (Fiala et al., 2002;Wang et al., 2001). While the response pattern of PNs is
stereotypy, the response spatial pattern in MB appears to be highly inconsistent between
individual flies. Consistent with this observation, a lack of stereotypy among individual
flies was presented in the branching pattern of individual PN axon within the MB (Wong
et al., 2002). Although the functional significance of this variability is unclear, the key
role of the MB in olfactory learning and memory (Heisenberg, 2003;Murthy et al., 2008)
raises the possibility that it might reflect experience dependent plasticity.
A spatial map of odor representation is also likely to exist in the lateral horn, although a
functional analysis of these neurons has not yet been reported. Genetic labeling of
individual PN axons using either the mosaic analysis with a repressible cell marker
(MARCM) (Lee and Luo, 1999) or FLP-out (Basler and Struhl, 1994) techniques has
revealed that PNs that connect to different glomeruli show stereotyped axon branching
patterns within the lateral horn that are distinct but overlapping, thus, allowing for the
integration of olfactory information from multiple AL glomeruli (Marin et al.,
2002;Wong et al., 2002).
13
Introduction
Figure 1.1.4 Molecular neuroanatomy of the adult AL annotated with the molecular and functional
identity of the glomeruli.
Glomeruli receiving projections from the ORNs expressing a given OR or GR are indicated, with antennal
basiconic projections indicated in black, antennal trichoid projections in yellow, and antennal coeloconic
projections in green. Maxillary palps projections are in cyan, and unmapped glomeruli in black dotted line.
Glomeruli innervated by fruitless positive neurons are indicated in pink. This figure was adapted from
Vosshall and Stocker, 2007.
1.1.3
New insights of Drosophila odorant receptor
Structural analysis in silico, in vitro and in vivo surprisingly showed that insect ORs have
a flipped topology compared with conventional GPCRs, presenting a cytoplasmic N-
14
Introduction
terminus and an extracellular C-terminus (Benton et al., 2006;Lundin et al., 2007).
Furthermore, a study published last year, pointed out that insect ORs are heteromeric
ligand-gated non-specific cation channels (Sato et al., 2008). The authors showed that
simultaneous measurements of whole-cell currents and Ca2+ influx in HeLa cells
expressing insect ORs have a response onset about 10-fold faster than what is usually
required by GPCRs. Moreover, general pharmacological inhibition of G-proteins did not
impair OR-evoked responses, as would be expected if they were GPCRs. Single-channel
recordings revealed that the response of insect ORs was not dependent on cellular
cytoplasmic components, including second messengers such as cAMP and cGMP.
Finally, different subunit compositions of the OR complex are able to shift the ion
selectivity of the measured current. This is an important finding as ion selectivity is a
unique property of ion channels, making it unlikely that ORs are associated with a
separate ion channel and suggesting that ORs themselves are necessary and sufficient to
produce an odorant-induced response.
However, a contemporary publication came to a hypothesis as regards content between
the provocative ion channel and classical GPCR theories (Wicher et al., 2008). Wicher
and colleagues showed that activation of recombinant expressed Drosophila receptor
OR22a induced opening of a cAMP-dependent CNG channel, suggesting the
involvement of Gs proteins downstream of OR22a activation. Patch clamp recordings in
whole cell and excised patch configurations from HEK293 cells expressing OR22a and
OR83b revealed a fast odor-dependent response independent of ATP and GTP (a likely
ionotropic response) as well as a slower ATP- and GTP-dependent component. In
contrast to the study by Sato and colleagues (Sato et al., 2008), pharmacological analysis
performed by Wicher and colleagues (Wicher et al., 2008) indicate that the later
component of the odor response is mediated by a G-protein dependent signaling cascade
that includes Gs, adenylate cyclase and cAMP. Moreover, the co-receptor OR83b alone
can generate currents after an increase of intracellular cAMP/cGMP, similar to the
currents recorded after ligand application. Finally, a mutation in OR83b can directly
modulate the ion permeability of the OR complex, showing that this protein probably
participates in the formation of the channel complex without the involvement of other ion
channels. Together, this study suggest a model in which the unique OR subunit
15
Introduction
contributes ligand selectivity and G-protein coupling, and can mediate fast activation of
the OR83b dependent conductance; OR83b is also stimulated by cAMP, but on a slower
time course.
Moreover, one of the latest studies on insect olfaction has unraveled a new class of
olfactory receptors in Drosophila melanogaster that belong to the ionotropic glutamate
receptor family (iGluRs) (Benton et al., 2009;Spletter and Luo, 2009). Since the
comprehensive analysis of the expression of classical ORs in the Drosophila has been
established (Couto et al., 2005), the lack odorant receptor on coeloconic sensilla (except
for the OR35a/OR83b-expression neuron) has hinted the existence of the other types of
insect chemosensory neurons, it seems that Benton and colleagues have discovered the
missing puzzle pieces of the whole receptor to sensillum map. This study revealed that
iGluR-like receptors (IRs) are expressed in antennal sensory neurons and confer odor
dependent responses to cells. IRs expression patterns are complementary to OR83bexpressing neurons and might explain the remaining olfactory-mediated responses in
OR83b-null fruit flies. More importantly, this discovery highlights how multiple receptor
families can be recruited to perform similar functions in the same organ but it is yet to be
determined if IRs play a special role in fruit fly olfaction.
1.2 Heterotrimeric G-proteins

Identified by the subunit, heterotrimeric G-proteins are composed of subunits. They
transduce, amplify and diversify the signal generated by the occupancy of a receptor by
its hormone or agonist into regulation of one or more effector systems. Receptors activate
G-proteins by increasing the affinity of the G-protein (GDP) complex for magnesium
ions. Bound magnesium causes GDP to dissociate and allows GTP to bind. This is
followed by a subunit dissociation reaction that yields active G (GTP) plus a G
complexed to agonist-occupied receptor. The latter can dissociate further to give free
G. As signaling molecules both the G (GTP) and dimer modulate positively or
negatively a variety of effector functions (McCudden et al., 2005) (Figure 1.2.5).
The G subunits remain active until GTP is hydrolyzed to GDP. G (GDP) has high
affinity for G and reconstitutes into trimeric G-protein (GDP), which is ready for
another round of nucleotide exchange, activation by GTP and effector modulation
16
Introduction
through subunit dissociation. Alternatively G (GDP) may recombine with the still
associated with the agonist-receptor complex to give a quaternary agonist-receptor-Gprotein (GDP) complex ready to be activated.
Figure 1.2.5 Standard model of the GDP/GTP cycle governing activation of heterotrimeric GPCR
signaling pathway.
(McCudden et al., 2005) Ligand binding induces the G GDP dissociation from the trimric G complex.
Without the ligand, G acts as a guanine nucleotide dissociation inhibitor (GDI) for GGDP, slowing the
spontaneous exchange of GDP for GTP. After G GDP dissociation, ligand-bound GPCRs act as guanine
nucleotide exchange factors (GEFs) by inducing a conformational change in the G subunit, allowing it to
exchange GTP for GDP. The cycle returns to the basal state when G hydrolyzes the gamma-phosphate
moiety of GFP, a reaction that is augmented by GFPase-accelerating proteins (GAP) such as the Regulator
of G-protein Signaling (RGS) proteins.
16 genes are identified in mammalian gene families encoding G subunits, 5 genes

encoding for G, and 12 genes encoding for G subunits (Downes and Gautam, 1999).
G subunit proteins can be divided into four classes based on sequence similarity: G(s /
olf),
G(i / o / t / gust / z), G(q / 11), G(12/13) (Simon et al., 1991). Sizes of G subunits range
from 39 to 45 kilodaltons (kDa) (Nurnberg et al., 1995). G subunits contain two

domains: a nucleotide binding domain with high structural homology to the Rassuperfamily GTPase that is involved in the binding and hydrolysis of GTP and a allalpha-helical domain that buries the GTP within the core of the protein (Sprang, 1997).
G subunits contain three flexible regions designated switch-I, -II and -III that change
conformation in response to GTP binding and hydrolysis (Lambright et al., 1994) (Figure
1.2.6). The helical domain is the most divergent domain among G families and may play
a role in directing specificity of receptor- and effector-G-protein coupling (Cabrera-Vera
et al., 2003).
17
Introduction
G-protein signaling cascade is crucial for three out of five primary senses in vertebrates
(Liman, 2006), namely vision, taste and smell. Gt1 and Gt2, which are also called
transducin, are specifically expressed in the outer segments of rod and cone
photoreceptor, couple to rhodopsin and mediate the phototransduction (Goc et al., 2008).
Ggust, also named gustducin, is expressed in the taste buds, mediate the taste signaling
pathways (McLaughlin et al., 1992). A subunit G13, is also involved in the
mammalian response to sweet and bitter compound (Huang et al., 1999). Golf and the
olfactory signal transduction pathway will be discussed in detail in the next section.
Figure 1.2.6 Structural features of heterotrimeric G-protein subunits.

(McCudden et al., 2005) (A) The crystal structure of G subunit illustrates the Ras-like GTPase domain,
the helical domain and the bound nucleotide at the interdomain interface. Switch regions I, II and III are
shown in yellow, GDP in green and the phosphate binding loop in blue. (B) Close view of the guanine
nucleotide-binding pocket of the G subunit.
In the Drosophila genome, 11 genes are predicted to encode for G subunits; three for
subunits and two for subunits (Ishimoto et al., 2005). Some of those G-proteins have
been cloned and assigned to different classes (Lee et al., 1990;Ray and Ganguly,
1994;Schmidt et al., 1989;Schulz et al., 1999;Talluri et al., 1995;Wolfgang et al., 1991).
The function of G-proteins, especially the subunits, has been studied thoroughly in the
field of asymmetric cell division and polarization (Knust, 2001;Matsuzaki, 2005). In
Drosophila sensory system as in the mammals, G-proteins are critical for the vision and
taste. A Gq and a G protein were reported as the visual specific G-proteins (Lee et al.,
1994;Schulz et al., 1999). Gs and G1 were thought to be critical in sugar perception
(Ishimoto et al., 2005;Ueno et al., 2006).
18
Introduction
1.3 Signal transduction pathway in odorant receptor neurons

The canonical mammalian olfactory signal transduction pathway (Figure 1.3.7), involves
a G-protein specific to the olfactory system, termed Golf (Jones and Reed, 1989). Odorant
receptors are probably subjected to conformational rearrangements upon odorant binding,
similar to the light-induced structural changes of rhodopsin (Grobner et al., 2000). In
general, this conformational change initiates GDP release from the G-subunit that in
turn binds GTP and thereby adopts its active state. This triggers a structural
rearrangement in the heterotrimeric protein (Bunemann et al., 2003) and release of the
-subunit (Hamm, 1998;Janetopoulos et al., 2001). Thereby transmitting the signal from
the extracellular to the intracellular side of the membrane (Paysan and Breer, 2001). Most
odorant activated ORs are linked to the stimulation of adenylate cyclase III (AC3)
(Bakalyar and Reed, 1990) via Golf (Jones and Reed, 1989). Activation of AC3 elicits the
increase of cyclic adenosine-3, 5-monophosphate (cAMP) level in the cilia (Jones, Jr. et
al., 1974;Sinnarajah et al., 1998), which then leads to the opening of cyclic-nucleotide
gated cation channels (Nakamura and Gold, 1987;Zufall et al., 1993;Zufall et al., 1994).
The CNG channels are highly permeable to Ca2+ (Dzeja et al., 1999). The influx of
cations through CNG channels depolarizes the cilia membrane, elevates the intracellular
Ca2+ concentration, and then triggers a Ca2+-activated chloride conductance that
significantly amplifies the electrical signal. The ORNs return to the inactive state
following GTP hydrolysis, which is catalyzed by the GTPase domain of the subunit
(Markby et al., 1993). This is a classic cyclic nucleotide-mediated transduction pathway
in which all of the proteins involved have been identified, cloned, expressed and
characterized. Additionally, many of them have been genetically deleted from strains of
mice, making this one of the most investigated and best understood second-messenger
pathways in the brain (Firestein, 2001).
19
Introduction
Figure 1.3.7 The canonical mammalian olfactory signal transduction pathway.

(Firestein, 2001) Within the compact cilia of the ORNs a cascade of enzymatic activity transduces the
binding of an odorant molecule to a receptor into an electrical signal that can be transmitted to the brain.
AC, adenylate cyclase; CNG channel, cyclic nucleotide-gated channel; PDE, phosphodiesterase; PKA,
protein kinase A; ORK, olfactory receptor kinase; RGS, regulator of G-proteins (but here acts on the AC);
CaBP, calmodulin-binding protein. Green arrows indicate stimulatory pathways; red indicates inhibitory
(feedback).
Until recently, much of our view of insect olfactory signal transduction pathway was
strongly influenced by this canonical pathway in mammals in addition with some
observations that were made in other vertebrates, crustaceans and nematodes (Hildebrand
and Shepherd, 1997;Krieger and Breer, 1999). Such as in nematodes (Figure 1.3.8 B),
binding of odorants to ORs induces activation of Gi and increases the level of cGMP. The
increase in cGMP levels then triggers the opening of a cyclic nucleotide gated Ca2+
channel (Krieger and Breer, 1999). In lobster olfactory receptor neurons, two second
messengers, namely IP3 and cAMP are produced in response to individual odors
(Boekhoff et al., 1994;Fadool and Ache, 1994;Hatt and Ache, 1994;Michel and Ache,
1992). In insect olfactory system, various second messengers, such as IP3, DAG, cAMP
and calcium, were found to moderate mutiple types of ion channels (Kaissling,
1996;Zufall et al., 1991a;Zufall et al., 1991b;Zufall et al., 1994;Zufall and Hatt, 1991).
Several of the regular molecular suspects have also been identified and in part
functionally characterized in insect olfactory organs. These include odorant binding
proteins (Pelosi and Maida, 1995;Tegoni et al., 2004;Ziegelberger, 1996), heterotrimeric
20
Introduction
G-proteins (Laue et al., 1997), arrestins (Merrill et al., 2002;Merrill et al., 2003;Merrill et
al., 2005) as well as a CNG (Baumann et al., 1994;Dubin et al., 1998b) and a IP3-gated
ion channel (Stengl, 1994).
Before the identification of Drosophila OR genes, there were several clues that suggested
the involvement of GPCR-mediated second messenger pathways based on biochemical
and electrophysiological evidence and the identification of the components of the cAMP
and IP3 signaling pathways in the Drosophila olfactory system. Stimulation with odorants
or pheromones on isolated ORNs increases the production of second messengers like IP3,
and in vivo recordings from antennal neurons showed that action potentials are generated
when IP3 is directly applied to the cells (Stengl et al., 1992a;Stengl, 1993;Talluri et al.,
1995). In addition, reduced expression of the Drosophila Gq gene, dgq, and other genes
involved in phospholipid signaling induces a decrease of ORNs odor-evoked responses
but not their complete abolishment (Kain et al., 2008;Kalidas and Smith, 2002).
Furthermore, Drosophila mutants in phospholipid signaling have reduced olfactory
responses (Kain et al., 2009). These observations led to the assumption that insect
odorant responses are mediated by Gq-coupled GPCRs. However, other groups reported
that altering the expression of the genes rut and dnc, both of which affect the cAMP
transduction cascade, showed abnormal electrophysiological and behavioral responses to
odorants, suggesting that Gs is also involved in the transduction mechanism (GomezDiaz et al., 2004;Martin et al., 2001).
21
Introduction
Figure 1.3.8 Models of signal transduction mechanisms in odorant receptor neurons (ORNs).
(Pellegrino and Nakagawa, 2009) (A,B) Signal transduction of non-insect olfactory receptors (ORs). (A)
Mammalian ORs are G-protein-coupled receptors (GPCRs) coupled to a stimulatory G-protein, Golf. After
binding to an odorant, the G-protein activates adenylate cyclase (AC), which increases the intracellular
concentration of cAMP. This leads to the opening of a cyclic nucleotide-gated (CNG) channel and the
depolarization of the neuron. (B) In nematodes, stimulation of ORs leads to the activation of guanylate
cyclase (GC) and an increase in cGMP levels. This leads to the opening of a CNG channel and the
depolarization of the ORN. (C,D) Divergent views on signal transduction of insect ORs. (C) Sato et al.,
provides evidence supporting a model in which the OR83b/ORX complex forms an ion channel that is
directly opened by the binding of the odorants and is permeable to cations (Sato et al., 2008). (D) In
contrast, in Wicher et al.s model the ligand-binding subunit (in green) is a GPCR that leads to the increase
in cAMP through a stimulatory G-protein (Wicher et al., 2008). This opens the CNG-like channel OR83b
(in yellow).
22
Objective
2 Objective
In all animals studied, odorant detection is mediated by seven transmembrane domain
receptors. In vertebrates, the seven transmembrane olfactory receptors are coupled to
Golf signaling activating ACIII, which in turn induces an increase in cAMP levels
leading to activation of cyclic nucleotide gated ion channels followed by opening of
chloride conducting channels. Compared to this well-established mechanism, the events
that follow odorant receptor interaction in insects are still controversial. The role of Gprotein-coupled olfactory signal transduction in insect is confounded by the fact that the
highly conserved OR83b receptor heterodimerizes with conventional receptors and both
Drosophila and Bombyx mori OR/OR83b dimmers directly confers ligand stimulated
nonselective channel activity in heterologous systems (Nakagawa et al., 2005;Sato et al.,
2008;Wicher et al., 2008). Cell culture and in vivo studies exhibit that Drosophila OR83b
has an inverse topology compared to the conventional GPCR (Benton et al., 2006). These
findings urged the analysis of the role of G-protein-coupled signaling in invertebrates
olfactory transduction.
The present work aimed (1) to elucidate whether and which heterotrimeric G-proteins are
involved in Drosophila olfactory signal transduction (2) to study the role of second
messengers and (3) to characterize the function of other channels in Drosophila ORNs
and to identify insect olfactory signal inhibitors.
23
Materials and Methods
3 Materials and Methods

3.1 Materials
3.1.1
Fly food
Cornmeal-molasses-yeast medium, Blommington fly food recipe

Semi-defined medium, Blommington fly food recipe
3.1.2
Enzymes and Kits and molecularbiological supplies
Agarose, Biozym
Calf Intestinal Alkaline Phosphatase, Fermentas
dNTPs, Invitrogen
Ethidium bromide, AppliChem
Gene Ruler 1kb DNA ladder, Fermentas
Go-Taq Polymerase, Promega
NuSieve 3:1 Agarose, Biozym
pCDNA3, Invitrogen
Plasmid Maxi Kit, Qiagen
pUAST, Brand und Perrimon; P-Element-Transformations vector with Miniwhitemarkergene.
Pure Yield Plasmid Maxi-Prep System, Promega
T4 Ligase, Fermentas
Wizard SV Gel & PCR Clean Up System, Promega
3.1.3 Immunoblotting and Immunohistochemistry
Acrylamide, Sigma
AlexaFluor546, goat anti-rabbit/mouse IgG, Molecular Probes
AlexaFluor488, goat anti-rabbit/mouse IgG, Molecular Probes
Ammonium persulfate, J.T. Baker
anti-Actin, mouse monoclonal, Sigma
anti-DM-Gi, rabbit polyclonal, Juergen Knoblich, (Institute of Molecular Biotechnology,
Austria)
24

anti-DM-Go, rabbit polyclonal, Andrew Tomlinson, (Columbia University, USA)
anti-DM-Gq, rabbit polyclonal, Zvi Selinger (The Hebrew University of Jerusalem)
anti-GFP, rabbit polyclonal, Abcam #ab290-50
anti-m-Golf, rabbit polyclonal IgG, Santa Cruz Biotechnology
anti-m-Gq, rabbit polyclonal IgG, Santa Cruz Biotechnology
Blotting Grade Blocker, Non-fat dry milk, BIO-RAD
Complete Mini protease inhibitor cocktail tabs, Roche
Dithiothreitol (DTT), BIO-RAD
ECL Advance Western Blotting Detection System, Amersham/GE Healthcare
ECL PLUS, Western Blotting Detection System, Amersham/ GE Healthcare
Fatty acid-free Bovine Serum Albumin, Sigma
Gelatin from cold water fish skin, Sigma-Aldrich
Goat serum, Gibco
Immobilion transfer membrane, PVDF, Millipore
Isopropanol, J.T. Baker
Lysoszyme, Sigma
Methanol, J.T. Baker
Opti-tran BA-s85 reinforced Nitrocellulose, Schleicher & Schuell
Page Ruler Prestained Protein Ladder, Fermentas
Page Ruler Prestained Protein Ladder Plus, Fermentas
Paraformaldehyde, Prolabo
Ponceau S, Fluka Biochemika
ProLong Antifade mounting medium, Molecular Probes
Roti-Block, Roth
Sucrose, AppliChem
Top-Block, Fluka Biochemika
TMEMD, Fluka Biochemika
Triton X-100, Amersham Life Sciences
Tween-20, Amersham Life Sciences
25

3.1.4 Cell culture
Bovine Serum Albumin (BSA), Sigma
DMEM, Gibco
FBS, BioChrom
Penicillin/streptomycin, PAA
Trypsin-EDTA, PAA
3.1.5
Mammalian cell lines
HEK293 (Human embryonic kidney cell 293)

3.1.6
Buffers and Solutions
2X HBS: 280 mM NaCl, 50 nM HEPES, 2,8 mM Na2HPO4, pH 7.2

Blocking solution: (immunohistochemistry) 1% gelatin, 0.01% TritonX, 1-10% goat
serum in PBS-/Cell lifting buffer: 10 mM HEPES, 0.9% NaCl, 0.2 % EDTA, pH 7.4
Coomassie blue staining: 50% methanol, 10% acetic acid, 39.95% distilled H2O, 0.05%
coomassie blue
Destaining solution: 5% methanol, 7% acetic acid, 88% distilled H2O
DNA loading buffer: 20% ficoll 400, 100 mM EDTA, 0.025% bromophenol blue,
0.025% xylen cyanol
Drosophila ringers solution: 3 mM CaCl2, 182 mM KCl, 46 mM NaCl, 10 mM Tris-Cl,
pH 7.2
Electrophoresis buffer: 250 mM Tris, 191.8 mM glycine, 0.1% SDS, pH 8.3
Filter-wash buffer: 10 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4
GTP binding assay buffer: 10 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4,
guanosine 5-diphosphate (GDP)
Homogenization buffers A: 10 mM HEPES, 10 mM EDTA, pH 7.4
Homogenization buffers B: 10 mM HEPES, 0.1 mM EDTA, pH 7.4
Laemmli buffer: 0.125 M Tris, 4% SDS, 20% glycerin, 0.02% bromophenol blue, pH
6.8
Larvae Whole Mount Fixative: 4% paraformaldehyde, 0.1% Triton X-100 in PBS-/-, pH
7.2
26

LB-Agar medium: 1.5% Agar, 1% bactotrypton, 0.5% yeast extract, 1% NaCl, 100
g/ml Ampicillin, pH 7.2
Luria-Bertani medium: 1% bactotrypton, 0.5% yeast extract, 1% NaCl, 100 g/ml
Ampicillin, pH 7.2
PBS+/+: 136 mM NaCl, 2.68 mM KCl, 0.9 mM CaCl2, 0.48 mM MgCl2, 1.47 mM
KH2PO4, 8.1 m M Na2HPO4, pH 7.4
PBS-/-: 136 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4
Ringers solution: 140 mM glucose, 5 mM KCl, 10 mM HEPES, 2 mM CaCl2, 1 mM
MgCl2, 10 mM glucose, pH 7.4
SDS-PAGE buffer A: 0,5 M Tris, 0.4% SDS, pH 6.8
SDS-PAGE buffer B: 1,5 M Tris, 0.4% SDS, pH 8.8
Sensillum lymph ringers solution: 150 mM KCl, 20 mM NaCl, 2 mM CaCl2, 5 mM
glucose, 10 mM HEPES, pH 7.2
TBS: 150 mM Tris, 50 mM NaCl, pH 7.4
TBS-T: 150 mM Tris, 50 mM NaCl, 0.1% Tween20, pH 7.4
Transfer buffer: 20 mM Tris, 150 mM glycine, 20% methanol, pH 8.6
Tricholine citrate solution: 0.05 M choline-dihydrogen citrat, adjusted to pH 7.0 by
choline base solution
3.1.7
Odorants
1-octen-3-ol
1-hexanol
2-heptanone Amyl butyrate
2,3-butanedione
Benzaldehyde
Cyclohexanol
E2-hexenal
Ethyl acetate
Ethyl butyrate
Geranyl acetate
27

Isoamyl butyrate
Methyl salicylate
Pentyl acetate
Odorants were from Sigma-Aldrich and were of the highest grade available (97%-99%).
3.1.8
Miscellaneous
[35S]GTPS (approx 1250 Ci/mmol), NEN Life Science Products

AlphaScreen cAMP Assay Kit, PerkinElmer Life and Analytical Sciences, Inc.
DMSO, J.T. Baker
Forskolin, Sigma
FURA-2, Invitrogen
IBMX, Sigma
3.2 Methods
3.2.1
Fly rearing and fly stocks
All fly stocks were maintained on conventional cornmeal-molasses-yeast medium or semi

defined medium under a 12-hr-light 12-hr-dark cycle at 18 C or 25 C. The following fly
stocks were kindly provided: Or83b-Gal4 (John Carlson, Yale University, USA), UASmCD8-GFP, Gr21a-Gal4, Or22a-Gal4 (Bloomingtom Stock Center), UAS-Gs-wt and
UAS-GsQ215L (Cahir OKane, University of Cambridge, England), UAS-Go-wt, UASGoQ205L, UAS-Go-G203T, UAS<[w+]<Ctx and UAS<[w+]<Ptx (Andrew Tomlinson,
Columbia University, USA); UAS-Gq and UAS-GqQ203L, also referred as UAS-AcGq3
(Gaiti Hasan, Tata Institute of Fundamental Research, India); UAS-Gi-wt, UASGiQ205L and UAS-GiRNAi (Juergen Knoblich, Institute of Molecular Biotechnology,
Austria); UAS-Pac (Martin Schwrzel, Saarland University, Germany); Or83b2 (Or83b
knock-out fly) (L. Vosshall, The Rockefeller University, USA); Ih-channel knock-out fly
(Isabelle Canal, Universidad Autonoma de Madrid, Spain). Transgenic constructs were
injected into yw embryo by VANEDIS Drosophila injection service (Oslo, Norway)
using standard procedures.
28

3.2.2
Molecular cloning and plasmid construction
All constructs were cloned according to regular cloning procedures, as outlined in

Methods in Molecular Cloning (Sambrook, 2001). Briefly, the polymerase chain reaction
(PCR) was carried out under various conditions of annealing temperature and elongation
time, using the primers outlined below. The resulting PCR products were restricted using
various restriction enzymes and ligated into the appropriate linearized plasmid vectors.
The ligation product was transformed into E.coli XL1 blue competent cells. Positive
colonies were selected for using ampicillin. Correct orientation of the insert was verified
via restriction analysis. Positive clones were then retransformed and a single colony used
to inoculate 200 ml cultures overnight at 37C, shaking at 180 rpm. The bacterial cells
were collected by centrifugation and plasmid constructs isolated using the Plasmid Maxi
Kit from Qiagen or Pure Yield Plasmid Maxi-Prep System from Promega. All
constructs were verified by sequencing.
The N-terminus of the human G-protein alpha subunit 16 (G16C44) was amplified
from an existing construct (pCISG16 (Amatruda, III et al., 1991)) by PCR using primers
5-cgg gat cca tgg ccc gct cgc tga cc-3; 5-cgg ata tcg ccc tcg ggg ccg tcc ac-3. The
PCR fragment was cloned into the BamHI and EcoRV sites of the mammalian expression
vector pCDNA3 (Invitrogen). Different cDNA fragments coding for the Drosophila Gprotein alpha subunits C-terminus 44 amino acids were generated from Drosophila head
cDNA by polymerase chain reaction and fused into the EcoRV and XbaI sites of the
pCDNA3-G16-C44 construct. The primers used for the PCR were: Gs-fw, 5-gct agc
gga gac gga aaa c-3, Gs-rw, 5-cgt cta gat agt aac aat tca tat tga cga agg-3; Gq-fw, 5cat tag ttt aga tac ata taa-3, Gq-rw, 5-cgt cta gat aga aac aga tta ctt tct ttt agg-3; Gofw, 5-aac aaa tca acc tca aaa g-3, Go-rw, 5-cgt cta gat agg tac agt cca cag ccg cg-3;
Gi-fw, 5-aac aag cga aaa gac caa aag g-3, Gi-rw, 5-cgt cta gat agg aat aag cca att tgt
ttc ag-3; G73B-fw, 5-ctg ggt acc tcg gaa agg gag-3, G73B-rw, 5-cgt cta gat agg aat
agg ccc atg ctg gac ac-3.
A Gs-GFP fusion construct was generated from GFP and Drosophila Gs using
polymerase chain reactions that produced DNA fragments with overlapping ends that
were combined subsequently in a fusion polymerase chain reaction. GFP was inserted
between Gs residues 71 and 72. A 6-residue linker sequence (SGGGGS) was inserted at
29

both of the junctions between Gs and GFP. The GFP with linkers was generated first by
polymerase chain reactions, then the linker GFP was used as template in the fusion
polymerase chain reactions. The primers used for the PCR were: GFP-linkerf: 5-tca ggt
gga gga ggt tct gtg agc aag ggc gag gag c-3, GFP-linkerr: 5-gct gcc gcc gcc gcc gct ctt
gta cag ctc ctc cat gc-3, Gs1f: 5-ggc cag atc tac cat ggg ttg ctt tgg gtc gcc c -3, Gs1r:
5-cac aga acc tcc tcc acc tga tcc gtc gac atg caa tat tcg c-3, GFPsf: 5-gaa tat tgc atg tcg
acg gat cag gtg gag gag gtt ctg tga gc-3, GFPsr: 5-ctg ttt ctt ttc cga gtc aga aaa gct gcc
gcc gcc gcc gct c-3, Gs2f: 5-gcg gcg gcg gcg gca gct ttt ctg act cgg aaa aga aac ag-3,
Gs2r: 5-gcg cgg ccg cct ata aca att cat att gac g-3. The fusion fragment of Gs-GFP was
inserted in the BglII/NotI site of pUAST. All constructs were verified by sequencing.
3.2.3
Cell culture and transfection
Reagents for cell culture use were purchased from Invitrogen, unless stated otherwise.
Human embryonic kidney (HEK) 293 cells were maintained in Dulbeccos Modified
Eagles Medium (DMEM), containing 10% FBS, 100 units/ml penicillin/streptomycin and
2 mM/l glutamine, to a confluency of ~70%. 5 - 10 g plasmid DNA was transfected per
35x10mm dish (Falcon). HEK293 cell transfections were performed using a standard
calcium phosphate precipitation technique: The appropriate volume of plasmid DNA
solution and CaCl2 was added to H2O to have a final volume of 100 l. 100 l 2XHBS
was then added drop-wise before the solution was gently mixed, incubated at room
temperature for 15 minutes and finally added drop-wise to the cells. 4 - 12 hours posttransfection, the medium was exchanged with fresh DMEM. Cells were subjected to
experimentation 48 hours post-transfection to allow for an optimum level of olfactory
receptor expression.
3.2.4
Ratiometric calcium imaging in HEK293 cells
HEK293 cells were washed once in PBS+/+ and loaded with a calcium-sensitive
fluorometric dye FURA-2-AM (Molecular Probes) for 45 minutes in the dark. FURA-2AM is a ratiometric calcium indicator and exhibits an absorption shift upon Ca2+ binding.
30

This shift can be observed by scanning the excitation spectrum between 300 and 400 nm,
while monitoring the emission at ~510 nm.
Ratiometric calcium imaging was performed as described (Spehr et al., 2003) using an
inverted microscope equipped for ratiometric imaging. Images were acquired from
randomly selected fields of view, and integrated fluorescence ratios (f 340/f 380 ratio)
were measured. WinNT based T.I.L.L Vision software was used to collect and quantify
spatiotemporal Ca2+-dependent fluorescence signals. At time-point 20 seconds, odorant
(500 M) was applied for 10 seconds using a specialized micro-capillary application
system, followed by washing with Ringers solution for 30 seconds. At the end of each
experiment, 20 M ATP was applied to the cells as a positive control.
3.2.5
RNA isolation, reverse transcription, RT-PCR and Real-time quantitative

PCR
The third antennal segments were collected from 100 flies (3-5 days old). RNA was
isolated with Trizol Reagent (Invitrogen) and cDNA was synthesized using MMLV
reverse transcriptase (Invitrogen).
Real time quantitative PCR was performed in Bio-Rad iQ5 Real-Time PCR Detection
System using Bio-Rad iQ SYBR Green Supermix kit, according to manufacturers
recommendations. The following amplification protocol was used: 40 cycles of 15 s at 95
C, 20 s at 60 C and 40 s at 72 C. Expression of the target gene was normalized to
Drosophila rp49 (ribosomal protein 49) RNA levels. The delta Ct (cycle threshold)
method was used to calculate relative expression levels, as previously described (Livak
and Schmittgen, 2001). Results are reported as fold change in gene expression relative to
control conditions.
The following primer pairs have been used in RT-PCR and real-time quantitative PCR:
CG2812f11:
5-AAT CGA GGG ACC TGG ATT TG-3
CG2812r255:
5-ATG GTG TTT CGT GCG CTT G-3
CG3004f47:
5-ACA CCA TTA AGG TGT GGC AGG-3
CG3004r203:
5-TTG GAC TCC AGG TCG TAC AGC-3
CG17760f406:
5-AAG GAG TGC TAC AAT CGT CG-3
31

CG17760r612:
5-TCG CTG ACC AGC GAC GTC CAC C-3
CG17766f392:
5-TCG AAC TCG GAT TCG GAA TTG-3
CG17766r611:
5-TGG TGC GCT TTA CCC AAT CTG-3
CG30054f833:
5-TGT ATT CGC ATT TGG TAG AC-3
CG30054r1019:
5-TCC TTA ACT GCA GCG AAC AC-3
Gsfa440:
5-TTC TTC AAA CCT ATG AGA GG-3
Gsra661:
5-TCC TAC GCT CGT CCC GCT GG-3
Gsbf428:
5-AAG ACA AGG GCG TTC TTC AA-3
Gsbr639:
5-ACC GAC ATC GAA CAT GTG AA-3
G49bQ3af471:
5-TCT CGA TCG TGT GGC TCA ACC-3
G49bQ3Ar768:
5-AGG GTA TGT AAT TAT AGT ACG-3
G49bretinalqaf805:
5-AAG AAG GAC TTG TTG GAA GAG-3
G49bretinalqar1028: 5-ATA ATT GTA TCT TTG ACA GC-3

G49BQ3bf534:
5-GCC CAC AAC AGG GAT AAT TG-3
G49BQ3br786:
5-TGA CGA ATT TTG AAA CCA AGG-3
G49bretinalqbf766:
5-CCT TGG TTT CAA AAT TCG TCA-3
G49bretinalqbr1019: 5-TCT TTG ACA GCG CAG AAC AC-3

Galpha73Bf323:
5-ATT TCG GCA GCT GTA CCA GCG-3
Galpha73Br567:
5-GTG CAG AAT GTC CTC GGT GC-3
CG7095f1588:
5-TTC TGG ATA GCA GTC AAT CG-3
CG7095r1709:
5-TTG CCA TCA ATA TTA ATC TCG-3
3.2.6
[35S] GTPS binding assay
ransfected cells were homogenized in 10 mM HEPES, 10 mM EDTA (pH 7.4) and

membrane preparation was performed as described previousely (Dowling et al., 2004).
[35S] GTPS binding was assayed in a final volume of 100 l (pH 7.4), containing 10
mM HEPES, 100 mM NaCl, 10 mM MgCl2, 100 mM guanosine 5-diphosphate (GDP)
and 0.1 nM [35S] GTPS. The incubation was started by the addition of membrane
suspension (about 50 g of membrane protein per reaction) and was carried out in
triplicate for 30 min at 30 C. The reaction was terminated by addition of 3 ml of ice-cold
32

filter wash buffer and rapidly vacuum filtered through glass fiber filters (Whatman
GF/C). Filters were washed three times with 3 ml of wash buffer and transferred to
scintillation vials. The scintillation cocktail was added and counted in a Packard Tri-Carb
liquid scintillation counter.
3.2.7 Larval chemotaxis assay

Chemotaxis assay was performed as previously described (Heimbeck et al., 1999). Daily,
flies were transferred into a fresh food vial where they could lay eggs until the following
day when they were transferred to a fresh vial once again. When third-instar larvae
appeared in the vial, usually at 108 h after beginning of the egg laying period in a 25C
culture condition, a 15% sucrose solution was used to wash out the larvae from the food.
After washing twice with distilled water, 50 larvae were used immediately.
Tests were done on Petri dishes (diameter, 85 mm) covered with a layer of 1.5% agarose.
Plates were air-dried before use to avoid diffusion of the test substance. Odorant and
control diluent (water or paraffin oil) were placed on lids of 1.5 ml eppendorf micro test
tubes to avoid diffusion through the agarose. 50 larvae were handpicked in the center of
the plate before adding the test substances. The dish was immediately covered with the
lid. After 5 min, larvae were counted as shown in Figure 3.2.9 A. Only larvae on
semicircular areas (radius, 30 mm) around the filter disks were included. Thus, the
animals had to move at least one body length toward the source. We then calculated a
response index (RI): (Ns Nc) /(Ns + Nc). Ns represents the number of animals at 30 mm
from the odorant source (inside area S in Figure 3.2.9); Nc is the number of larvae found
inside an identical surface on the opposite (control) side. Positive RIs indicate attraction;
negative RIs indicate avoidance; and RI = 0 indicates indifferent behavior.
33
Figure 3.2.9 Larval chemotaxis assay

Schematic representation of the test setup. Small filter paper containing a test chemical (S) and control
diluent (C) are placed on opposite sides of a Petri dish covered with a layer of agarose. Fifty animals are
transferred to the start point and counted after 5 min in indicated semicircular areas. (Heimbeck et al.,
1999)
3.2.8 Electroantennogram (EAG)

The EAG responses of wt and mutant flies were recorded as previously described
(Neuhaus et al., 2005;Stortkuhl and Kettler, 2001). Briefly, 2 or 3-day-old flies were
mounted in truncated micropipette tips with the anterior portion of the head protruding
from the end of the tip. The reference electrode was inserted into the haemolymph of the
head capsule. The recording electrode was placed on the frontal surface of the anterior
aspect of the antenna. After obtaining a stable baseline, EAG recordings were initiated by
a short odor pulse (0.3 s, precisely controlled by the odorant application system), applied
into an air stream that was directed toward the antenna (Figure 3.2.10). All odorants were
dissolved in paraffin oil at given concentrations. Odorants used were cyclohexanol,
benzaldehyde, heptanone and ethyl acetate, etc.
34
Figure 3.2.10 Schematic overview of the EAG recording

(A) The reference electrode was inserted into the haemolymph of the head capsule. The recording electrode
was placed on the frontal surface of the anterior aspect of the antenna. (B) Illustration of a typical EAG
recording setup.
3.2.9
Antennal single sensillum recording
The procedures of extracellular single sensillum recording was essentially similar as

previously reported (Yao et al., 2005). Canton-S flies aged < 1 week were used for wildtype recordings. hs-flp; Or83b-Gal4, UAS<[w+]<CTX flies were recorded after standard
heat shock (one hour, 38C) at age 4-5 days. Electrical activity of the neurons was
recorded extracellularly by placing an electrode filled with sensillum lymph ringer
solution in the base of the sensillum. A reference electrode filled with the same ringer
solution was placed in the eye (Figure 3.2.11). Signals were amplified using a patchclamp amplifier (L/M-PC, List-Medical Electronic, Darmstadt, Germany) set in voltageclamp mode and fed into a computer via a 16-bit analog/digital converter (Digidata
1200A; Molecular Devices, CA, USA). Electrophysiology data were recorded by the
WinEDR software (Strathclyde Electrophysiology Software, University of Strathclyde).
Impulses during the 1 s period before stimulation and the 1 s during stimulation were
counted off-line using the Mini Analysis software (Synaptosoft, Decatur, GA, USA). The
spikes were sorted into different groups according to their amplitude as previously
described (deBruyne et al., 2001). Each sensillum was tested with multiple odorants, and
no more than two sensilla were analyzed per fly.
35
Figure 3.2.11 Schematic overview of single sensillum recording

Electrode positions for the extracellular recording of voltage differences between the sensillum lymph (L)
and the haemolymph
AC, auxiliary cells; EC, epidermal cells. Figure adapted from de Bruyne et al., 1999
3.2.10 Labellar single sensillum tip recording

The single sensillum tip recordings from fly labellum were preformed on the same setup
as the recording from the antenna. Except for substance application and electrode placing,
most of the experimental procedures and data analysis were similar to the antennal single
sensillum recording. Particularly, the recording electrode was placed on the tip of the
sensillum, and the gustatory substance was applied in the recording electrode. A
tricholine citrate solution was used to dissolve the stimulation substance to inhibit the
water response of gustatory receptor neurons (Ishimoto and Tanimura, 2004) (Figure
3.2.12).
Figure 3.2.12 Schematic overview of single unit tip recording from GRNs
The electrode is used for both stimulating and recording (Ishimoto and Tanimura, 2004).
36

3.2.11 Odorant application system in EAG and single sensillum recording
Flies were under a constant stream of humidified charcoal-filtered air (2.4 l/min, 22-25
C) to prevent any potential environmental odors from inducing activity during these
studies. Pure odorant substance was diluted in paraffin oil, and 50 l was applied to a
filter paper and put into a 5 ml syringe, the volume of the syringe was adjusted to 4ml,
and an air stream of 0.4 l/min was applied to go through the syringe for one or three
second. The odorant was then delivered to the fly with the background air. At the same
time, an equal amount of air was cut from the background to make the input air pressure
on the fly constant before and after the stimulation. An electronic device controlled this
application system, and this device is connected to the digital converter, thus the
application process was recorded together with the electrophysiological recording.
Since this odorant application system was controlled by the flowmeter, and the odorant
was contained in a sealed syringe, by weighting the syringe before and after the
application, combined with the whole air volume that pass though the fly, the exact
concentration of the odorant that delivered to the fly could be calculated.
3.2.12 Immunohistochemistry
Fly heads were cut, fixed for 3 hours in 4% paraformaldehyde at 4 C and subsequently
incubated overnight in 25% sucrose in Drosophila Ringers solution. Cryosectioning was
performed to produce 12 m sections. After blocking with 5% goat serum in Drosophila
Ringers, the primary antibody was applied to the sections overnight at 4 C or 3 hours at
room temperature. Subsequently slices was washed with PBS containing 0.01% triton X100 (30 min, 3 times) and the secondary antibody coupled to A546 or A488 (Invitrogen)
was applied for 1 hr at room temperature. After washing again (30 min, 3 times), slices
were mounted in ProLong anti-fading mounting medium (Molecular Probes). Pictures
were taken with a Zeiss confocal microscope (LSM510 Meta; Zeiss).
37

3.2.13 Whole mount fluorescent antibody staining of Drosophila larvae
Larvae were removed from the culture vial and transferred into a clean plastic Petri dish
with fixative after food residues were rinsed away. The larvae were dissected in the
fixative solution, the anterior tips, including the brain, imaginal discs, salivary gland and
mouth hooks, were used for further staining. Fixation was carried out on ice for 1 hour.
The larvae pieces were washed with 0.1% Triton X-100 (PBS-Triton), 3 times 10 minutes
at room temperature. The tissue was then blocked for 30 min at RT (1XPBS, 0.1% Triton
X-100, 5% heat inactivated normal goat serum). Afterwards the block solution was
replaced with primary antibody solution, incubated at RT for 2 hours, followed by
washing 3 times 10 minutes with 0.1% Triton PBS. Secondary antibody was then applied
for 1 hour at RT in the dark and samples were washed subsequently three times for 10
minutes with 0.1% Triton PBS. Finally, the tissue was mounted and pictures were taken
with a Zeiss confocal microscope (LSM510 Meta; Zeiss).
3.2.14 SDS gel electrophoresis and Western-blotting

200 or more antennae were homogenized in Laemmli buffer (30% glycerol, 3% SDS, 125
mM Tris/Cl, pH 6.8), resolved by 10% SDS-PAGE and transferred to nitrocellulose
membrane (Protran; Schleicher & Schuell). The membranes were blocked with 5% nonfat dried milk (Bio-Rad) in TBS-T and incubated with primary antibody (diluted in TBST). After washing and incubation with HRP coupled secondary antibodies, detection was
performed with ECL plus on Hyperfilm (Amersham).
3.2.15 cAMP assay
PerkinElmer alpha screen cAMP assay kit was used for the measurement. Antennae were
cut manually from 2-5 days old adult flies, and immediately homogenized. The
homogenized tissue of approximately 10-15 antennae was finally aliquoted into each
reaction well. The assay was then performed according to the manufacturers instruction.
38
Results
4 Results
4.1 Different G subunits are expressed in Drosophila antenna.
Drosophila ORs were originally classified as G-protein-coupled receptors, assuming
expression of G-proteins in Drosophila antennae. We therefore set out to determine Gprotein expression and designed RT-PCR, western blot, and immunostaining experiments
to study the expression level and expression pattern of G-protein alpha subunits in the
antenna.
4.1.1
RT-PCR analysis of G subunits in Drosophila antenna
By searching the database of Drosophila genome, we collected the sequences of 10

transcript variants translated from 9 genes that were classified to encode for
heterotrimeric G-protein alpha subunits, designed primers against those variants and
performed RT-PCR. G q3 and the retina specific G q1 are transcribed from Dmel\G49B
(CG17759) (Talluri et al., 1995). Dmel\Gs60A has two transcript variants, which differ
by inclusion or deletion of three amino acids and substitution of a Ser for a Gly (Quan
and Forte, 1990). We could show that both of the transcripts are expressed in the antenna,
however primers used here were designed for the identical sequence of these two
transcript variants. About 200 antennae were cut manually; RNA isolation and cDNA
synthesis were subsequently performed. RT-PCR results revealed expression of all G
subunits transcripts in the antenna, with a higher expression level of Gi, Gq1-retinal,
Gq3, Gs, CG17766, CG17760 and Go as compared to that of Gf, CG3004 and
CG30054 (Figure 4.1.13 A).
Heterotrimeric G-protein subunits are divided into 4 classes based on their sequence
similarity and downstream effectors. Together with some well-identified G subunits
from other species, a phylogenetic tree of G subunits was generated by the MegAlign
program using the Clustal V method (Figure 4.1.13 B). As shown, 3 classes of G can
also be identified from Drosophila. The ungrouped G-proteins are Gf, CG3004 and
CG17766, which show a low similarity with those classified G. These proteins also have
relatively low expression level in antenna from the RT-PCR result, they were not directly
investigated in my study.
39
Results
Figure 4.1.13 PT-PCR analysis of G subunit expression in Drosophila antenna.

(A) RT-PCR analysis showed that most of the G subunits found in the Drosophila genome are expressed
in the antenna. (B). The phylogenetic tree of G subunits from Drosophila. melanogaster, Mus. musculus,
Homo sapiens, Anopheles gambiae, Panulirus argus and Rattus norvegicus.
4.1.2
Western-blot analysis showed that different classes of G are expressed in

the antenna
Fly antennae were collected manually as described before, followed by a standard

western-blotting procedure. Antibodies against Gs, Gq, Gi and Go were used. Each
lane contained protein of approximately 50 antennae. The blot shown in Figure 4.1.14 A
confirms expression of Gs, Gq, Gi and Go in the antenna. Gs was detected by a
mouse Golf antibody, since Drosophila Gs shows strong homology to vertebrate Golf,
especially at the C-terminus (Figure 4.1.14 B). The anti-Gq antibody purchased from
Santa Cruz Biotechnology has been raised
against
the
C-terminal
peptide
(FAAVKDTILQLNLKE YNLV) of mammalian Gq. This differs from the corresponding

Drosophila Gq3 sequence by a single residue (FAAVKDTILQSNLKEYNLV)
(Ratnaparkhi et al., 2002). Anti-DM-Go was provided by Zvi Selinger (The Hebrew
University of Jerusalem), and anti-DM-Gi was provided by Juergen Knoblich (Institute of
Molecular Biotechnology, Austria).
40
Results
Figure 4.1.14 Western blot analysis of G-protein subunits expression in antenna.

(A). Western blot analysis detected the expression of Gs, Gq, Gi and Go in antenna. (B). Alignment of
Drosophila Gs, human Gs, and human Golf shows a high degree of homology between these sequences,
especially at the C-terminus.
4.1.3
Immunohistochemical analysis of the expression patterns of different G

proteins in Drosophila antenna
Although RT-PCR and western-blot analysis strongly supported the idea that G-proteins
are present in fly antenna, we next attempted to study their cellular localization in the
41
Results
antenna. Therefore, we performed immunostaining experiments. As mentioned before,
ORNs are not the only cell type in the antenna; cell bodies of the ORNs are surrounded
by specialized auxiliary cells and epidermal cells. For the purpose of marking the ORNs,
the UAS-GAL4 system was used to generate a fly line that expresses a membrane
targeted GFP (mCD8-GFP) under the control of an OR83b driver. As described
previously, OR83b serves as a general ORN marker. In this fly line, OR83b positive cells
are all labelled with GFP (Figure 4.1.15 B). The UAS-GAL4 system is a biochemical
method used for the ectopic overexpression of transgenes. GAL4 encodes a protein
identified in the yeast Saccharomyces cerevisiae as a transcription activator and UAS
(Upstream Activation Sequence) is a short section of the promoter region to which the
Gal4 protein specifically binds to activate gene transcription. For studies in Drosophila,
the GAL4 gene is placed under the control of a native gene promoter, or driver gene,
while the UAS controls the expression of a target gene. Gal4 is then only expressed in
cells where the driver gene is usually active. In turn, Gal4 should only activate gene
transcription where a UAS has been introduced (Figure 4.1.15 A).
Immunostaining was performed on the antennae cryosection of the OR83b-GAL4, UASmCD8-GFP fly. Go and Gi were homogeneously expressed in the whole antenna (Go
data not shown), while Gq was detected in a subset of ORNs, and Gs was found to be
expressed in the dendritic part of the ORNs, where the initial olfactory signal
transduction takes place, suggesting that Gs may play an essential role in the olfactory
signal transduction pathway (Figure 4.1.15 C).
42
Results
Figure 4.1.15 Immunohistochemical analysis of G-protein subunit expression patterns in antenna.

(A) The UAS-GAL4 system. (B) Front view of the OR83b-GAL4; UAS-mCD8-GFP fly. Antennae and
maxillary palps express GFP. Scale bar = 100 m. (C) Immunostaining showing different expression
patterns for different G proteins. Scale bar = 20 m.
4.1.4
Real-time quantitative PCR analysis of Gs and Gq expression in Drosophila

antenna and head
Since both Gs and Gq show an interesting expression pattern in the antenna, a real-time
quantitative PCR was performed to compare the mRNA level of those two proteins in the
antennae and the head. Antennae and fly head cDNA were synthesized and qPCR was
carried out. The delta ct method was used to analyze the relative gene expression level.
The G-protein expression levels were first normalized to the endogenous control gene
rp49, using the expression level in the female head as control. The data shown here are
presented as percentage of controls. The results indicate that Gs expressed in the
43
Results
antennae was about 40% of the amount expressed in the female head, and the Gq3 in
antenna was about 80% compared with the female head (Figure 4.1.16). However, a
student t-test showed that the differences between the antenna, male head and female
head are not significant.
Figure 4.1.16 RT-qPCR analysis of Gs and Gq expression level in antenna.

(A) The relative Gs expression level in Drosophila antenna, male head and female head. Gs expression
level in the antenna was about 40% of that expressed in the female head. The Gs expression level of the
male head is significantly higher than that of the female head (p = 0.024). The difference between antenna
and male, female head are significant (p < 0.01). (B) The relative Gq3 expression level in antenna, male
head and female head. The expression level of Gq3 expression in those tissues showed no significant
difference. Error bars represent S.E.M.
4.2 Functional study of G subunit in Drosophila olfactory signal

transduction pathway
4.2.1
Overexpression study of different G subunits and G-protein specific protein

toxins in Drosophila ORNs
Due to the rapid expansion of the use of the UAS-GAL4 system in the last 20 years, it
becomes fairly easy to target gene expression in Drosophila. In our study, we collected
44
Results
most of the available UAS fly lines of G-proteins and toxins (Hampoelz et al.,
2005;Katanaev et al., 2005;Ratnaparkhi et al., 2002), crossing those flies with the
OR83b-GAL4 driver line, to generate different G-protein or G-protein specific toxin
overexpression flies. With the use of the OR83b-GAL4 driver, protein overexpression
was targeted specifically to the ORNs. If the G-proteins were essential for the olfactory
signal transduction, we expected olfactory aberration to occur in some of those transgenic
flies. We screened those flies for olfactory deficits by using EAG recordings, and it
turned out that the only fly line showing a severe defect in odorant induced signaling was
a fly line expressing CTX, which is an ADP-ribosyltransferase that typically activates
Gs proteins (Figure 4.2.17 A). Data are shown for ethyl acetate stimulation. Overexpression of wt Go, GTPase deficient (active) Go, constitutively GDP bound
(inactive) Go, pertussis toxin (PTX), GTPase deficient (active) Gq3, wt Gs, GTPase
deficient (active) Gs, wt Gi, GTPase deficient (active) Gi, and Gi RNAi did not show
any defect in EAG recordings. Other odors, for example cyclohexanol, benzaldehyde and
heptanone were also tested on those flies and similar results were obtained from EAG
recordings.
4.2.2 Dose-response curves of EAG responses from CTX and control flies.
By measuring the EAG response amplitude of G-protein, mutant G-protein and toxin
overexpression flies, we found that the expression of CTX in ORNs can cause a reduction
in EAG amplitude. CTX is a protein complex secreted by the bacterium Vibrio cholerae.
The cholera toxin is an oligomeric complex made up of six protein subunits: a single
copy of the A subunit, and five copies of the B subunit. Subunit B is responsible for the
toxin binding to the cell surface, and induces endocytosis of subunit A, which acts as a
toxin: once inside the cell, it ribosylates Gs and leads to a constitutive cAMP production
(Zhang et al., 1995). The cholera toxin subunit A is generally used in heterotrimeric Gprotein studies as a non-cytotoxic and irreversible activator of Gs (Burton et al., 1991).
Due to its potential toxicity, the protein toxins CTX and PTX were expressed under an
inducible heat-shock promoter based on the yeast Flippase/FLP recognition target
(FLP/FRT) recombination strategy (Strapps and Tomlinson, 2001). The CTX or PTX
gene in the UAS response line was intruded by a marker gene and a stop codon, thus the
45
Results
toxin expression can start only after the removal of the insertion part. The insertion part
was cut out by the activation of the flippase gene, which was under the control of a heat
shock promoter (Figure 4.2.17 G). To ensure that the observed defects were not caused
by the heat shock process itself, we tested wt flies, OR83b-Gal4; UAS-CTX, hs-flp flies
(CTX), heat shocked OR83b-Gal4; UAS-CTX, hs-flp flies, (CTX hs), and heat shocked
wt flies with four different odorants of different concentrations. We found that only heat
shocked CTX expressing flies showed EAG response deficits (Figure 4.2.17 C-F). CTX
expression did not alter the expression level or cellular distribution of Gs, and did not
disturb the general cellular morphology in the antenna (Figure 4.2.17 B).
Figure 4.2.17 Screening of G-proteins for participation in the olfactory signal transduction
(A) UAS constructs of different G-proteins, mutated G-proteins and G-protein affecting toxins were
expressed in the sensory neurons of the third antennal segment using an OR83b-Gal4 driver line. EAG
amplitudes (mV) in response to application of ethyl acetate (pure) were recorded. Error bars represent SD.
(B) Expression pattern of Gs in the antenna of wt and CTX expression flies. (C-F) EAG amplitudes (mV)
upon exposure to different concentrations of 4 odorants in wt flies, OR83b-Gal4; UAS-CTX flies (CTX),
heat shocked OR83b-Gal4; UAS-CTX flies (CTX hs), and heat shocked wt flies (wt hs). Error bars
represent S.E.M. (G) Schematic representation of the CTX expression strategy. The white gene and a stop
codon were inserted upstream of the toxin gene. Once the FLP is activated, it will induce the recombination
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between two FPT sites, the white gene plus stop code will be cut out, and then the protein toxin will be
expressed in the target cell.
4.2.3 CTX blocks the olfactory response of ORNs in Drosophila

To further investigate how CTX affects the odorant response on a cellular level, we
performed single sensillum recordings from CTX flies. Single sensillum recording
usually monitors all of the neuronal activities from one sensillum, and the spikes from
different neurons can be resolved into different populations. The sensillum we focused on
here was ab1 (antenna basiconic sensillum 1), which compartments 4 neurons: 3 ORNs
(neuron A, B and D) and one GRN (neuron C). 4 classes of spikes could be distinguished
in the recording traces (Figure 4.2.18).
Figure 4.2.18 Single sensillum recording from wild type ab1 sensillum.
4 populations of spikes can be resolved in the recording trace.
The response of the ab1 sensillum is shown in Figure 4.2.19 A-E. The ab1 sensillum
contains three neurons that express OR83b, and the one expressing the gustatory
receptors Gr21a and Gr63a, which are thought to respond to CO2 (Jones et al.,
2007;Kwon et al., 2007). In our recordings we found that expression of CTX not only
inhibits the response to odorant stimuli, but also blocks the spontaneous activity of ORNs
(Figure 4.2.19 B, G). Only one population of spikes could be observed in the CTX
expressing ab1 sensillum, and odorant responses were abrogated (Figure 4.2.19 B). In
addition to the comprehensive ORNs driver OR83b-GAL4, other OR drivers that only
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direct the expression of one class of ORNs were also used for this study. As expected,
when CTX was expressed in a single class of ORNs, for example OR22a neurons, the
neuronal activity of the target ORNs was disrupted (Figure 4.2.19 F, G). The ab3
sensillum normally houses two ORNs, both of which respond to 2-heptanone (Figure
4.2.19 F). Expression of CTX in ab3A neurons causes loss of spontaneous activity and
only one group of neuronal activity can be observed in the recordings, which is that of the
ab3B neuron still responding to the odorant (Figure 4.2.19 G).
Figure 4.2.19 Single sensillum recordings from CTX and control flies
(A) ab1 sensillum response to ethyl acetate. (B) CTX expressing ab1 sensillum showed only one group of
neuronal activity, and does not respond to ethyl acetate. (C) Wt ab1 neuron responses to CO2. (D) The CTX
expressing ab1 sensillum showed only one group of neuronal activity and this neuron also responds to CO2.
(E) Expression of CTX in Gr21a neurons does not influence the CO2 response. (F) The ab3 sensillum
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responds to 2-heptanone. (G) In the ab3 sensillum, OR22a-GAL4 driven expression of CTX leads to
silencing of the OR22a neuron (ab3a neuron). The ab3A neuron activity was gone, however the ab3B
neuron still responded to 2-heptanone.
4.2.4
CTX does not act as a non-specific toxin on cells
We already showed that CTX expressing sensilla have a normal morphology and results
from two further experiments also support the idea that CTX does not act as a toxin in
cells.
In the first set of experiments we were able to show that CTX expression in gustatory
sensory neurons does not impair their normal responses to the stimuli tested. The driver
lines we used were Gr21a-GAL4 and Gr5a-GAL4, and no response alterations could be
observed in the recording traces (Figure 4.2.19 C, E and Figure 4.2.20 A, B), which as
well means that the gustatory response of Gr21a and Gr5a neurons is independent of a
Gs-coupled pathway.
Additionally we performed larval chemotaxis assays on the OR83b-GAL4; UAS-CTX
larvae. Unlike in adult flies, CTX expression in larvae did not influence their olfactory
response. Details of the assay are described in part 5.4.2.
Figure 4.2.20 Gr5a-CTX GRNs response to sucrose

(A, B) Labellar single sensillum tip recordings showed both Gr5a-CTX fly and wt fly respond to sucrose.
4.2.5 Functional study of OR and G protein interactivity in the recombinant

HEK293 expression system
4.2.5.1 Chimera Gq16s protein improves the OR signaling cascade.
To further investigate the functional interactivity between ORs and G-proteins, we
expressed Drosophila OR43a and OR83b in HEK293 cells, and performed single cell
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calcium imaging. In this assay, ligand-mediated OR activation triggers an endogenous
Gq dependent calcium cascade, and this signal can be monitored and recorded. When the
odorant receptors were transfected without any G-proteins, only a weak response to the
odorant stimulation (1 mM cyclohexanone) could be detected. This might be due to the
weak coupling of Drosophila receptors to the endogenous calcium pathway, or as
demonstrated in the previous experiments be based on OR coupling to Gs rather than
Gq. The C-terminus of the G-protein subunit is a key determinant for the fidelity of
receptor coupling. The Drosophila and human G-protein chimera Gq16x (human Gq16,
C-terminus 44 amino acids replaced by Drosophila Gx C-terminus 44 aa) is supposed to
couple to Drosophila odorant receptor and initiate the HEK293 cell endogenous Gqprotein cascade. Accordingly, we cotransfected HEK293 cells with constructs encoding
OR43a, OR83b and one of the five different chimaeric G-Proteins (Gq16q, Gq16i, Gq16o,
Gq16-73b, or Gq16s) (Figure 4.2.21 A) designed to divert Drosophila Gq, Gi, Go,
G73b, or Gs dependent signaling to the HEK293 cells calcium pathway (Conklin et al.,
1993;Conklin et al., 1996;Yapici et al., 2008). Calcium imaging experiments were
performed on transfected cells and the number of cells responding to odorant stimulation
was counted. The ratios of the responding cell number from the chimera G-protein
transfected cells to the responding cell number from the cells transfected without the
chimera G-protein were calculated. A higher value of the ratio indicated an increased
coupling efficiency with the transfection of the chimera G-protein. The experiments
revealed that coexpression of Gq16s leads to the most effectice 7-fold increase in
response to the odorant stimulus (Figure 4.2.21 B). This experiment proves that in a
recombinant system the combination of OR43a/OR83b couples much better to Gs than
to any other G-protein tested.
4.2.5.2 OR stimulation can induce an increase of GTP-binding to the membrane.

Agonist-stimulated [35S] guanylyl-5-O-(-thio)-triphosphate ([35S] GTPS) binding is
used to measure receptor activation of G-proteins in isolated membranes (Hilf et al.,
1989). The [35S] GTPS assay is based on the fact that the inactive state of the G-protein
subunit has a relatively high affinity for GDP over GTP, whereas activation of a
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receptor by its agonist shifts the subunit into a higher affinity for GTP versus GDP.
Therefore, the [35S] GTPS assay utilizes excess GDP to shift the G-proteins into the
inactive state and lower basal activity. Addition of agonist decreases the affinity of the
subunit for GDP and increases its affinity for GTP, so that the receptor-stimulated Gprotein binds GTP. [35S] GTPS is a hydrolysis-resistant form of GTP allowing to assess
the degree to which an agonist stimulates [35S] GTPS binding in membranes. We
performed the assay on HEK293 cell membranes that were transfected with ORs and
Drosophila Gs. Four to five transfections were carried out for each odorant receptor, and
the assay was run in triplicate for each transfection. OR83b was cotransfected with
conventional odorant receptors. Control cells were transfected with empty vector, and the
membranes were stimulated by a combination of the respective odorants. Particularly, 1hexanol, cyclohexanol and 1-octen-3-ol were mixed and used for OR43a, ethyl butyrate,
pentyl acetate and 1-octen-3-ol were mixed and used for OR22a (Hallem et al., 2004).
The counting result of the OR transfected sample was normalized to the empty vector
transfected sample. Students T-test analysis for the stimulation ratio between transfected
cell membranes and untransfected cell membranes revealed a significant difference,
indicating that activated receptors can stimulate Gs and induce an increase in GTP
binding to the membrane (Figure 4.2.21 C).
Figure 4.2.21 Functional study of ORs and G proteins interactivity in the recombinant HEK293
expression system.
(A). Chimera construct with N-terminus of human G16 and the C-terminus of Drosophila G-proteins.
(B). Ratio of transfected HEK293 cells responding to cyclohexanone in calcium imaging experiments; cells
express OR43a, OR83b and the respective G-protein chimera or OR43a, OR83b and the full length human
G16. An increase in the ratio means that more cells responded upon co-expression of the G-protein
chimera. (C). [35S]GTPS binding assay on transfected HEK293 cell membranes. Error bars represent
S.E.M. (** p <0.01, n = 5-7)
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4.2.6
Overexpression of a GTPase deficient Gs mutant in Drosophila antenna

leads to a change in response dynamics
Although the response amplitude of EAG recordings from the OR83b-GAL4; UAS-GsGTP flies showed no significant difference to the wt flies, we could clearly observe a
prolonged spike activity in the single sensillum recordings (Figure 4.2.22 A, B). By
counting the spikes in an interval of 200 ms, a significant prolongation of the response
period could be observed (Figure 4.2.22 C, D).
Figure 4.2.22 Overexpression of a GTPase deficient Gs mutant in Drosophila antenna lead to a

change in response dynamics
(A) Single sensillum recording from wt fly. (B) Single sensillum recording from OR83b-GAL4; UAS-GsGTP fly. (C, D) Spike distribution of the SSRs (n = 5-6). All the recordings are from the ab2 sensillum.
Error bars represent S.E.M. (**: p < 0.01, *: 0.01 < p <0.05)
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4.2.7
Odorant exposure caused Gs redistribution in antenna
The immunostaining of the antenna section shown in Figure 4.2.23 A reveals that Gs is
expressed at the base of the sensillum but not in the dendrites of ORNs where olfactory
transduction gets initiated. If however Gs is involved in olfactory signaling, what
explains its location at the base of the sensillum? We looked over our procedure of
immunostaining, and noticed that flies were always fixed immediately after taken out
from the food vials. We decided to repeate the experiment this time keeping flies in a 2%
agar vial for 2 hours before fixation. Interestingly, we observed that Gs diffused into the
dendrite of ORNs in food odorant deprived flies (Figure 4.2.23 B).
For the purpose of monitoring this translocation process in the living flies, we generated a
GFP-Gs fusion construct. Because of the importance of both the amino and carboxyl
termini for localization and function of the protein, we adapted a strategy to express a
functional expressed GFP-tagged Gs (Hughes et al., 2001;Sunahara et al., 1997;Yu and
Rasenick, 2002). Specifically, the GFP sequence was inserted in-between the residues 71
and 72 of Gs, a 6-residue linker sequence (SGGGGS) was inserted at both of the
junctions between Gs and GFP (Figure 4.2.23 E, F). The fusion cDNA was first cloned
into the pCDNA3 vector, and the protein expression was tested in transfected HEK293
cells (Figure 4.2.23 G). The fusion cDNA was subsequently cloned into the pUAST
construct and transgenic flies were generated. Afterwards OR83b-GAL4; UAS-Gs-GFP
flies were produced, and Gs translocation was studied before and after odorant exposure.
Gs was spread in the dendrites of the ORNs when the experiment fly was segregated
from food odors (Figure 4.2.23 C), and clustered at the base of the dendrites when the fly
was exposed to the odors (Figure 4.2.23 D).
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Figure 4.2.23 Odorant exposure caused Gs redistribution in antenna.

(A, B) Immunostaining of Gs on antenna section. A, + food odor. B, - food odor. (C, D) immunostaining
of GFP on the antenna section. C, + food odor. D, - food odor. (E) Schematic representation of the GFPGs fusion construct. GFP sequence was inserted in-between Gs residues 71 and 72, a 6-residue linker
sequence (SGGGGS) was inserted at both of the junctions between Gs and GFP. (F) Model of Gs-GFP.
GFP is depicted as an insert into the Gs structure. (G) Expression of GFP-Gs fusion protein in HEK293
cells. Scale bar in B and G = 10 m.
4.2.8
Generation of a Gs knockout or knockdown fly
To further investigate the function of Gs in odorant receptor neurons, we performed

experiments to generate Gs knockdown or knockout flies. It is know that Gs is essential
for the initial stages of larval development, the homozygote Gs null mutant dgsB19
results in late embryonic-early larval lethality (Wolfgang et al., 2001). We examined the
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olfactory responses of the heterozygote dgsB19 by EAG and SSR and found no significant
phenotype in response amplitude and response kinetics. Therefore, we decided to
generate flies in which the knockdown or knockout was only targeted to the olfactory
sensory organ.
4.2.8.1 Usage of the UAS-RNAi fly line
RNA interference (RNAi) is a system within living cells that helps to control the
activation of genes; it is now a widely used tool for post-transcriptional gene silencing.
To target the RNAi expression in odorant receptor neurons, we used the OR83-GAL4 to
drive the UAS-RNAi expression. UAS-GsRNAi was obtained from three different
sources: Vienna Drosophila RNAi center, NIG fly center and fly lines generated by our
own lab.
Performing EAG and SSR, we observed no obvious olfaction deficiency in all those flies.
We therefore tested RNAi efficiency by expressing them under a different driver, the elav
driver. Unfortunately, all RNAi lines have generated robust offspring. It seems that all
Gs RNAi lines tested do not work the way they were designed, or at least, do not down
regulate the post Gs transcription to a level that would affect either neuronal
development at embryonal stages or influence signal transduct in ORNs.
4.2.8.2 Generation of mosaic Gs knockout flies
To avoid lethality in the early stage of embryonic development, we used a strategy based
on the FLP-induced mitotic recombination (Xu and Rubin, 1993) to generate genetic
mosaic flies that only have the homozygote dgsB13 cells in a part of their sensory organ.
FLP-induced somatic clones appear to result from a simple reciprocal recombination
event between FRT sites. A mitotic recombination event in a cell heterozygous for a
marker gene would produce one daughter cell with two copies of the marker and a sibling
cell with no copies (Figure 4.2.24). Each of these daughter cells will divide to give a
clone of cells in the adult (twin-spot clones). If neither cell is defective in proliferation or
differentiation, the twin-spot clones will be of similar size. In our experiment, a ey-FLP
construct (Jefferis et al., 2004;Sweeney et al., 2007) was used to induce the chromosome
recombination between the P[neoFRT]42D dgsB13 and the P[neoFRT]42D UbiGPF(S56T). If the homozygote dgsB13 cells in the eye-antennal disc could still develop
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similar to wild type cells, the adult fly could have some Gs empty clusters in the
antennae.
The flies were generated and the cryosection of the antenna was examined. Cells without
GFP expression were not observed, arguing that either recombination did not occur or
that Gs is essential for antenna cell development. Since this ey-Flp construct was well
described in several studies (Hummel et al., 2003;Jefferis et al., 2004;Sweeney et al.,
2007), it is very likely that the Gs plays a crucial role in the antennal neuron
development.
Figure 4.2.24 Schematic overview of genetic mosaic fly generation.

Homozygous mutant cells are identified as unstained cells if the marker gene is placed distal to the FRT
site on the homologous chromosome arm in trans to the mutant gene.
4.3 Secondary messengers and other components in the ORNs signal

transduction cascade
4.3.1
The cAMP pathway is involved in odorant signaling in fly olfactory neurons
4.3.1.1 A cAMP level increase in ORNs causes a rise in neuronal activity

A photoactivated adenylate cyclase (PAC) was expressed in ORNs to manipulate the
cAMP level. This photoactivated adenylate cyclase was identified in the unicellular
flagellate Euglena gracilis (Iseki et al., 2002). It is composed of two PAC and two
PAC subunits, which exhibit adenylate cyclase activity that is enhanced by blue light.
Each subunit harbors two BLUE-type photoreceptor domains, binding flavin adenine
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dinucleotide (Anderson et al., 2005;Gendre et al., 2004), and two catalytic domains that
are homologous to class III adenylate cyclase. Studies showed that in a heterologous
expression system, the PAC subunit exhibits a strong cAMP synthesis efficiency
(Schroder-Lang et al., 2007). Similar results were obtained from Xenopus oocytes, HEK
cells and transgenic Drosophila (Schroder-Lang et al., 2007). The same transgenic fly
was used in our experiment. By crossing the UAS-PAC responder line with the OR83b
driver line, flies expressing the photoactivated adenylate cyclase in odorant receptor
neurons were generated. During the whole fly culture, the animals were kept in the dark,
or shortly exposed to red light for the requirement of handling. During single sensillum
recording flies were alternately exposed to light stimulation and dark rest. The neuronal
activity increased upon light stimulation and turned back to normal in the dark (Figure
4.3.25 A C). Light stimulation was carried out for 10 seconds (Figure 4.3.25 A), 30
seconds (Figure 4.3.25 C), or even several minutes (similar data not shown). After
stimulation, neuronal activity always returned to basal level. The longest stimulation time
tested was 5 minutes. Spike numbers were counted for ab1, ab2 and ab3 sensilla
recordings. Except for the ab1C neurons, which could not be covered by the OR83b
driver, all other neurons analyzed showed a statistically significant increase in spiking
frequency (Figure 4.3.25 B).
Figure 4.3.25 cAMP level moderate the activity of ORNs

(A) Representative recording of an OR83b-PAC ab2 sensillum. The whole trace represents a 30 seconds
recording. (B) Comparison of spike number between light and dark stimulations. Error bars represent S.D.,
n = 5-7. (C) Repetitive model of PAC-ORNs in response to light stimulation. Data were obtained from the
ab2A neuron. Error bars represent S.D., n = 5-7.
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4.3.1.2 Activation of PAC recovers the spontaneous neuronal activity in OR83b
knockout ORNs.
To test whether an increase in cAMP levels would affect odorant receptor neurons of
OR83b knock-out flies (OR83b-/-), we generated a mutant fly expressing PAC in the
odorant receptor neurons on a OR83b knock-out background (Figure 4.3.26 A). Single
sensillum recordings from the ab1 sensillum of OR83b-/- flies have only one group of
spikes (Figure 4.3.27 A), representing the Gr21a neuron activity, instead of four from the
wt ab1 sensillum (Figure 4.3.26 C). When the PAC was expressed by the OR83b-GAL4
driver, and the fly antenna was exposed to blue light, additional neuronal activity could
be recorded (Figure 4.3.26 B). We sorted all spikes in a given time and analyzed the
spike amplitude distribution (Figure 4.3.26 D, E). 453 spikes were analyzed from 8
seconds of recording of an OR83b PAC (OR83b-/-) fly (Figure 4.3.26 E), 229 spikes
from wt fly were as well analyzed (Figure 4.3.26 D). We observed a similar spike
distribution pattern, indicating that PAC expression in olfactory receptor neurons does
efficiently recover spontaneous neuronal activity.
Figure 4.3.26 An increase in cAMP levels can recover the spontaneous activity of ORNs.
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(A) Crossing scheme of the OR83b-GAL4 / UAS-PAC; OR83b2 / OR83b2 fly. (B) Single sensillum
recording from the OR83b-GAL4 / UAS-PAC; OR83b2 / OR83b2 fly with blue light illumination. The
whole trace is 3 seconds in duration. (C) SSR recording from a wt fly exposed to blue light. The whole
trace is 3 seconds in duration. (D) Spike amplitude distribution of the wt ab1 sensillum. (E) Spike
amplitude distribution of the blue light illuminated OR83b-GAL4 / UAS-PAC; OR83b2 / OR83b2 ab1
sensillum.
Figure 4.3.27 SSR of the OR83b-GAL4 / UAS-PAC; OR83b2 / OR83b2 and control flies
This figure shows orginal single sensillum recordings of flies: (A) OR83b2 / OR83b2 (B) OR83b2 / OR83b2
with blue light (C) OR83b-GAL4 / UAS-PAC; OR83b2 / OR83b2 (D) OR83b-GAL4 / UAS-PAC; OR83b2
/ OR83b2 with CO2 stimulation. (E) OR83b-GAL4 / UAS-PAC; OR83b2 / OR83b2 with odorant
stimulation. (F) OR83b-GAL4 / UAS-PAC; OR83b2 / OR83b2 with blue light stimulation. (G) OR83bGAL4 / UAS-PAC; OR83b2 / OR83b2 with blue light stimulation, then plus odorant stimulation. All of the
traces are 3 seconds in duration.
4.3.2
Expression of activated Gq in ORNs leads to resensitization deficiency.
In general, the continuously activated Gq3 functions as a dominant gain-of function allele
in a tissue and cell specific manner (Ratnaparkhi et al., 2002). The UAS-AcGq3; OR83bGAL4 fly was used in an EAG screen for olfactory deficient flies. As described in section
4.2.1, this fly showed no olfactory defect in EAG amplitude. However, while studying
desensitization, we observed a deficiency in resensitization of EAG responses.
Two sets of assays were performed to evalute desensitization. At first, short pulse odorant
stimuli (pure odorant) were applied every minuteand EAG response were recorded. In
wild type flies, EAG amplitudes of consecutive responses were unaltered, while EAG
responses of UAS-AcGq3;OR83b-GAL4 flies declined upon repetitive stimulation
eventually reaching a steady level (Figure 4.3.28 A). In a second previously decribed
assay (Stortkuhl et al., 1999), flies were pre-treated with a given odorant (pure odorant)
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for one minute followed by a short pulse odorant stimulation every minute (1:100
dilution). Normalizing EAG amplitudes to the none pre-treated condition, UASAcGq3;OR83b-GAL4 flies appeared to exhibit a resensitization deficiency (Figure 4.3.28
B, C).
Figure 4.3.28 Expression of an AcGq in ORNs leads to a resensitization deficiency

(A) The UAS-AcGq3;OR83b-GAL4 fly showed an abnormal desensitization to a continuous odorant
stimulation. EAG amplitudes are normalized to the response of the first stimulation. (B, C) Ethyl acetate
and benzaldehyde were used in B and C. The UAS-AcGq3; OR83b-GAL4 fly showed an abnormal
resensitization after constant odorant stimulation. Error bars represent S.E.M. n = 5-7.
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4.3.3
Effect of MIA on Drosophila odorant receptors
MIA, an amiloride derivative, was reported as a lobster TRP channel blocker (Bobkov
and Ache, 2007) (Figure 4.3.29 D). As demonstrated by Dr. Gnter Gisselmann, MIA
can block odorant-induced currents in oocyte expressing OR47a and OR83b (Figure
4.3.29 A-C). To further investigate whether this substance works on the fly as well, we
applied the substance on the surface of the antenna and measured olfactory responses.
Different approaches of substance application were tested, and finally we chose to mount
the antenna in a micropipet tip filled with 1 mM MIA solution. 20 minutes after MIA
mounting, a filter paper fiber was used to wipe up the antenna surface. The fly was
subsequently exposed to a constant airflow to allow the antenna surface to dry. After 20
minutes, the fly was subjected to single sensillum recording. Since the antenna surface is
covered by cuticle, the efficiency of substance delivery by using this approach is unclear.
From all single sensillum recordings, only a few sensilla show a phenotype in olfactory
response. About one tenth of the recorded sensilla showed a lack of spontaneous activity
and a reduction of odorant responses (Figure 4.3.30). On one hand, we observed spike
traces from the sensilla missing only one group of neuronal activity, on the other hand, a
sensillum which losing the neuronal spontaneous activity of all neurons is hard to access
by the recording electrode in the SSR. As a control, the solvent of MIA (DMSO) was also
applied directly on the antenna, but no significant olfactory response alteration could be
observed.
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Figure 4.3.29 MIA blocks odorant-induced currents in oocytes

(A) Pentyl acetate induced currents could be observed in oocytes expressing OR47a and OR83b. (B) MIA
blocked the pentyl acetate induced current in oocytes. (C) Dose-inhibition curve showing the blocking
efficiency of MIA. (D) The chemical structure of MIA (5-(N-methyl-Nisobutyl) amiloride). Error bars
represent S.E.M. These data were produced by Dr. Gnter Gisselmann (shown with permission).
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Figure 4.3.30 Direct application of MIA on antenna blocks the spontaneous neuronal activity
(A, C) MIA treated antenna, ab2 sensillum, the spontaneous activity of the ab2A neuron was gone after
MIA treatment. Although the ab2A neuron still respond to odorant stimulation (1:100 diluted ethyl acetate
and 1:10000 diluted ethyl butyrate), the response is largely decreased. (B, D) 1% DMSO treated antenna,
DMSO was used as the solvent for MIA.
4.3.4
Functional study of Ih channels in Drosophila odorant receptor neurons
Ih (HCN, Hyperpolarization-activated, cyclic-nucleotide-gated ion channel) channels are

widely expressed in the mammalian heart and brain (Brown et al., 1979;Halliwell and
Adams, 1982). They are responsible for a variety of physiological functions ascribed to
the depolarizing cation current (Ih) in the cellular context. In physiological context Ih
channels are involved in control of pacemaker activity in both heart and brain
(DiFrancesco, 1993;Pape and McCormick, 1989), determination of the resting membrane
potential (Williams and Stuart, 2000), control of membrane resistance and synaptic
integration in dendrites (Magee, 1999) and primary sensory transduction (Stevens et al.,
2001). Ih channels have also been cloned form arthropod, for example Drosophila (Marx
et al., 1999), Apis mellifera (Gisselmann et al., 2003;Gisselmann et al., 2004), Heliothis
(Krieger et al., 1999), and Panulirus (Gisselmann et al., 2005b). The sequence of these
channels exhibits homology to both CNG channels (Finn et al., 1996;Santoro et al., 1997)
and voltage-gated potassium channels (Catterall, 1995). In addition, mRNA of Ih-channel
was detected in the antennae of different insects and lobster (Gisselmann et al.,
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2005a;Gisselmann et al., 2005b;Krieger et al., 1999;Marx et al., 1999;Roeper et al.,
1998), suggesting a possible role of this channel in olfactory signal transduction.
Two mutant fly lines have been used in our study: UAS-DoIh was generated by Martina
Kper as previously described (Kper M, 2008), and Ih-/- mutant was obtained from
Inmaculada Canal (Universidad Autonoma de Madrid, Spain). The Ih-/- fly was measured
directly. The dominant negative Ih construct encoding a truncated protein was expressed
by OR83b-GAL4. Single sensillum recordings from both mutants has been performed,
and the recording results were analyzed by counting the spikes in an interval of 200 ms.
Both of the mutants showed a response dynamic change compared to control flies. The
odorant-induced firing rate exhibited a faster decline in Ih-/- flies as compared to control
flies (Figure 4.3.31 A). The UAS-DoIh; OR83b-GAL4 ORNs showed a spontaneous
neuronal activity increase, and a prolonged activity after odorant stimulation (Figure
4.3.31 B).
Figure 4.3.31 Single sensillum recording analysis from Ih channel mutants

(A) Single sensillum recordings on Ih channel knockout flies. Odorant: ethyl butyrate, 1:10000 diluted.
Odorant application lasted for 1 second. The sensillum measured here is ab3. The response of neuron A is
shown here. (B) Single sensillum recordings on UAS-DoIh, OR83b-GAL4 ab2 sensillum. Odorant: ethyl
acetate, 1:100 diluted. Odorant application lasted for 1 second. The response of neuron A was analyzed.
Error bars represent S.E.M. n = 5-7. **: p < 0.01, *: 0.01 < p < 0.05.
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4.4 Larva study

4.4.1
Gs and Gq are expressed in larva olfactory receptor neurons
To investigate whether G-proteins are also involved in the larval olfactory system, we
carried out similar experiments in larvae as we did in adult flies. First, we checked the
expression of Gs and Gq in larval olfactory receptor neurons. OR83b-GAL4; UASmCD8-GFP larvae were used for the whole mount and cryosection staining. As described
earlier in section 1.1.1, the OR83b expression cells are the only ORNs in larvae.
Therefore, OR83b-GAL4 driver can be used to mark the ORNs in larvae as well as in the
adult olfactory organs. Immunohistochemical stainings revealed that both Gs and Gq
are expressed in the cell body of larval odorant receptor neurons. The cilia of the larval
odorant receptor neurons are housed in the dorsal organ, as shown in Figure 4.4.32 A.
The dorsal organ exhibits strong autofluorescence, which makes the detection of the Gs
and Gq almost impossible.
Figure 4.4.32 Immunostaining of larval ORNs

(A) Whole mount staining of the OR83b-GAL4; UAS-mCD8-GFP larval olfactory sensory neurons. Gs is
expressed in the cell bodies of the ORNs. Since dorsal organs (DO) have a strong autofluorescence, Gs is
expression in the dendritic area cannot be assessed. (B, C) Cryosection of the larva (cross section of the
head tip). Both Gs and Gq are expressed in the cell body of the ORNs. Scale bar = 20 m.
65
Results
4.4.2
CTX larvae have normal chemotaxis behavior
Larval chemotaxis assays were performed with OR83b-CTX larvae, which have the same
genotype as the CTX adult described before (Chapter 4.2.2). It turned out that OR83bCTX larvae behave completely normal like wt larvae in the chemotaxis assay (Figure
4.4.33). It has already been shown that OR83b neurons are essential for the larval
chemosensation, and OR83b knock out larvae are anosmic to a wide range of fly odors
(Larsson et al., 2004). Our result showed that larval ORNs expressing CTX function
normally, which substantiate that the signal transduction pathways in larvae olfactory
receptor neurons is Gs independent.
4.4.3
Expression of AcGq3 in larval olfactory receptor neurons causes olfactory

aberration
For the expression of AcGq3, females of the genotype UAS-AcGq3/FM7-GFP were

crossed to homozygous males of OR83b-GAL4. The non-GFP expressing larvae were
picked up manually under the fluorescence microscope and used for the larval
chemotaxis assay. Ethyl acetate, cyclohexanol and benzaldehyde were applied as odorous
stimuli.
As indicated in Figure 4.4.33, AcGq larvae have a reduced response to ethyl acetate as
well as a high concentration of cyclohexanol. To benzaldehyde and low concentration of
cyclohexanol behavior responses remained unaltered.
Figure 4.4.33 Chemotaxis assays on AcGq and CTX larvae

(A, B, C) Ethyl acetate (A), cyclohexanol (B) and benzaldehyde (C) are used for the test. OR83b-GAL4;
UAS -CTX larvae have a normal chemotaxis behavior. UAS-AcGq3; OR83b-GAL4 larvae showed a response
decrease to ethyl acetate and high concentration of cyclohexanol. Error bars represent S.E.M.
66
Discussion
5 Discussion
5.1 The role of Gs in the olfactory signal transduction pathway
In recent years, it has been realized that Drosophila ORs are not classical GPCRs since
these proteins neither show a homology to conventional GPCRs nor have the
extracellular N- terminus and intracellular C- terminus membrane topology (Benton,
2006;Clyne et al., 1999;Gao and Chess, 1999;Lundin et al., 2007;Vosshall et al., 1999).
Moreover, two studies published last year provided evidence that heterologously
expressed Drosophila ORs have an ion channel capacity (Sato et al., 2008;Wicher et al.,
2008). These studies raise the question of whether heterotrimeric G-proteins are involved
in the signal transduction pathway of odorant receptor neurons. In the present study, we
investigated whether Drosophila ORs couple to G-proteins to trigger downstream signal
transduction events.
Previous studies provide plenty of evidence indicating that G-protein signaling is
involved in invertebrate olfactory signal transduction. Two G-protein signaling
transduction pathways depending on the second messengers 1,4,5-inositol triphosphate
and cAMP, respectively, appeared to be involved in olfactory perception (Breer,
1994;Ronnett and Moon, 2002;Stengl et al., 1992a). The existence of both signaling
cascades has been proven by using different approaches: at the molecular level, by
expression of genes encoding for intermediate products of both signal transduction
pathways in ORNs (Baumann et al., 1994;Dubin et al., 1998a;Hasan and Rosbash,
1992;Martin et al., 2001;Marx et al., 1999;Riesgo-Escovar et al., 1997;Yoshikawa et al.,
1992) and at the cellular level by electrophysiological measurements of vertebrate and
invertebrate ORNs (Fadool and Ache, 1994;Hatt and Ache, 1994;Martin et al.,
2001;McClintock et al., 1997). In our study, convincing evidence is provided to
substantiate the involvement of Gs-mediated cAMP signaling in Drosophila olfactory
signal transduction.
67
Discussion
First of all, the expression levels and expression patterns of different G-proteins in the
antennae of Drosophila were investigated. Two proteins of interest were specified due to
their expression pattern in ORNs. The most suspicious goes to Gs, which is expressed in
the dendrites of odorant receptor neurons, where the olfactory signal transduction cascade
is initiated. Further investigation on Gs localization revealed that food odor exposure
causes Gs protein translocation from the end of the dendrite to the base of the dendrite.
This redistribution is similar to the light induced transducin, the eye specific Gt,
translocation in rod photoreceptors (Philp et al., 1987;Whelan and McGinnis, 1988),
suggesting that Gs might serve a similar function in ORNs as transducin in rod
photoreceptors. Gq was found to be expressed in some of the ORNs. Quantitative PCR
also showed that Gq was highly expressed in the antennae compared to brain tissue, but
the expression pattern of Gq in antennae showed that Gq protein is localized in the cell
body of ORNs but not in the dendrite. Although a recent publication revealed that a
mutation in the Gq gene can cause a reduction in amplitude and spiking frequency,
respectively, in EAG and single sensillum recordings (Kain et al., 2008), it is not likely
that Gq directly couples to ORs as the protein is not located in the cellular location
where the receptor and the downstream factor should interact. Also, it is important to
notice that although homozygote Gq null mutant neurons were generated, no anosmic
phenotype has been found. Precisely how is involved in OR signal transduction interacts
requires further investigation.
Secondly, a functional screening of distinct G-proteins as to be involved in olfactory
transduction was performed in both the fly antennae as well as heterologous expression
system. In Drosophila, different wt G-proteins, mutated G-proteins, and G-protein
affecting toxins were expressed in the ORNs. Only CTX flies showed a strong reduction
of odorant-induced signals in EAG recordings. Further investigation by single sensillum
recordings on CTX flies showed that odorant-induced spikes in CTX expressing neurons
are completely gone. Since CTX is an ADP-ribosyltransferase that typically activates Gs
proteins (Moss and Vaughan, 1988), the strong olfactory deficiency of CTX expressing
flies indicated that Gs, the only stimulative type of G-proteins in Drosophila, is crucially
important for odorant induced signal transduction in olfactory sensory neurons. Although
68
Discussion
other G-protein over-expressing flies exhibited no change in EAG amplitude, olfactory
response aberration could be demonstrated by other experimental procedures. For
example, single sensillum recordings of Gs-GTP flies, which express a mutated Gsprotein with decreased GTPase activity and therefore can be regarded as constitutively
active, revealed a significant prolongation of spike activity after odorant stimulation as
compared to control flies. The absence of phenotype in EAG recordings might be
reflected by the fact that the initial increase in neuronal firing was not significantly
different in these flies. Changes were observed in the duration of the period of increased
firing in the mutant flies, which may be caused by the deficiency in GTPase activity of
the activated G-protein subunit.
For the screening of odorant receptor coupled G-proteins in the heterologous expression
system, we used the G-protein chimera strategy to switch the Drosophila odorant
receptor signals to the endogenous calcium signal in HEK293 cells (Conklin et al.,
1993;Conklin et al., 1996;Mody et al., 2000). Using calcium imaging, HEK293 cells
cotransfected with OR and Gq16s showed stronger odorant-induced signals as compared
to other chimeric G-proteins (Gq16q, Gq16i, Gq16o and Gq16-73b). The increased
coupling efficiency emphasizes that Gs serves as downstream G-protein of ORs.
Conventional odorant receptors were cotransfected with OR83b to increase the
expression efficiency (Neuhaus et al., 2005), albeit the conventional odorant receptor
alone expressed in HEK cells or Xenopus oocytes responds to odorant stimulation
(Neuhaus et al., 2005;Wetzel et al., 2001).
To further examine our hypothesis that Drosophila odorant receptors are coupling to Gs,
the classical [35S]GTPS binding assay was carried out to test whether activation of ORs
could increase GTP binding to the G-protein. We coexpressed Gs and odorant receptors
in HEK293 cells. As expected, the binding assay showed a significant increase of
[35S]GTPS binding in OR transfected cell membranes, indicating the odorant-treated
Drosophila ORs can interact with Gs. Although further speculations have to await
investigations on the mode of receptor G-protein coupling in the olfactory system, it is
tempting to speculate that Drosophila ORs couple to heterotrimeric G-proteins although
69
Discussion
bearing an inverse membrane topology compared to other GPCRs. In fact, there are other
reversed GPCR like receptors that show a possibility of coupling to G-proteins, for
example the human adiponectin receptors (AdipoRs). Human adiponectin receptors and
membrane progestin receptors (mPRs) belong to the PAQR (Progestin, AdipoQReceptor) family of proteins, which are seven transmembrane receptors of a novel type,
that similar to Drosophila OR83b share little sequence homology with other GPCRs
(Yamauchi et al., 2003;Zhu et al., 2003). As Drosophila OR83b (Benton et al., 2006),
AdipoRs have been shown to have intracellular N- and extracellular C-termini (Deckert
et al., 2006;Yamauchi et al., 2003), but location of the termini of mPR has yet to be
confirmed. Nevertheless, the fish mPR has been shown to be a plasma membrane protein
whose activation leads to inhibition of adenylate cyclase in a pertussis toxin-sensitive
manner, consistent with mPR being a novel type of GPCR (Zhu et al., 2003).
Gs typically activates adenylate cyclases to produce cAMP. To test whether odorant
stimulation could induce a cAMP increase in Drosophila antennae, a cAMP assay was
performed. Fly antennae were cut manually and the PerkinElmer alpha screen kit was
used for the assay. Most of the time no significant cAMP increase could be observed.
This may either be due to the fact that a too small amount of tissue was used for each
assay, or be due to the nature of the odorant receptor neurons in Drosophila, in a way that
certain odorants can cause excitation in some ORNs and inhibition in other ORNs
(deBruyne et al., 2001;Hallem et al., 2004). If an odorant can induce both excitation and
inhibition at the same time in the antenna, this may lead to an unchanged cAMP level
before and after odorant stimulation. Even though no cAMP increase could be observed
after odorant application, a cAMP increase in odorant receptor neurons can certainly
cause neuronal excitement.
Expression of a light-activated adenylate cyclase (PAC) (Schroder-Lang et al., 2007) in
olfactory neurons enabled us to show that a cAMP increase in these cells results in
increased firing rates, providing hints for the existence of a cAMP dependent excitatory
signaling pathway. Also, overexpression of a cAMP-phosphodiesterase in olfactory
neurons was indicative for an excitatory role of cAMP in the Drosophila olfactory system
70
Discussion
(Gomez-Diaz et al., 2004). Moreover, cyclic nucleotidegated channels and Ih channels
as potential cAMP targets are known to be expressed in the antennae of Drosophila
(Baumann et al., 1994;Gisselmann et al., 2005a;Marx et al., 1999) and mutants of a cyclic
nucleotide-modulated potassium channel show olfactory deficits (Dubin et al., 1998a). To
further understand the function of cAMP in the olfactory signal transduction pathway, the
light-activated adenylate cyclase was expressed in the OR83b-/- odorant receptor neurons.
By lightening up the antenna, neuronal activity can be recorded by single sensillum
recording. After grouping the spikes by their amplitude, similar spontaneous activity
patterns can be observed. From previous publications (Dobritsa et al., 2003;Larsson et al.,
2004;Neuhaus et al., 2005) and our own recording data, it is clear that the ORNs of
OR83b-/- flies show a complete lack of spontaneous activity, but the halo fly, which
have no functional OR expressed in the ab3a neuron, still maintains a spontaneous
neuronal activity of the ab3a neuron. Our data suggest that the spontaneous activity is
largely dependent on the cellular level of cAMP.
As the gain of function of Gs has already been investigated, studying the loss of function
of Gs became critical. It is known that the complete knockout of Gs in the entire animal
causes early embryo lethality (Wolfgang et al., 2001). Two strategies were used for
generating the olfactory organ targeted Gs knockdown or knockout flies. One is based
on the GAL4-UAS RNAi method; the other one is based on the genetic mosaic fly
generation. Unfortunately, none of these efforts achieved the predicted effect. For the
RNAi method, three sources of UAS-Gs-RNAi were used, but none of them could induce
an efficient post-transcriptional gene silencing. The recordings from those flies also did
not show any obvious olfactory deficiency. For the mosaic fly, the prerequisite of
successfully generating such a fly is that the homozygote mutant cells are healthy enough
to generate offspring cells. In our case, it seems that the homozygote dgsB13 cells in the
embryo stage cannot survive through the development. No homozygote dgsB13 cells could
be observed in cryosections of the antenna. Since the driver line used (ey-Flp) was well
described and examined in several studies (Hummel et al., 2003;Jefferis et al.,
2004;Sweeney et al., 2007), it is very likely that the Gs plays a crucial role in antennal
neuron development.
71
Discussion
Putting aside the Gs coupling, the odorant receptor ion channel hypothesis raises a series
of new questions. First, there is no clear consensus on the position where the pore of the
channel is located and to what extent different subunits in the OR complex contribute to
the pore itself. Only a weak similarity was found between the suspected odorant receptor
pore motif and the well-characterized potassium channel pores (Wicher et al., 2008).
Second, there is little data on the exact stoichiometry of the OR complex. Although it was
showed by Benton et al. that at least two subunits each of OR83b and the conventional
odorant detecting OR are included in the receptor complex (Benton et al., 2006), the
composition of the functional complex is still unknown and it might even vary for
different OR83b/OR combinations. Third, if Drosophila odorant receptors form a
heteromeric ligand-gated ion channel, and raise action potentials immediately after
odorant binding, the ablation of any one of the channel subunits should lead to a similar
phenotype. The fact is that the OR83b knockout fly results in a lack of spontaneous
action potential firing of the ORNs (Larsson et al., 2004), while conventional OR knock
out ORNs still generate spontaneous action potentials (Dobritsa et al., 2003). Of course
none of these ORNs respond to odorants any more. In addition, it is still not well
understood whether classical GPCRs form dimers or higher ordered oligomers, and the
general functional significance of this polymerization or dimerization is not clear.
Vertebrate class C GPCRs, such as metabotropic glutamate receptors and -aminobutyric
acid type B receptors clearly form homo- and heterodimeric structures, essential for both
trafficking of receptors to the cell surface and G-protein coupling (Pin et al., 2005). The
relevance of the monomeric or dimeric state for G-protein activation for other GPCRs is
currently under debate. For example, NTS1, a dimerizing class A receptor, was recently
shown to alter the mode of the receptor G-protein interaction (White et al., 2007). The
fact that two distinct families of seven transmembrane domain receptors, namely
vertebrate ORs as classical GPCRs and Drosophila ORs seem to make use of similar
intracellular signaling cascades could demonstrate an interesting case of convergent
evolution (Benton, 2006). It moreover demonstrates that olfactory signaling pathways
seem to be principally conserved between species.
72
Discussion
5.2 Larval olfactory system

We have demonstrated the possible role of Gs in the adult olfactory system. Since the
larval olfactory neurons also coexpress conventional odorant receptors and OR83b, it is
of great interest to understand if their olfactory signal transduction pathways are similar.
For this purpose, we examined all those different wt G-proteins, mutated G-proteins, and
G-protein affecting toxins expressing lines using the larval chemotaxis assay.
Surprisingly, the CTX expressing larvae show normal chemotaxis behavior. It has
already been proven that OR83b neurons in the Drosophila larvae dorsal organ ganglia
are the only essential odorant receptor neurons that are responsible for larval olfaction
(Fishilevich et al., 2005;Heimbeck et al., 1999;Larsson et al., 2004), suggesting that Gs
is not required for the larval olfactory signal transduction pathway. The OR83b-GAL4;
UAS-AcGq larvae showed a reduced sensitivity to some of the odorants that were used,
indicating that Gq may be important for the larval olfactory signal transduction.
Immunostaining of larval olfactory organ was performed with Gs and Gq antibodies on
OR83b-GAL4; UAS-mCD8-GFP larvae. Adults of the same fly line were used for the
immunostaining of the antennae. The immunohistochemistry showed that both Gs and
Gq are expressed in the cell body of larvae ORNs. The dendrites of the ORNs in larvae
are housed in the dorsal organ, which exhibits a strong autofluorescence, making it very
difficult by immunostaining to detect whether the Gs and Gq are expressed in the
dendrites.
It is known that olfactory organs of adults and larvae have different developmental
origins. During metamorphosis, the larval dorsal organ is histolysed (Stocker, 1994), and
the antennae and palps develop de novo from the eye-antenna imaginal disc (Postlethwait
and Schneiderman, 1971). Larvae and adults inhabit in different phases. The demands of
olfaction are also different: for the larvae, the major purpose is foraging; while for the
adult, foraging, courtship and oviposition become equally important. It seems likely that
Drosophila larvae and adult depend on different signal transduction pathways.
73
Discussion
5.3 Other signaling components in the olfactory signal transduction

pathway
Despite the recent discovery of the ion channel capacity of Drosophila odorant receptors,
it has long been assumed that the canonical model of olfactory signal transduction would
also hold true in insects. Indeed, most of the molecules involved in mammalian OR or
GPCR signal transduction cascade have been identified in insect, including OBPs (Pelosi
and Maida, 1995;Tegoni et al., 2004), arrestins (Merrill et al., 2002;Merrill et al.,
2003;Merrill et al., 2005), AC (Gomez-Diaz et al., 2004;Martin et al., 2001), CNG
(Baumann et al., 1994;Dubin et al., 1998b;Marx et al., 1999), and IP3 gated ion channel
(Stengl, 1994). In the first part of this chapter, we have already discussed the
participation of cAMP in the transduction. Therefore, we will only discuss the function of
Gq, Ih channels, and the amiloride derivative MIA as OR83b blocker in this section.
Although Gq3 is not expressed in the cilia of odorant receptor neurons, and overexpression of the continuously activated Gq3 in the antenna does not induce any
olfactory response aberrations in the single pulse EAG measurement, the UASAcGq3;OR83b-GAL4 fly did show a phenotype in the desensitization test. Two sets of
tests were performed for the desensitization evaluation by the EAG recordings. The
difference between these two assays is whether the fly had a pretreatment of
desensitization. With or without pre-exposure, the short pulse odorant stimulations were
applied every minute, and the amplitudes of the olfactory response were recorded. The
test without odorant pre-exposure showed that at the frequency of one stimulation per
minute, ethyl acetate, benzaldehyde and cyclohexanol all evoked a response
desensitization in the UAS-AcGq3; OR83b-GAL4 fly. While in the wt fly, the same pattern
of repetitive stimulation did not induce such phenotype. In the odorant pre-exposure test,
similar results could be observed, compared to the wt fly, the AcGq3 fly showed a
deficiency in resensitization. In the Drosophila visual system, it was described that a
prolonged Gq activity could trigger the rhodopsin endocytosis and degradation, thus
resulting in a reduced photoreceptor sensitivity (Han et al., 2007). Albeit the Drosophila
odorant receptor are not likely coupled to the Gq as the light receptor Rh1, similar
receptor endocytosis and degradation could be induced by the continuously activated Gq
74
Discussion
and lead to this resensitization defect. The same UAS-AcGq3 construct used by us was
expressed under the hs-GAL4 driver by Kain and colleagues, since the activated Gq can
cause an intercellular calcium level increase and probably lead to up or down regulation
of other genes. This strategy of conditional expression has the advantage of avoiding this
complication in the development of odorant receptor neurons. The EAG recording from
these heat-shocked mutant flies showed a mildly reduced or similar amplitude compared
to the control flies (Kain et al., 2008). This observation further supports the idea that Gq
is not the direct downstream effector of the odorant receptor. A lack of odorant induced
EAG amplitude aberration and the phenotype in the desensitization assays indicate that
Gq is not essential for the depolarization of odorant receptor neurons, but somehow
crosstalks with the Gs signaling cascade to regulate the olfactory transduction pathway.
The presence of both transduction system depending on Gq and Gs in the same
olfactory receptor neurons has been suggested using pharmacological approaches in
vertebrates (Noe and Breer, 1998), the crosstalk between both systems had also been
reported (Vogl et al., 2000). All these data reinforced previous hypotheses of a functional
meaning of this coexistence and of the role of the olfactory receptor cell as primary
complex integrating unit in the olfactory system (Ache, 1994).
It is clear that cyclic nucleotide-gated ion channels serve as downstream targets of
signaling pathways in vertebrate olfactory sensory neurons. Since the first identification
of CNG channes in Drosophila, it has been proposed that CNG channels are involved in
signal transduction cascade of invertebrate olfactory neurons (Baumann et al., 1994). The
CNG channels we discussed here belong to a heterogeneous gene superfamily of ion
channels that share a common transmembrane topology and pore structure and that
harbor in their C-terminal region a binding domain for nucleoside 3,5-cyclic
monophosphates, Ih channels are also members of this superfamily. CNG channels form
heterotetrameric complexes consisting of two or three different types of subunits. Six
genes encording CNG channels have been found in human genome (four A subunits, A1
to A4, and 2 B subunits, B1 and B3), while the Drosophila genome contains 4 CNG
channel genes (dmA, dmB, dm3, dm4) (Kaupp and Seifert, 2002). Although CNG
75
Discussion
channels are widely expressed in the Drosophila olfactory tissue, their function is still
largely unknown.
Like CNG channels, Ih channels (HCN, hyperpolarization-activated, cyclic-nucleotidegated ion channel) have also been detected in the antennae of different insect and lobster
(Gisselmann et al., 2005a;Gisselmann et al., 2005b;Krieger et al., 1999;Marx et al.,
1999;Roeper et al., 1998). Signal sensillum recording from the Ih channel knockout fly
showed a response dynamic change compared to the control fly. The firing rate of the
odorant stimulated ORNs showed a relatively faster decline in the Ih channel knockout fly
than in the control fly. In addition to the knockout fly, a truncated Ih channel mutant
construct has also been generated, and this truncated Ih channel protein was supposed to
act as a dominant negative mutant when expressed in the flies. The UAS-DoIh, OR83bGAL4 ORNs showed a spontaneous neuron activity increase. Both of these mutant fly
lines had no obvious EAG amplitude variation in response to the odorant stimulation. The
response dynamic change in the Ih channel knockout fly is in accordance with the
phenotype of the continuously activated Gs overexpression fly, which showed a
prolonged activation in the same type of measurement, indicating that Ih channel is one of
the downstream effectors of the cAMP in Drosophila ORNs.
Since the molecular mechanism of insect repellent DEET has been published (Ditzen et
al., 2008), our lab started a screening of a series of chemicals as potential novel insect
odorant repellent on the oocyte recombinant system. To achieve a high-throughput
screening, the insect odorant receptor and OR83b or OR83b homologue have been
expressed in the oocyte. Odorant was applied to maintain the current; the chemicals were
then applied to check if they could block the odorant-induced current. By this procedure,
an amiloride derivative, MIA, was identified as a novel odorant receptor blocker. This
substance was published before as a lobster TRP channel blocker (Bobkov and Ache,
2007). To understand if MIA worked on the natural system as well, it was applied
directly on the antenna, and followed by the single sensillum recording. In the single
sensillum recordings, only a part of the sensilla showed a missing spontaneous activity
and a reduction in odorant evoked spike frequency. This relative low number of affected
76
Discussion
sensilla observed in the living animal indicated the low delivery efficiency of the MIA
substance through the antennae cuticle, or, there are molecules other than conventional
OR
and
OR83b
essential
for
the
ORNs
to
generate
depolarization.
77
Conclusion
6 Conclusion
6.1 Summary
Drosophila melanogaster has 60 odorant receptor (OR) genes, which are expressed in the
third antenna segments as well as in the maxillary palps. The receptor proteins have well
understood odor response profiles. A given odorant receptor gene is expressed only in a
small fraction of olfactory neurons and each neuron expresses a very small number of
odorant receptor genes. This arrangement is similar to the mammalian olfactory system.
On a cellular level, olfactory signal transduction starts with the activation of olfactory
receptors, which are known to recognize a wide range of structurally highly variable
substances. While vertebrate olfactory receptors are 7-transmembrane proteins, which
activate heterotrimeric G-proteins after ligand binding, olfactory receptor proteins in
Drosophila were reported to have an inverse membrane topology compared to classical
G-protein-coupled receptors, presenting an intracellular N- and an extracellular Cterminus. Moreover, it was shown recently that the Drosophila odorant receptors could
function as ligand gated ion channels. Controversial findings were reported concerning
the additional involvement of heterotrimeric G-proteins in olfactory receptor signaling.
One arising question is therefore, whether these 7-transmembrane receptors also couple
to heterotrimeric G-proteins, in addition to the reported novel direct activation. Our data
involving in vivo pharmacological studies, electrophysiological recordings and protein
redistribution analysis, as well as investigations using recombinantly expressed olfactory
receptors, now demonstrate that odorant receptor signaling in Drosophila indeed involves
G-proteins for signal transduction. Moreover, our results provide compelling evidence
that the stimulatory Gs protein is involved in the olfactory signaling cascade of odorant
receptor neurons. In conformity with Gs signaling we could show that increased cAMP
levels lead to excitation of olfactory sensory neurons. Furthermore, the manipulation of
cellular cAMP level can lead to a spontaneous neuronal activity restoration in the OR83b
knockout fly, which shows no spontaneous activity of ORNs, indicating that OR83b
might play a role in cAMP-dependent modulation. Results on the Ih channel knockout fly
let us suggest that Ih channels serve as one of the downstream effectors of cAMP.
78
Conclusion
A previous study on Gq, PLC and DAG mutant flies also revealed a possible
involvement of the IP3 pathway in insect olfactory transduction. In the present study,
Gq3 was found highly expressed in Drosophila antennae, but not in the cilia of olfactory
cells. Expression of a continuously activated Gq3 in the olfactory neurons led to a
resensitization deficiency. These results suggest that Gq3 is not the downstream target of
the odorant receptor, but might play a role in the receptor endocytosis and degradation or
for a crosstalk with the Gs signaling cascade to regulate the olfactory signal
transduction.
A preliminary study showed that in larvae olfaction a different cellular signaling pathway
might be engaged with a participation of Gq but not Gs.
79
Conclusion
6.2 Zusammenfassung
Drosophila melanogaster hat 60 Geruchsrezeptorgene, die in den dritten antennalen
Segementen sowie den Maxillarpalpen exprimiert werden. Die Rezeptorproteine weisen
ein gut charakterisiertes Antwortprofil fr Geruchsstoffe auf. Ein bestimmtes
Geruchsrezeptorgen wird nur in einer kleinen Population von olfaktorischen Neuronen
exprimiert.
Jedes
Neuron
exprimiert
nur
eine
sehr
kleine
Anzahl
an
Geruchsrezeptorgenen vergleichbar mit dem olfaktorischen System von Sugetieren.

Auf zellulrer Ebene beginnt die olfaktorische Signaltransduktion mit der Aktivierung
von olfaktorischen Rezeptoren, die in der Lage sind, ein breites Spektrum von strukturell
unterschiedlichen Substanzen zu erkennen. Whrend die olfaktorischen Rezeptoren der
Vertebraten 7-Transmembranproteine sind, die nach der Bindung von Liganden
heterotrimere G-Proteine aktivieren, wurde fr die olfaktorischen Rezeptoren von
Drosophila eine inverse Membrantopologie im Vergleich zu den klassischen G-Protein
gekoppelten Rezeptoren festgestellt, mit einem intrazellulren N- und einem
extrazellulren C-Terminus. Darber hinaus wurde krzlich gezeigt, dass die
olfaktorischen
Rezeptoren
von
Drosophila
als
ligandengesteuerte
Ionenkanle
funktionieren knnen. Ergebnisse anderer Arbeitsgruppen lassen jedoch auf eine

zustzliche Beteiligung von heterotrimeren G-Proteinen in der Geruchsrezeptorabhngigen Signalweiterleitung schlieen. Es stellt sich daher die Frage, ob diese 7Transmembranrezeptoren ungeachtet der Tatsache, dass diese direkt aktiviert werden
knnen, auch an heterotrimere G-Proteine koppeln. Die von uns erzielten Ergebnisse aus
pharmakologischen in vivo Studien, elektrophysiologischen Messungen, ProteinUmverteilungsanalysen
olfaktorischen
sowie
Rezeptoren
Untersuchungen
zeigen,
dass
fr
an
die
rekombinant
exprimierten
Geruchsrezeptor-abhngige
Signaltransduktion in Drosophila tatschlich G-Proteine bentigt werden. Unsere Daten

erbringen klare Beweise fr eine Beteiligung des stimulatorischen Gs-Proteins in der
Signalkaskade von olfaktorischen Rezeptorneuronen. Passend zu einer Gs-abhngigen
Signalkaskade konnte gezeigt werden, dass ein erhhtes Niveau von cAMP zu einer
Erregung von olfaktorischen Neuronen fhrt. Weiterhin bewirkte die Manipulation des
zellulren cAMP Niveaus eine Wiederherstellung der spontanen neuronalen Aktivitt in
80
Conclusion
OR83b-knockout-Fliegen, die prinzipiell keine spontane Aktivitt von olfaktorischen
Rezeptorneuronen zeigen, was mglicherweise auf eine Rolle von OR83b in der cAMPabhngigen Modulation schlieen lsst. Die Messungen an Ih Kanal knockout-Fliegen
lassen vermuten, dass Ih-Kanle als ein Typ von Effektoren stromabwrts der cAMPBildung dienen knnten.
Vorangegangene Studien mit Gq, PLC und DAG Mutanten deuten auf eine mgliche
Beteiligung des IP3-Signalweges in der olfaktorischen Signaltransduktion von Insekten
hin. In der vorliegenden Studie wurde ein hoher Expressionslevel von Gq3 in den
Antennen von Drosophila gefunden, jedoch nicht in den Zilien der olfaktorischen Zellen.
Die Expression eines kontinuierlich aktivierten Gq3 in den olfaktorischen Neuronen
fhrte zu einer Verminderung in der Resensitisierung. Diese Ergebnisse weisen darauf
hin, dass Gq3 nicht an der primren Signaltransduktionskaskade beteiligt ist, aber
mglicherweise eine Rolle in der Endozytose und Degradation der Rezeptoren spielt oder
die olfaktorische Signaltransduktion in Abstimmung mit dem Gs Signalweg reguliert.
Erste Untersuchungen zur Geruchswahrnehmung von Larven zeigten, dass hier ein
unterschiedlicher zellulrer Signalweg vorliegen knnte, an dem Gq und nicht Gs
beteiligt ist.
81
References
References
Reference List
Ache,B.W. (1994). Towards a common strategy for transducing olfactory information.
Semin. Cell Biol. 5, 55-63.
Adams,M.D., Celniker,S.E., Holt,R.A., Evans,C.A., Gocayne,J.D., Amanatides,P.G.,
Scherer,S.E., Li,P.W., Hoskins,R.A., Galle,R.F., George,R.A., Lewis,S.E., Richards,S.,
Ashburner,M., Henderson,S.N., Sutton,G.G., Wortman,J.R., Yandell,M.D., Zhang,Q.,
Chen,L.X., Brandon,R.C., Rogers,Y.H., Blazej,R.G., Champe,M., Pfeiffer,B.D.,
Wan,K.H., Doyle,C., Baxter,E.G., Helt,G., Nelson,C.R., Gabor,G.L., Abril,J.F.,
Agbayani,A., An,H.J., Andrews-Pfannkoch,C., Baldwin,D., Ballew,R.M., Basu,A.,
Baxendale,J., Bayraktaroglu,L., Beasley,E.M., Beeson,K.Y., Benos,P.V., Berman,B.P.,
Bhandari,D., Bolshakov,S., Borkova,D., Botchan,M.R., Bouck,J., Brokstein,P.,
Brottier,P., Burtis,K.C., Busam,D.A., Butler,H., Cadieu,E., Center,A., Chandra,I.,
Cherry,J.M., Cawley,S., Dahlke,C., Davenport,L.B., Davies,P., de,P.B., Delcher,A.,
Deng,Z., Mays,A.D., Dew,I., Dietz,S.M., Dodson,K., Doup,L.E., Downes,M., DuganRocha,S., Dunkov,B.C., Dunn,P., Durbin,K.J., Evangelista,C.C., Ferraz,C., Ferriera,S.,
Fleischmann,W., Fosler,C., Gabrielian,A.E., Garg,N.S., Gelbart,W.M., Glasser,K.,
Glodek,A., Gong,F., Gorrell,J.H., Gu,Z., Guan,P., Harris,M., Harris,N.L., Harvey,D.,
Heiman,T.J., Hernandez,J.R., Houck,J., Hostin,D., Houston,K.A., Howland,T.J.,
Wei,M.H., Ibegwam,C., Jalali,M., Kalush,F., Karpen,G.H., Ke,Z., Kennison,J.A.,
Ketchum,K.A., Kimmel,B.E., Kodira,C.D., Kraft,C., Kravitz,S., Kulp,D., Lai,Z.,
Lasko,P., Lei,Y., Levitsky,A.A., Li,J., Li,Z., Liang,Y., Lin,X., Liu,X., Mattei,B.,
McIntosh,T.C., McLeod,M.P., McPherson,D., Merkulov,G., Milshina,N.V., Mobarry,C.,
Morris,J., Moshrefi,A., Mount,S.M., Moy,M., Murphy,B., Murphy,L., Muzny,D.M.,
Nelson,D.L., Nelson,D.R., Nelson,K.A., Nixon,K., Nusskern,D.R., Pacleb,J.M.,
Palazzolo,M., Pittman,G.S., Pan,S., Pollard,J., Puri,V., Reese,M.G., Reinert,K.,
Remington,K., Saunders,R.D., Scheeler,F., Shen,H., Shue,B.C., Siden-Kiamos,I.,
Simpson,M., Skupski,M.P., Smith,T., Spier,E., Spradling,A.C., Stapleton,M., Strong,R.,
Sun,E., Svirskas,R., Tector,C., Turner,R., Venter,E., Wang,A.H., Wang,X., Wang,Z.Y.,
Wassarman,D.A., Weinstock,G.M., Weissenbach,J., Williams,S.M., WoodageT,
Worley,K.C., Wu,D., Yang,S., Yao,Q.A., Ye,J., Yeh,R.F., Zaveri,J.S., Zhan,M.,
Zhang,G., Zhao,Q., Zheng,L., Zheng,X.H., Zhong,F.N., Zhong,W., Zhou,X., Zhu,S.,
Zhu,X., Smith,H.O., Gibbs,R.A., Myers,E.W., Rubin,G.M., and Venter,J.C. (2000). The
genome sequence of Drosophila melanogaster. Science 287, 2185-2195.
Amatruda,T.T., III, Steele,D.A., Slepak,V.Z., and Simon,M.I. (1991). G alpha 16, a G
protein alpha subunit specifically expressed in hematopoietic cells. Proc. Natl. Acad. Sci.
U. S. A 88, 5587-5591.
Anderson,S., Dragnea,V., Masuda,S., Ybe,J., Moffat,K., and Bauer,C. (2005). Structure
of a novel photoreceptor, the BLUF domain of AppA from Rhodobacter sphaeroides.
Biochemistry 44, 7998-8005.
82
References
Ayyub,C., Paranjape,J., Rodrigues,V., and Siddiqi,O. (1990). Genetics of olfactory
behavior in Drosophila melanogaster. J. Neurogenet. 6, 243-262.
Bakalyar,H.A., and Reed,R.R. (1990). Identification of a specialized adenylyl cyclase
that may mediate odorant detection. Science 250, 1403-1406.
Basler,K., and Struhl,G. (1994). Compartment boundaries and the control of Drosophila
limb pattern by hedgehog protein. Nature 368, 208-214.
Baumann,A., Frings,S., Godde,M., Seifert,R., and Kaupp,U.B. (1994). Primary structure
and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes
and antennae. EMBO J. 13, 5040-5050.
Beaty,B.J., and Marquardt,W.C. (1996). The biology of disease vectors University Press
of Colorado).
Benton,R. (2006). On the ORigin of smell: odorant receptors in insects. Cell Mol. Life
Sci. 63, 1579-1585.
Benton,R., Sachse,S., Michnick,S.W., and Vosshall,L.B. (2006). Atypical membrane
topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS. Biol.
4, e20.
Benton,R., Vannice,K.S., Gomez-Diaz,C., and Vosshall,L.B. (2009). Variant ionotropic
glutamate receptors as chemosensory receptors in Drosophila. Cell 136, 149-162.
Bestmann,H.J., Roth,K.D., Rehefeld,C., Leinemann,B., Kern,F., and Vostrowsky,O.
(1996). Ab initio calculations on (Z)-5-decenyl acetate, a component of the pheromone
complex of Agrotis segetum (Lepidoptera: Noctuidae) and electrophysiological studies
with chain elongated analogues. Bioorg. Med. Chem. 4, 473-477.
Bobkov,Y.V., and Ache,B.W. (2007). Block by amiloride derivatives of odor-evoked
discharge in lobster olfactory receptor neurons through action on a presumptive TRP
channel. Chem. Senses 32, 149-159.
Boekhoff,I., Michel,W.C., Breer,H., and Ache,B.W. (1994). Single odors differentially
stimulate dual second messenger pathways in lobster olfactory receptor cells. J. Neurosci.
14, 3304-3309.
Breer,H. (1994). Odor recognition and second messenger signaling in olfactory receptor
neurons. Semin. Cell Biol. 5, 25-32.
Brown,H.F., DiFrancesco,D., and Noble,S.J. (1979). How does adrenaline accelerate the
heart? Nature 280, 235-236.
Buck,L., and Axel,R. (1991). A novel multigene family may encode odorant receptors: a
molecular basis for odor recognition. Cell 65, 175-187.
83
References
Bunemann,M., Frank,M., and Lohse,M.J. (2003). Gi protein activation in intact cells
involves subunit rearrangement rather than dissociation. Proc. Natl. Acad. Sci. U. S. A
100, 16077-16082.
Burton,F.H., Hasel,K.W., Bloom,F.E., and Sutcliffe,J.G. (1991). Pituitary hyperplasia
and gigantism in mice caused by a cholera toxin transgene. Nature 350, 74-77.
Cabrera-Vera,T.M.,
Vanhauwe,J.,
Thomas,T.O.,
Medkova,M.,
Preininger,A.,
Mazzoni,M.R., and Hamm,H.E. (2003). Insights into G protein structure, function, and
regulation. Endocr. Rev. 24, 765-781.
Catterall,W.A. (1995). Structure and function of voltage-gated ion channels. Annu. Rev.
Biochem. 64, 493-531.
Clyne,P.J., Warr,C.G., Freeman,M.R., Lessing,D., Kim,J., and Carlson,J.R. (1999). A
novel family of divergent seven-transmembrane proteins: candidate odorant receptors in
Drosophila. Neuron 22, 327-338.
Cobb,M., Bruneau,S., and Jallon,J.M. (1992). Genetic and developmental factors in the
olfactory response of Drosophila melanogaster larvae to alcohols. Proc. Biol. Sci. 248,
103-109.
Conklin,B.R., Farfel,Z., Lustig,K.D., Julius,D., and Bourne,H.R. (1993). Substitution of
three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature
363, 274-276.
Conklin,B.R., Herzmark,P., Ishida,S., Voyno-Yasenetskaya,T.A., Sun,Y., Farfel,Z., and
Bourne,H.R. (1996). Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the
fidelity of receptor activation. Mol. Pharmacol. 50, 885-890.
Couto,A., Alenius,M., and Dickson,B.J. (2005). Molecular, anatomical, and functional
organization of the Drosophila olfactory system. Curr. Biol. 15, 1535-1547.
deBruyne,M., Foster,K., and Carlson,J.R. (2001). Odor coding in the Drosophila antenna.
Neuron 30, 537-552.
Deckert,C.M., Heiker,J.T., and Beck-Sickinger,A.G. (2006). Localization of novel
adiponectin receptor constructs. J. Recept. Signal. Transduct. Res. 26, 647-657.
DiFrancesco,D. (1993). Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol 55,
455-472.
Ditzen,M., Pellegrino,M., and Vosshall,L.B. (2008). Insect odorant receptors are
molecular targets of the insect repellent DEET. Science 319, 1838-1842.
Dobritsa,A.A., van der Goes van Naters, Warr,C.G., Steinbrecht,R.A., and Carlson,J.R.
(2003). Integrating the molecular and cellular basis of odor coding in the Drosophila
antenna. Neuron 37, 827-841.
84
References
Dowling,M.R., Nahorski,S.R., and John Challis,R.A. (2004). Measurement of agoniststimulated [35S]GTPgammaS binding to assess total G-protein and Galpha-subtypespecific activation by GPCRs. In Receptor signal transduction protocols, pp. 197-206.
Downes,G.B., and Gautam,N. (1999). The G protein subunit gene families. Genomics 62,
544-552.
Dubin,A.E., Liles,M.M., and Harris,G.L. (1998a). The K+ channel gene ether a go-go is
required for the transduction of a subset of odorants in adult Drosophila melanogaster. J.
Neurosci. 18, 5603-5613.
Dubin,A.E., Liles,M.M., Seligman,F., Le,T., Tolli,J., and Harris,G.L. (1998b).
Involvement of genes encoding a K+ channel (ether a go-go) and a Na+ channel
(smellblind) in Drosophila olfaction. Ann. N. Y. Acad. Sci. 855, 212-222.
Dzeja,C., Hagen,V., Kaupp,U.B., and Frings,S. (1999). Ca2+ permeation in cyclic
nucleotide-gated channels. EMBO J. 18, 131-144.
Fadool,D.A., and Ache,B.W. (1994). Inositol 1,3,4,5-tetrakisphosphate-gated channels
interact with inositol 1,4,5-trisphosphate-gated channels in olfactory receptor neurons.
Proc. Natl. Acad. Sci. U. S. A 91, 9471-9475.
Fiala,A., Spall,T., Diegelmann,S., Eisermann,B., Sachse,S., Devaud,J.M., Buchner,E.,
and Galizia,C.G. (2002). Genetically expressed cameleon in Drosophila melanogaster is
used to visualize olfactory information in projection neurons. Curr. Biol. 12, 1877-1884.
Finn,J.T., Grunwald,M.E., and Yau,K.W. (1996). Cyclic nucleotide-gated ion channels:
an extended family with diverse functions. Annu. Rev. Physiol 58, 395-426.
Firestein,S. (2001). How the olfactory system makes sense of scents. Nature 413, 211218.
Fishilevich,E., Domingos,A.I., Asahina,K., Naef,F., Vosshall,L.B., and Louis,M. (2005).
Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila. Curr.
Biol. 15, 2086-2096.
Fishilevich,E., and Vosshall,L.B. (2005). Genetic and functional subdivision of the
Drosophila antennal lobe. Curr. Biol. 15, 1548-1553.
Freitag,J., Beck,A., Ludwig,G., von,B.L., and Breer,H. (1999). On the origin of the
olfactory receptor family: receptor genes of the jawless fish (Lampetra fluviatilis). Gene
226, 165-174.
Galindo,K., and Smith,D.P. (2001). A large family of divergent Drosophila odorantbinding proteins expressed in gustatory and olfactory sensilla. Genetics 159, 1059-1072.
Gao,Q., and Chess,A. (1999). Identification of candidate Drosophila olfactory receptors
from genomic DNA sequence. Genomics 60, 31-39.
85
References
Gao,Q., Yuan,B., and Chess,A. (2000). Convergent projections of Drosophila olfactory
neurons to specific glomeruli in the antennal lobe. Nat. Neurosci. 3, 780-785.
Gendre,N., Luer,K., Friche,S., Grillenzoni,N., Ramaekers,A., Technau,G.M., and
Stocker,R.F. (2004). Integration of complex larval chemosensory organs into the adult
nervous system of Drosophila. Development 131, 83-92.
Gerber,B., and Stocker,R.F. (2007). The Drosophila larva as a model for studying
chemosensation and chemosensory learning: a review. Chem. Senses 32, 65-89.
Gisselmann,G., Gamerschlag,B., Sonnenfeld,R., Marx,T., Neuhaus,E.M., Wetzel,C.H.,
and Hatt,H. (2005a). Variants of the Drosophila melanogaster Ih-channel are generated
by different splicing. Insect Biochem. Mol. Biol. 35, 505-514.
Gisselmann,G., Marx,T., Bobkov,Y., Wetzel,C.H., Neuhaus,E.M., Ache,B.W., and
Hatt,H. (2005b). Molecular and functional characterization of an I(h)-channel from
lobster olfactory receptor neurons. Eur. J. Neurosci. 21, 1635-1647.
Gisselmann,G., Warnstedt,M., Gamerschlag,B., Bormann,A., Marx,T., Neuhaus,E.M.,
Stoertkuhl,K., Wetzel,C.H., and Hatt,H. (2003). Characterization of recombinant and
native Ih-channels from Apis mellifera. Insect Biochem. Mol. Biol. 33, 1123-1134.
Gisselmann,G., Wetzel,C.H., Warnstedt,M., and Hatt,H. (2004). Functional
characterization of Ih-channel splice variants from Apis mellifera. FEBS Lett. 575, 99104.
Goc,A., Angel,T.E., Jastrzebska,B., Wang,B., Wintrode,P.L., and Palczewski,K. (2008).
Different properties of the native and reconstituted heterotrimeric G protein transducin.
Biochemistry 47, 12409-12419.
Godfrey,P.A., Malnic,B., and Buck,L.B. (2004). The mouse olfactory receptor gene
family. Proc. Natl. Acad. Sci. U. S. A 101, 2156-2161.
Gomez-Diaz,C., Martin,F., and Alcorta,E. (2004). The cAMP transduction cascade
mediates olfactory reception in Drosophila melanogaster. Behav. Genet. 34, 395-406.
Grobner,G., Burnett,I.J., Glaubitz,C., Choi,G., Mason,A.J., and Watts,A. (2000).
Observations of light-induced structural changes of retinal within rhodopsin. Nature 405,
810-813.
Hallem,E.A., and Carlson,J.R. (2004). The odor coding system of Drosophila. Trends
Genet. 20, 453-459.
Hallem,E.A., Ho,M.G., and Carlson,J.R. (2004). The molecular basis of odor coding in
the Drosophila antenna. Cell 117, 965-979.
Halliwell,J.V., and Adams,P.R. (1982). Voltage-clamp analysis of muscarinic excitation
in hippocampal neurons. Brain Res. 250, 71-92.
86
References
Hamm,H.E. (1998). The many faces of G protein signaling. J. Biol. Chem. 273, 669-672.
Hampoelz,B., Hoeller,O., Bowman,S.K., Dunican,D., and Knoblich,J.A. (2005).
Drosophila Ric-8 is essential for plasma-membrane localization of heterotrimeric G
proteins. Nat. Cell Biol. 7, 1099-1105.
Han,J., Reddig,K., and Li,H.S. (2007). Prolonged G(q) activity triggers fly rhodopsin
endocytosis and degradation, and reduces photoreceptor sensitivity. EMBO J. 26, 49664973.
Hasan,G., and Rosbash,M. (1992). Drosophila homologs of two mammalian intracellular
Ca(2+)-release channels: identification and expression patterns of the inositol 1,4,5triphosphate and the ryanodine receptor genes. Development 116, 967-975.
Hatt,H., and Ache,B.W. (1994). Cyclic nucleotide- and inositol phosphate-gated ion
channels in lobster olfactory receptor neurons. Proc. Natl. Acad. Sci. U. S. A 91, 62646268.
Heimbeck,G., Bugnon,V., Gendre,N., Haberlin,C., and Stocker,R.F. (1999). Smell and
taste perception in Drosophila melanogaster larva: toxin expression studies in
chemosensory neurons. J. Neurosci. 19, 6599-6609.
Heisenberg,M. (2003). Mushroom body memoir: from maps to models. Nat. Rev.
Neurosci. 4, 266-275.
Hildebrand,J.G., and Shepherd,G.M. (1997). Mechanisms of olfactory discrimination:
converging evidence for common principles across phyla. Annu. Rev. Neurosci. 20, 595631.
Hilf,G., Gierschik,P., and Jakobs,K.H. (1989). Muscarinic acetylcholine receptorstimulated binding of guanosine 5'-O-(3-thiotriphosphate) to guanine-nucleotide-binding
proteins in cardiac membranes. Eur. J. Biochem. 186, 725-731.
Hill,C.A., Fox,A.N., Pitts,R.J., Kent,L.B., Tan,P.L., Chrystal,M.A., Cravchik,A.,
Collins,F.H., Robertson,H.M., and Zwiebel,L.J. (2002). G protein-coupled receptors in
Anopheles gambiae. Science 298, 176-178.
Huang,L., Shanker,Y.G., Dubauskaite,J., Zheng,J.Z., Yan,W., Rosenzweig,S.,
Spielman,A.I., Max,M., and Margolskee,R.F. (1999). Ggamma13 colocalizes with
gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium. Nat.
Neurosci. 2, 1055-1062.
Hughes,T.E., Zhang,H., Logothetis,D.E., and Berlot,C.H. (2001). Visualization of a
functional Galpha q-green fluorescent protein fusion in living cells. Association with the
plasma membrane is disrupted by mutational activation and by elimination of
palmitoylation sites, but not be activation mediated by receptors or AlF4-. J. Biol. Chem.
276, 4227-4235.
87
References
Hummel,T., Vasconcelos,M.L., Clemens,J.C., Fishilevich,Y., Vosshall,L.B., and
Zipursky,S.L. (2003). Axonal targeting of olfactory receptor neurons in Drosophila is
controlled by Dscam. Neuron 37, 221-231.
Iseki,M., Matsunaga,S., Murakami,A., Ohno,K., Shiga,K., Yoshida,K., Sugai,M.,
Takahashi,T., Hori,T., and Watanabe,M. (2002). A blue-light-activated adenylyl cyclase
mediates photoavoidance in Euglena gracilis. Nature 415, 1047-1051.
Ishimoto,H., Takahashi,K., Ueda,R., and Tanimura,T. (2005). G-protein gamma subunit
1 is required for sugar reception in Drosophila. EMBO J. 24, 3259-3265.
Ishimoto,H., and Tanimura,T. (2004). Molecular neurophysiology of taste in Drosophila.
Cell Mol. Life Sci. 61, 10-18.
Janetopoulos,C., Jin,T., and Devreotes,P. (2001). Receptor-mediated activation of
heterotrimeric G-proteins in living cells. Science 291, 2408-2411.
Jefferis,G.S., Marin,E.C., Stocker,R.F., and Luo,L. (2001). Target
prespecification in the olfactory map of Drosophila. Nature 414, 204-208.
neuron
Jefferis,G.S., Vyas,R.M., Berdnik,D., Ramaekers,A., Stocker,R.F., Tanaka,N.K., Ito,K.,

and Luo,L. (2004). Developmental origin of wiring specificity in the olfactory system of
Drosophila. Development 131, 117-130.
Jin,X., Ha,T.S., and Smith,D.P. (2008). SNMP is a signaling component required for
pheromone sensitivity in Drosophila. Proc. Natl. Acad. Sci. U. S. A 105, 10996-11001.
Jones,D.T., and Reed,R.R. (1989). Golf: an olfactory neuron specific-G protein involved
in odorant signal transduction. Science 244, 790-795.
Jones,W.D., Jr., Beam,R.E., and David,H.L. (1974). Transduction of a streptomycin Rfactor from Mycobacterium smegmatis to Mycobacterium tuberculosis H37Rv. Tubercle.
55, 73-80.
Jones,W.D., Cayirlioglu,P., Kadow,I.G., and Vosshall,L.B. (2007). Two chemosensory
receptors together mediate carbon dioxide detection in Drosophila. Nature 445, 86-90.
Kain,P., Chakraborty,T.S., Sundaram,S., Siddiqi,O., Rodrigues,V., and Hasan,G. (2008).
Reduced odor responses from antennal neurons of G(q)alpha, phospholipase Cbeta, and
rdgA mutants in Drosophila support a role for a phospholipid intermediate in insect
olfactory transduction. J. Neurosci. 28, 4745-4755.
Kain,P., Chandrashekaran,S., Rodrigues,V., and Hasan,G. (2009). Drosophila mutants in
phospholipid signaling have reduced olfactory responses as adults and larvae. J.
Neurogenet. 23, 303-312.
Kaissling,K.E. (1996). Peripheral mechanisms of pheromone reception in moths. Chem.
Senses 21, 257-268.
88
References
Kalidas,S., and Smith,D.P. (2002). Novel genomic cDNA hybrids produce effective RNA
interference in adult Drosophila. Neuron 33, 177-184.
Katanaev,V.L., Ponzielli,R., Semeriva,M., and Tomlinson,A. (2005). Trimeric G proteindependent frizzled signaling in Drosophila. Cell 120, 111-122.
Kaupp,U.B., and Seifert,R. (2002). Cyclic nucleotide-gated ion channels. Physiol Rev.
82, 769-824.
Knust,E. (2001). G protein signaling and asymmetric cell division. Cell 107, 125-128.
Komiyama,T., Johnson,W.A., Luo,L., and Jefferis,G.S. (2003). From lineage to wiring
specificity. POU domain transcription factors control precise connections of Drosophila
olfactory projection neurons. Cell 112, 157-167.
Kper M. Olfactory perception and olfactory prossessing of Drosophila melanogaster.
2008. IGB, Ruhr-University Bochum.
Ref Type: Thesis/Dissertation
Kreher,S.A., Kwon,J.Y., and Carlson,J.R. (2005). The molecular basis of odor coding in
the Drosophila larva. Neuron 46, 445-456.
Krieger,J., and Breer,H. (1999). Olfactory reception in invertebrates. Science 286, 720723.
Krieger,J., Strobel,J., Vogl,A., Hanke,W., and Breer,H. (1999). Identification of a cyclic
nucleotide- and voltage-activated ion channel from insect antennae. Insect Biochem. Mol.
Biol. 29, 255-267.
Kurtovic,A., Widmer,A., and Dickson,B.J. (2007). A single class of olfactory neurons
mediates behavioural responses to a Drosophila sex pheromone. Nature 446, 542-546.
Kwon,J.Y., Dahanukar,A., Weiss,L.A., and Carlson,J.R. (2007). The molecular basis of
CO2 reception in Drosophila. Proc. Natl. Acad. Sci. U. S. A 104, 3574-3578.
Laissue,P.P., Reiter,C., Hiesinger,P.R., Halter,S., Fischbach,K.F., and Stocker,R.F.
(1999). Three-dimensional reconstruction of the antennal lobe in Drosophila
melanogaster. J. Comp Neurol. 405, 543-552.
Lambright,D.G., Noel,J.P., Hamm,H.E., and Sigler,P.B. (1994). Structural determinants
for activation of the alpha-subunit of a heterotrimeric G protein. Nature 369, 621-628.
Larsson,M.C., Domingos,A.I., Jones,W.D., Chiappe,M.E., Amrein,H., and Vosshall,L.B.
(2004). Or83b encodes a broadly expressed odorant receptor essential for Drosophila
olfaction. Neuron 43, 703-714.
89
References
Laue,M., Maida,R., and Redkozubov,A. (1997). G-protein activation, identification and
immunolocalization in pheromone-sensitive sensilla trichodea of moths. Cell Tissue Res.
288, 149-158.
Lee,T., and Luo,L. (1999). Mosaic analysis with a repressible cell marker for studies of
gene function in neuronal morphogenesis. Neuron 22, 451-461.
Lee,Y.J., Dobbs,M.B., Verardi,M.L., and Hyde,D.R. (1990). dgq: a drosophila gene
encoding a visual system-specific G alpha molecule. Neuron 5, 889-898.
Lee,Y.J., Shah,S., Suzuki,E., Zars,T., O'Day,P.M., and Hyde,D.R. (1994). The
Drosophila dgq gene encodes a G alpha protein that mediates phototransduction. Neuron
13, 1143-1157.
Liman,E.R. (2006). Use it or lose it: molecular evolution of sensory signaling in primates.
Pflugers Arch. 453, 125-131.
Livak,K.J., and Schmittgen,T.D. (2001). Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408.
Lundin,C., Kall,L., Kreher,S.A., Kapp,K., Sonnhammer,E.L., Carlson,J.R., Heijne,G.,
and Nilsson,I. (2007). Membrane topology of the Drosophila OR83b odorant receptor.
FEBS Lett. 581, 5601-5604.
Magee,J.C. (1999). Dendritic lh normalizes temporal summation in hippocampal CA1
neurons. Nat. Neurosci. 2, 508-514.
Malnic,B., Godfrey,P.A., and Buck,L.B. (2004). The human olfactory receptor gene
family. Proc. Natl. Acad. Sci. U. S. A 101, 2584-2589.
Marin,E.C., Jefferis,G.S., Komiyama,T., Zhu,H., and Luo,L. (2002). Representation of
the glomerular olfactory map in the Drosophila brain. Cell 109, 243-255.
Markby,D.W., Onrust,R., and Bourne,H.R. (1993). Separate GTP binding and GTPase
activating domains of a G alpha subunit. Science 262, 1895-1901.
Martin,F., Charro,M.J., and Alcorta,E. (2001). Mutations affecting the cAMP
transduction pathway modify olfaction in Drosophila. J. Comp Physiol A 187, 359-370.
Marx,T., Gisselmann,G., Stortkuhl,K.F., Hovemann,B.T., and Hatt,H. (1999). Molecular
cloning of a putative voltage- and cyclic nucleotide-gated ion channel present in the
antennae and eyes of Drosophila melanogaster. Invert. Neurosci. 4, 55-63.
Matsuzaki,F. (2005). Drosophila G-protein signalling: intricate roles for Ric-8? Nat. Cell
Biol. 7, 1047-1049.
McClintock,T.S.,
Landers,T.M.,
Gimelbrant,A.A.,
Fuller,L.Z.,
Jackson,B.A.,
Jayawickreme,C.K., and Lerner,M.R. (1997). Functional expression of olfactory-
90
References
adrenergic receptor chimeras and intracellular retention of heterologously expressed
olfactory receptors. Brain Res. Mol. Brain Res. 48, 270-278.
McCudden,C.R., Hains,M.D., Kimple,R.J., Siderovski,D.P., and Willard,F.S. (2005). Gprotein signaling: back to the future. Cell Mol. Life Sci. 62, 551-577.
McLaughlin,S.K., McKinnon,P.J., and Margolskee,R.F. (1992). Gustducin is a taste-cellspecific G protein closely related to the transducins. Nature 357, 563-569.
Merrill,C.E., Pitts,R.J., and Zwiebel,L.J. (2003). Molecular characterization of arrestin
family members in the malaria vector mosquito, Anopheles gambiae. Insect Mol. Biol.
12, 641-650.
Merrill,C.E., Riesgo-Escovar,J., Pitts,R.J., Kafatos,F.C., Carlson,J.R., and Zwiebel,L.J.
(2002). Visual arrestins in olfactory pathways of Drosophila and the malaria vector
mosquito Anopheles gambiae. Proc. Natl. Acad. Sci. U. S. A 99, 1633-1638.
Merrill,C.E., Sherertz,T.M., Walker,W.B., and Zwiebel,L.J. (2005). Odorant-specific
requirements for arrestin function in Drosophila olfaction. J. Neurobiol. 63, 15-28.
Michel,W.C., and Ache,B.W. (1992). Cyclic nucleotides mediate an odor-evoked
potassium conductance in lobster olfactory receptor cells. J. Neurosci. 12, 3979-3984.
Mody,S.M., Ho,M.K., Joshi,S.A., and Wong,Y.H. (2000). Incorporation of Galpha(z)specific sequence at the carboxyl terminus increases the promiscuity of galpha(16)
toward G(i)-coupled receptors. Mol. Pharmacol. 57, 13-23.
Mombaerts,P., Wang,F., Dulac,C., Chao,S.K., Nemes,A., Mendelsohn,M., Edmondson,J.,
and Axel,R. (1996). Visualizing an olfactory sensory map. Cell 87, 675-686.
Monte,P., Woodard,C., Ayer,R., Lilly,M., Sun,H., and Carlson,J. (1989).
Characterization of the larval olfactory response in Drosophila and its genetic basis.
Behav. Genet. 19, 267-283.
Moss,J., and Vaughan,M. (1988). ADP-ribosylation of guanyl nucleotide-binding
regulatory proteins by bacterial toxins. Adv. Enzymol. Relat Areas Mol. Biol. 61, 303379.
Murthy,M., Fiete,I., and Laurent,G. (2008). Testing odor response stereotypy in the
Drosophila mushroom body. Neuron 59, 1009-1023.
Nakagawa,T., Sakurai,T., Nishioka,T., and Touhara,K. (2005). Insect sex-pheromone
signals mediated by specific combinations of olfactory receptors. Science 307, 16381642.
Nakamura,T., and Gold,G.H. (1987). A cyclic nucleotide-gated conductance in olfactory
receptor cilia. Nature 325, 442-444.
91
References
Nef,S., Allaman,I., Fiumelli,H., De,C.E., and Nef,P. (1996). Olfaction in birds:
differential embryonic expression of nine putative odorant receptor genes in the avian
olfactory system. Mech. Dev. 55, 65-77.
Neuhaus,E.M., Gisselmann,G., Zhang,W., Dooley,R., Stortkuhl,K., and Hatt,H. (2005).
Odorant receptor heterodimerization in the olfactory system of Drosophila melanogaster.
Nat. Neurosci. 8, 15-17.
Ng,M., Roorda,R.D., Lima,S.Q., Zemelman,B.V., Morcillo,P., and Miesenbock,G.
(2002). Transmission of olfactory information between three populations of neurons in
the antennal lobe of the fly. Neuron 36, 463-474.
Noe,J., and Breer,H. (1998). Functional and molecular characterization of individual
olfactory neurons. J. Neurochem. 71, 2286-2293.
Nurnberg,B., Gudermann,T., and Schultz,G. (1995). Receptors and G proteins as primary
components of transmembrane signal transduction. Part 2. G proteins: structure and
function. J. Mol. Med. 73, 123-132.
Oppliger,F.Y., Guerin,M., and Vlimant,M. (2000). Neurophysiological and behavioural
evidence for an olfactory function for the dorsal organ and a gustatory one for the
terminal organ in Drosophila melanogaster larvae. J. Insect Physiol 46, 135-144.
Pape,H.C., and McCormick,D.A. (1989). Noradrenaline and serotonin selectively
modulate thalamic burst firing by enhancing a hyperpolarization-activated cation current.
Nature 340, 715-718.
Park,S.K., Shanbhag,S.R., Dubin,A.E., de,B.M., Wang,Q., Yu,P., Shimoni,N.,
D'Mello,S., Carlson,J.R., Harris,G.L., Steinbrecht,R.A., and Pikielny,C.W. (2002).
Inactivation of olfactory sensilla of a single morphological type differentially affects the
response of Drosophila to odors. J. Neurobiol. 51, 248-260.
Park,S.K., Shanbhag,S.R., Wang,Q., Hasan,G., Steinbrecht,R.A., and Pikielny,C.W.
(2000). Expression patterns of two putative odorant-binding proteins in the olfactory
organs of Drosophila melanogaster have different implications for their functions. Cell
Tissue Res. 300, 181-192.
Paysan,J., and Breer,H. (2001). Molecular physiology of odor detection: current views.
Pflugers Arch. 441, 579-586.
Pellegrino,M., and Nakagawa,T. (2009). Smelling the difference: controversial ideas in
insect olfaction. J. Exp. Biol. 212, 1973-1979.
Pelosi,P., and Maida,R. (1995). Odorant-binding proteins in insects. Comp Biochem.
Physiol B Biochem. Mol. Biol. 111, 503-514.
Philp,N.J., Chang,W., and Long,K. (1987). Light-stimulated protein movement in rod
photoreceptor cells of the rat retina. FEBS Lett. 225, 127-132.
92
References
Pin,J.P., Kniazeff,J., Liu,J., Binet,V., Goudet,C., Rondard,P., and Prezeau,L. (2005).
Allosteric functioning of dimeric class C G-protein-coupled receptors. FEBS J. 272,
2947-2955.
Postlethwait,J.H., and Schneiderman,H.A. (1971). Pattern formation and determination in
the antenna of the homoeotic mutant Antennapedia of Drosophila melanogaster. Dev.
Biol. 25, 606-640.
Python,F., and Stocker,R.F. (2002). Adult-like complexity of the larval antennal lobe of
D. melanogaster despite markedly low numbers of odorant receptor neurons. J. Comp
Neurol. 445, 374-387.
Quan,F., and Forte,M.A. (1990). Two forms of Drosophila melanogaster Gs alpha are
produced by alternate splicing involving an unusual splice site. Mol. Cell Biol. 10, 910917.
Ratnaparkhi,A., Banerjee,S., and Hasan,G. (2002). Altered levels of Gq activity modulate
axonal pathfinding in Drosophila. J. Neurosci. 22, 4499-4508.
Ray,K., and Ganguly,R. (1994). Organization and expression of the Drosophila
melanogaster D-G gamma 1 gene encoding the G-protein gamma subunit. Gene 148,
315-319.
Ressler,K.J., Sullivan,S.L., and Buck,L.B. (1994). Information coding in the olfactory
system: evidence for a stereotyped and highly organized epitope map in the olfactory
bulb. Cell 79, 1245-1255.
Riesgo-Escovar,J.R., Piekos,W.B., and Carlson,J.R. (1997). The Drosophila antenna:
ultrastructural and physiological studies in wild-type and lozenge mutants. J. Comp
Physiol A 180, 151-160.
Robertson,H.M., Warr,C.G., and Carlson,J.R. (2003). Molecular evolution of the insect
chemoreceptor gene superfamily in Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S.
A 100 Suppl 2, 14537-14542.
Rodrigues,V. (1988). Spatial coding of olfactory information in the antennal lobe of
Drosophila melanogaster. Brain Res. 453, 299-307.
Roeper,J., Sewing,S., Zhang,Y., Sommer,T., Wanner,S.G., and Pongs,O. (1998). NIP
domain prevents N-type inactivation in voltage-gated potassium channels. Nature 391,
390-393.
Ronnett,G.V., and Moon,C. (2002). G proteins and olfactory signal transduction. Annu.
Rev. Physiol 64, 189-222.
Rutzler,M., and Zwiebel,L.J. (2005). Molecular biology of insect olfaction: recent
progress and conceptual models. J. Comp Physiol A Neuroethol. Sens. Neural Behav.
Physiol 191, 777-790.
93
References
Sakurai,T., Nakagawa,T., Mitsuno,H., Mori,H., Endo,Y., Tanoue,S., Yasukochi,Y.,
Touhara,K., and Nishioka,T. (2004). Identification and functional characterization of a
sex pheromone receptor in the silkmoth Bombyx mori. Proc. Natl. Acad. Sci. U. S. A
101, 16653-16658.
Santoro,B., Grant,S.G., Bartsch,D., and Kandel,E.R. (1997). Interactive cloning with the
SH3 domain of N-src identifies a new brain specific ion channel protein, with homology
to eag and cyclic nucleotide-gated channels. Proc. Natl. Acad. Sci. U. S. A 94, 1481514820.
Sato,K., Pellegrino,M., Nakagawa,T., Nakagawa,T., Vosshall,L.B., and Touhara,K.
(2008). Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452,
1002-1006.
Schmidt,C.J., Garen-Fazio,S., Chow,Y.K., and Neer,E.J. (1989). Neuronal expression of
a newly identified Drosophila melanogaster G protein alpha 0 subunit. Cell Regul. 1,
125-134.
Schroder-Lang,S., Schwarzel,M., Seifert,R., Strunker,T., Kateriya,S., Looser,J.,
Watanabe,M., Kaupp,U.B., Hegemann,P., and Nagel,G. (2007). Fast manipulation of
cellular cAMP level by light in vivo. Nat. Methods 4, 39-42.
Schulz,S., Huber,A., Schwab,K., and Paulsen,R. (1999). A novel Ggamma isolated from
Drosophila constitutes a visual G protein gamma subunit of the fly compound eye. J.
Biol. Chem. 274, 37605-37610.
Selbie,L.A., Townsend-Nicholson,A., Iismaa,T.P., and Shine,J. (1992). Novel G proteincoupled receptors: a gene family of putative human olfactory receptor sequences. Brain
Res. Mol. Brain Res. 13, 159-163.
Sengupta,P., Chou,J.H., and Bargmann,C.I. (1996). odr-10 encodes a seven
transmembrane domain olfactory receptor required for responses to the odorant diacetyl.
Cell 84, 899-909.
Shanbhag,S.R., Muller,B., and Steinbrecht,R.A. Atlas of olfactory organs of Drosophila
melanogaster 1. Types, external organization, innervation and distribution of olfactory
sensilla. International Journal of Insect Morphology and Embryology 28. 1999.
Ref Type: Generic
Shanbhag,S.R., Muller,B., and Steinbrecht,R.A. (2000). Atlas of olfactory organs of
Drosophila melanogaster 2. Internal organization and cellular architecture of olfactory
sensilla. Arthropod. Struct. Dev. 29, 211-229.
Simon,M.I., Strathmann,M.P., and Gautam,N. (1991). Diversity of G proteins in signal
transduction. Science 252, 802-808.
Sinnarajah,S., Ezeh,P.I., Pathirana,S., Moss,A.G., Morrison,E.E., and Vodyanoy,V.
(1998). Inhibition and enhancement of odorant-induced cAMP accumulation in rat
94
References
olfactory cilia by antibodies directed against G alpha S/olf- and G alpha i-protein
subunits. FEBS Lett. 426, 377-380.
Smart,R., Kiely,A., Beale,M., Vargas,E., Carraher,C., Kralicek,A.V., Christie,D.L.,
Chen,C., Newcomb,R.D., and Warr,C.G. (2008). Drosophila odorant receptors are novel
seven transmembrane domain proteins that can signal independently of heterotrimeric G
proteins. Insect Biochem. Mol. Biol. 38, 770-780.
Smith,D.P. (2007). Odor and pheromone detection in Drosophila melanogaster. Pflugers
Arch. 454, 749-758.
Spehr,M., Gisselmann,G., Poplawski,A., Riffell,J.A., Wetzel,C.H., Zimmer,R.K., and
Hatt,H. (2003). Identification of a testicular odorant receptor mediating human sperm
chemotaxis. Science 299, 2054-2058.
Spletter,M.L., and Luo,L. (2009). A new family of odorant receptors in Drosophila. Cell
136, 23-25.
Sprang,S.R. (1997). G protein mechanisms: insights from structural analysis. Annu. Rev.
Biochem. 66, 639-678.
Stengl,M. (1993). Intracellular-messenger-mediated cation channels in cultured olfactory
receptor neurons. J. Exp. Biol. 178, 125-147.
Stengl,M. (1994). Inositol-trisphosphate-dependent calcium currents precede cation
currents in insect olfactory receptor neurons in vitro. J. Comp Physiol A 174, 187-194.
Stengl,M., Hatt,H., and Breer,H. (1992a). Peripheral processes in insect olfaction. Annu.
Rev. Physiol 54, 665-681.
Stengl,M., Zufall,F., Hatt,H., and Hildebrand,J.G. (1992b). Olfactory receptor neurons
from antennae of developing male Manduca sexta respond to components of the speciesspecific sex pheromone in vitro. J. Neurosci. 12, 2523-2531.
Stensmyr,M.C., Giordano,E., Balloi,A., Angioy,A.M., and Hansson,B.S. (2003). Novel
natural ligands for Drosophila olfactory receptor neurones. J. Exp. Biol. 206, 715-724.
Stevens,D.R., Seifert,R., Bufe,B., Muller,F., Kremmer,E., Gauss,R., Meyerhof,W.,
Kaupp,U.B., and Lindemann,B. (2001). Hyperpolarization-activated channels HCN1 and
HCN4 mediate responses to sour stimuli. Nature 413, 631-635.
Stocker,R.F. (1994). The organization of the chemosensory system in Drosophila
melanogaster: a review. Cell Tissue Res. 275, 3-26.
Stocker,R.F., Lienhard,M.C., Borst,A., and Fischbach,K.F. (1990). Neuronal architecture
of the antennal lobe in Drosophila melanogaster. Cell Tissue Res. 262, 9-34.
95
References
Stortkuhl,K.F., Hovemann,B.T., and Carlson,J.R. (1999). Olfactory adaptation depends
on the Trp Ca2+ channel in Drosophila. J. Neurosci. 19, 4839-4846.
Stortkuhl,K.F., and Kettler,R. (2001). Functional analysis of an olfactory receptor in
Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A 98, 9381-9385.
Strapps,W.R., and Tomlinson,A. (2001). Transducing properties of Drosophila Frizzled
proteins. Development 128, 4829-4835.
Sunahara,R.K., Tesmer,J.J., Gilman,A.G., and Sprang,S.R. (1997). Crystal structure of
the adenylyl cyclase activator Gsalpha. Science 278, 1943-1947.
Sweeney,L.B., Couto,A., Chou,Y.H., Berdnik,D., Dickson,B.J., Luo,L., and
Komiyama,T. (2007). Temporal target restriction of olfactory receptor neurons by
Semaphorin-1a/PlexinA-mediated axon-axon interactions. Neuron 53, 185-200.
Talluri,S., Bhatt,A., and Smith,D.P. (1995). Identification of a Drosophila G protein
alpha subunit (dGq alpha-3) expressed in chemosensory cells and central neurons. Proc.
Natl. Acad. Sci. U. S. A 92, 11475-11479.
Tegoni,M., Campanacci,V., and Cambillau,C. (2004). Structural aspects of sexual
attraction and chemical communication in insects
1. Trends Biochem. Sci. 29, 257-264.
Tissot,M., Gendre,N., Hawken,A., Stortkuhl,K.F., and Stocker,R.F. (1997). Larval
chemosensory projections and invasion of adult afferents in the antennal lobe of
Drosophila. J. Neurobiol. 32, 281-297.
Ueno,K., Kohatsu,S., Clay,C., Forte,M., Isono,K., and Kidokoro,Y. (2006). Gsalpha is
involved in sugar perception in Drosophila melanogaster. J. Neurosci. 26, 6143-6152.
van der Goes van Naters, and Carlson,J.R. (2007). Receptors and neurons for fly odors in
Drosophila. Curr. Biol. 17, 606-612.
Vassar,R., Chao,S.K., Sitcheran,R., Nunez,J.M., Vosshall,L.B., and Axel,R. (1994).
Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981-991.
Vogl,A., Noe,J., Breer,H., and Boekhoff,I. (2000). Cross-talk between olfactory second
messenger pathways. Eur. J. Biochem. 267, 4529-4535.
Vosshall,L.B., Amrein,H., Morozov,P.S., Rzhetsky,A., and Axel,R. (1999). A spatial
map of olfactory receptor expression in the Drosophila antenna. Cell 96, 725-736.
Vosshall,L.B., Wong,A.M., and Axel,R. (2000). An olfactory sensory map in the fly
brain. Cell 102, 147-159.
96
References
Wang,J.W., Wong,A.M., Flores,J., Vosshall,L.B., and Axel,R. (2003). Two-photon
calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112, 271282.
Wang,Y., Wright,N.J., Guo,H., Xie,Z., Svoboda,K., Malinow,R., Smith,D.P., and
Zhong,Y. (2001). Genetic manipulation of the odor-evoked distributed neural activity in
the Drosophila mushroom body. Neuron 29, 267-276.
Wellerdieck,C., Oles,M., Pott,L., Korsching,S., Gisselmann,G., and Hatt,H. (1997).
Functional expression of odorant receptors of the zebrafish Danio rerio and of the
nematode C. elegans in HEK293 cells
1. Chem. Senses 22, 467-476.
Wetzel,C.H., Behrendt,H.J., Gisselmann,G., Stortkuhl,K.F., Hovemann,B., and Hatt,H.
(2001). Functional expression and characterization of a Drosophila odorant receptor in a
heterologous cell system. Proc. Natl. Acad. Sci. U. S. A 98, 9377-9380.
Whelan,J.P., and McGinnis,J.F. (1988). Light-dependent subcellular movement of
photoreceptor proteins. J. Neurosci. Res. 20, 263-270.
White,J.F., Grodnitzky,J., Louis,J.M., Trinh,L.B., Shiloach,J., Gutierrez,J., Northup,J.K.,
and Grisshammer,R. (2007). Dimerization of the class A G protein-coupled neurotensin
receptor NTS1 alters G protein interaction. Proc. Natl. Acad. Sci. U. S. A 104, 1219912204.
Wicher,D., Schafer,R., Bauernfeind,R., Stensmyr,M.C., Heller,R., Heinemann,S.H., and
Hansson,B.S. (2008). Drosophila odorant receptors are both ligand-gated and cyclicnucleotide-activated cation channels. Nature 452, 1007-1011.
Williams,S.R., and Stuart,G.J. (2000). Site independence of EPSP time course is
mediated by dendritic I(h) in neocortical pyramidal neurons. J. Neurophysiol. 83, 31773182.
Wilson,R.I., Turner,G.C., and Laurent,G. (2004). Transformation of olfactory
representations in the Drosophila antennal lobe. Science 303, 366-370.
Wojtasek,H., Hansson,B.S., and Leal,W.S. (1998). Attracted or repelled?--a matter of
two neurons, one pheromone binding protein, and a chiral center. Biochem. Biophys.
Res. Commun. 250, 217-222.
Wolfgang,W.J., Hoskote,A., Roberts,I.J., Jackson,S., and Forte,M. (2001). Genetic
analysis of the Drosophila Gs(alpha) gene. Genetics 158, 1189-1201.
Wolfgang,W.J., Quan,F., Thambi,N., and Forte,M. (1991). Restricted spatial and
temporal expression of G-protein alpha subunits during Drosophila embryogenesis.
Development 113, 527-538.
97
References
Wong,A.M., Wang,J.W., and Axel,R. (2002). Spatial representation of the glomerular
map in the Drosophila protocerebrum. Cell 109, 229-241.
Xu,T., and Rubin,G.M. (1993). Analysis of genetic mosaics in developing and adult
Drosophila tissues. Development 117, 1223-1237.
Yamauchi,T., Kamon,J., Ito,Y., Tsuchida,A., Yokomizo,T., Kita,S., Sugiyama,T.,
Miyagishi,M., Hara,K., Tsunoda,M., Murakami,K., Ohteki,T., Uchida,S., Takekawa,S.,
Waki,H., Tsuno,N.H., Shibata,Y., Terauchi,Y., Froguel,P., Tobe,K., Koyasu,S., Taira,K.,
Kitamura,T., Shimizu,T., Nagai,R., and Kadowaki,T. (2003). Cloning of adiponectin
receptors that mediate antidiabetic metabolic effects. Nature 423, 762-769.
Yao,C.A., Ignell,R., and Carlson,J.R. (2005). Chemosensory coding by neurons in the
coeloconic sensilla of the Drosophila antenna. J. Neurosci. 25, 8359-8367.
Yapici,N., Kim,Y.J., Ribeiro,C., and Dickson,B.J. (2008). A receptor that mediates the
post-mating switch in Drosophila reproductive behaviour. Nature 451, 33-37.
Yoshikawa,S., Tanimura,T., Miyawaki,A., Nakamura,M., Yuzaki,M., Furuichi,T., and
Mikoshiba,K. (1992). Molecular cloning and characterization of the inositol 1,4,5trisphosphate receptor in Drosophila melanogaster. J. Biol. Chem. 267, 16613-16619.
Yu,J.Z., and Rasenick,M.M. (2002). Real-time visualization of a fluorescent G(alpha)(s):
dissociation of the activated G protein from plasma membrane. Mol. Pharmacol. 61, 352359.
Zhang,R.G., Scott,D.L., Westbrook,M.L., Nance,S., Spangler,B.D., Shipley,G.G., and
Westbrook,E.M. (1995). The three-dimensional crystal structure of cholera toxin. J. Mol.
Biol. 251, 563-573.
Zhang,X., and Firestein,S. (2002). The olfactory receptor gene superfamily of the mouse.
Nat. Neurosci. 5, 124-133.
Zhao,H., Ivic,L., Otaki,J.M., Hashimoto,M., Mikoshiba,K., and Firestein,S. (1998).
Functional expression of a mammalian odorant receptor. Science 279, 237-242.
Zhu,Y., Bond,J., and Thomas,P. (2003). Identification, classification, and partial
characterization of genes in humans and other vertebrates homologous to a fish
membrane progestin receptor. Proc. Natl. Acad. Sci. U. S. A 100, 2237-2242.
Ziegelberger,G. (1996). The multiple role of the pheromone-binding protein in olfactory
transduction. Ciba Found. Symp. 200, 267-275.
Zufall,F., Firestein,S., and Shepherd,G.M. (1994). Cyclic nucleotide-gated ion channels
and sensory transduction in olfactory receptor neurons. Annu. Rev. Biophys. Biomol.
Struct. 23, 577-607.
98
References
Zufall,F., and Hatt,H. (1991). Dual activation of a sex pheromone-dependent ion channel
from insect olfactory dendrites by protein kinase C activators and cyclic GMP. Proc.
Natl. Acad. Sci. U. S. A 88, 8520-8524.
Zufall,F., Hatt,H., and Firestein,S. (1993). Rapid application and removal of second
messengers to cyclic nucleotide-gated channels from olfactory epithelium. Proc. Natl.
Acad. Sci. U. S. A 90, 9335-9339.
Zufall,F., Hatt,H., and Keil,T.A. (1991a). A calcium-activated nonspecific cation channel
from olfactory receptor neurons of the silkmoth Antheraea polyphemus.
2. J. Exp. Biol. 455-468.
Zufall,F., Stengl,M., Franke,C., Hildebrand,J.G., and Hatt,H. (1991b). Ionic currents of
cultured olfactory receptor neurons from antennae of male Manduca sexta. J. Neurosci.
11, 956-965.
99
Abbreviations
Abbreviations
AC
Adenylate Cyclase
AL
Antennal Lobe
ATP
Adenosine 5-triphosphate
BSA
Bovine Serum Albumin
cAMP
Adenosine 3,5-cyclic monophosphate
CNG
Cyclic Nucleotide-Gated Channel
CTX
Cholera Toxin
DMEM
Dulbeccos Modified Eagles Medium
DMSO
Dimethylsulfoxide
DO
Dorsal organ
EAG
Electroantennogram
EDTA
Ethylene-diamine-tetra-acetic acid
FBS
Foetal Bovine Serum
GFP
Green Fluorescent Protein
GPCR
G-protein-coupled receptor
GRN
Gustatory Receptor Neuron
GTP
Guanosine-5-triphosphate
GTPS
guanosine 5-O-[gamma-thio]triphosphate
HEK
Human Embryonic Kidney Cells
HEPES
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
IBMX
3-isobutyl-1-Methylxanthine
IP3
Inositol (1,4,5)-trisphosphate
LH
lateral horn
MB
mushroom body
MIA
5-(N-methyl-Nisobutyl) amiloride
OBP
Odorant-Bingding Protein
OR
Odorant Receptor
ORN
Odorant Receptor Neuron
100
Abbreviations
OSN
Olfactory Sensory Neuron
PBP
Pheromone-Binding Protein
PCR
Polymerase chain reaction
PLC
Phospholipase C
PN
Projection Neuron
SDS
sodium dodecyl sulfate
SEM
Standard Error of the Mean
SSR
Single sensillum recording
TO
Terminal Organ
UAS
Upstream Activation Squence
VO
Ventral Organ
wt
wild-type
101
List of Figures and Tables
List of Figures and Tables

Figure 1.1.1 The Drosophila olfactory organ .................................................................... 3
Figure 1.1.2 Organization of the Drosophila olfactory system. .........................................6
Figure 1.1.3. Analysis of odor response spectra of individual odorant receptors..............11
Figure 1.1.4 Molecular neuroanatomy of the adult AL annotated with the molecular and
functional identity of the glomeruli. .................................................................................14
Figure 1.2.1 Standard model of the GDP/GTP cycle governing activation of
heterotrimeric GPCR signaling pathway. ..........................................................................17
Figure 1.2.2 Structural features of heterotrimeric G-protein subunits...............................18
Figure 1.3.1 The canonical mammalian olfactory signal transduction pathway. ..............20
Figure 1.3.2 Models of signal transduction mechanisms in odorant receptor neurons
(ORNs)...............................................................................................................................22
Figure 3.2.1 Larval chemotaxis assay ...............................................................................34
Figure 3.2.2 Schematic overview of the EAG recording...................................................35
Figure 3.2.3 Schematic overview of single sensillum recording .......................................36
Figure 3.2.4 Schematic overview of single unit tip recording from GRNs .......................36
Figure 4.1.1 PT-PCR analysis of G subunit expression in Drosophila antenna..............40
Figure 4.1.2 Western blot analysis of G-protein subunits expression in antenna...........41
Figure 4.1.3 Immunohistochemical analysis of G-protein subunit expression patterns in
antenna. ..............................................................................................................................43
Figure 4.1.4 RT-qPCR analysis of Gs and Gq expression level in antenna. .................44
Figure 4.2.1 Screening of G-proteins for participation in the olfactory signal transduction46
Figure 4.2.2 Single sensillum recording from wild type ab1 sensillum. ...........................47
Figure 4.2.3 Single sensillum recordings from CTX and control flies..............................48
Figure 4.2.4 Gr5a-CTX GRNs response to sucrose...........................................................49
Figure 4.2.5 Functional study of ORs and G proteins interactivity in the recombinant
HEK293 expression system. ..............................................................................................51
Figure 4.2.6 Overexpression of a GTPase deficient Gs mutant in Drosophila antenna
lead to a change in response dynamics ..............................................................................52
102
Figure 4.2.7 Odorant exposure caused Gs redistribution in antenna................................54

Figure 4.2.8 Schematic overview of genetic mosaic fly generation. ................................56
Figure 4.3.1 cAMP level moderate the activity of ORNs..................................................57
Figure 4.3.2 An increase in cAMP levels can recover the spontaneous activity of ORNs.58
Figure 4.3.3 SSR of the OR83b-GAL4 / UAS-PAC; OR83b2 / OR83b2 and control flies59
Figure 4.3.4 Expression of an AcGq in ORNs leads to a resensitization deficiency ......60
Figure 4.3.5 MIA blocks odorant-induced currents in oocytes ........................................62
Figure 4.3.6 Direct application of MIA on antenna blocks the spontaneous neuronal
activity................................................................................................................................63
Figure 4.3.7 Single sensillum recording analysis from Ih channel mutants.......................64
Figure 4.4.1 Immunostaining of larval ORNs ...................................................................65
Figure 4.4.2 Chemotaxis assays on AcGq and CTX larvae ..............................................66
Table 1 Expression patterns of the complete repertoire of Drosophila odorant receptors. .8
Curriculum Vitae
Curriculum Vitae
Personal:
name:
data of birth, place of birth:
nationality:
Ying Deng
09.09.1980, Wuhan
China
EDUCATION
PhD
Department of Cellphysiology,
Ruhr-Universitt Bochum, Bochum, Germany
2005-present
Pre-PhD
Department of Cellphysiology,
Ruhr-Universitt Bochum, Bochum, Germany
2004-2005
Master
Department of Biology,
Xiamen University, Xiamen, China
2002-2004
Bachelor
Department of Biology,
1998-2002
RESEARCH EXPERIENCE
International Max-Planck Chemical Biology Program
2005-present
Ruhr-Universitt Bochum, Germany
Doctoral thesis research conducted with Prof. Dr. Dr. Dr. Hanns Hatt
Thesis: Molecular mechanisms of olfactory signal transduction in Drosophila
melanogaster
International Graduate School of Neuroscience
2004-2005
Ruhr-Universitt Bochum, Germany
Pre-PhD training with Prof. Dr. Dr. Dr. Hanns Hatt
Thesis: Interaction partners of odorant receptors from Drosophila melanogaster
Regulatory Biology, Department of Biology,
2002-2004
Master study conducted with Prof. Dr. Shengcai Lin
Thesis: The structural basis of Axin function in the JNK MAPK pathway.
Regulatory Biology, Department of Biology,
Bachelor degree thesis study conducted with Prof. Dr. Shengcai Lin
Thesis: Identification of genes required for TNF-induced cell death.
2001-2002
104
Curriculum Vitae
LIST of PUBLICATIONS
Wong CK, Luo W, Deng Y, Zou H, Ye Z, Lin SC. J Biol Chem. 2004 Sep
17;279(38):39366-73.
The DIX domain protein coiled-coil-DIX1 inhibits c-Jun N-terminal kinase activation by
Axin and dishevelled through distinct mechanisms.
Neuhaus EM, Zhang W, Gelis L, Deng Y, Noldus J, Hatt H. J Biol Chem. 2009 Jun
12;284(24):16218-25.
Activation of an olfactory receptor inhibits proliferation of prostate cancer cells.
Deng Y, Zhang W, Farhat K, Hatt H, Gisselmann G, Neuhaus EM.
The stimulatory heterotrimeric G-protein Gs mediates olfactory signal transduction in
Drosophila. (In preparation)
Gisselmann G, Deng Y, Neuhaus EM, Werner M, Hatt H,
Pharmacological characterization of MIA as an insect odorant receptor blocker. (In
preparation)
Kper M, Deng Y, Schreiner B, Strtkuhl K, Hatt H, Gisselmann G
Molecular and functional characterization of an Ih-channel in Drosophila olfactory
receptor neurons. (In preparation)
POSTERS
Deng Y, Gisselmann G, Zhang W, Hatt H, Neuhaus EM.
Role of heterotrimeric G-proteins in the olfactory signal transduction cascade in
Drosophila. CSHL Meeting on Neurobiology of Drosophila. Oct. 3 7, 2007.Cold spring
harbor, New York (poster)
Deng Y, Gisselmann G, Zhang W, Hatt H, Neuhaus EM.
The stimulatory heterotrimeric G-protein Gs is involved in olfactory signal transduction
in Drosophila. 12th European Drosophila Neurobiology Conference. Sep. 6-10, 2008.
Wrzburg, Germany (poster)
Deng Y, Zhang W, Gisselmann G, Hatt H, Neuhaus EM.
105
Curriculum Vitae
The stimulatory heterotrimeric G-protein Gs is involved in olfactory signal transduction
in Drosophila. 8th Goettingen metting of the German neuroscience society. Mar. 25-29,
Gttingen, Germany (poster)
REFERENCES
Prof. Dr. Dr. Hanns Hatt
Ruhr-Universitt Bochum, 44780
Institute of Cellphysiology
+49 234 32 24586
Hanns.Hatt@ruhr-uni-bochum.de
PD. Dr. Eva Neuhaus
Ruhr-Universitt Bochum, 44780
Institute of Cellphysiology
+49 234 32 24315
Eva.Neuhaus@ruhr-uni-bochum.de
Prof. Dr. Martin Engelhard
Max Planck Institute of Molecular Physiology
Dept. of Physical Biochemistry
Otto-Hahn-Strasse 11
44227 Dortmund
Phone: +49 231 133 2302
Email: martin.engelhard@mpi-dortmund.mpg.de
106
Acknowledgements
Acknowledgements
My sincerest gratitude goes to my supervisor Prof. Dr. Dr. Dr. Hanns Hatt for giving me
the opportunity to become a member of his successful and dynamic team at the
Department of Cell Physiology.
I would like to thank my direct supervisors PD. Dr. Eva Neuhaus and Dr. Gnter
Gisselmann for their endless energy and enthusiasm for my work. Their constant
guidance in the right direction, rapid development of new ideas in order to unravel
important questions, clear and concise experimental design and unceasing determination
were invaluable to me.
For their technical support I would like to thank Mr. Harry Bartel, Ms. Farideh Salami,
Ms. Jasmin Gerkrath, Mr. Thomas Lichtleitner, Ms. Andrea Stoeck, Ms. Ute Mller, and
Mr. Grabowski.
A special thanks goes to Julia Drner, Weiyi Zhang, Ruth Dooley, Nico Bredendiek,
Sabrina Baumgart, Nicole Schbel, Lain Gelis, Stefan Kurtenbach and Sebastian Rasche
for their friendship and support, interesting discussions, unconditional help during my
thesis writing and ideas for problem-solving.
To my parents I am forever indebted, for their unwavering support throughout my years
of study.
This work was financially supported by The International Max-Planck Research School
in Chemical Biology and The Deutsche Forschungsgemeinschaft.
107

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INTERNATIONAL GRADUATE SCHOOL OF BIOSCIENCES

MOLECULAR MECHANISM OF OLFACTORY SIGNAL

Department of Cell Physiology

First thesis advisor: Prof. Dr. Dr. Dr. Hanns Hatt

INTERNATIONALEN GRADUIERTENSCHULE BIOWISSENSCHAFTEN

DER MOLEKULAR MECHANISMUS DER OLFAKTORISCHEN

Referent: Prof. Dr. Dr. Dr. Hanns Hatt

Error! Reference source not found.

The Drosophila olfactory system........................................................................ 1

Anatomy of the Drosophila olfactory system............................................. 1

The odor coding system in Drosophila....................................................... 7

New insights of Drosophila odorant receptor........................................... 14

Signal transduction pathway in odorant receptor neurons................................ 19

Materials and Methods.............................................................................................. 24

Enzymes and Kits and molecularbiological supplies................................ 24

Immunoblotting and Immunohistochemistry............................................ 24

Mammalian cell lines................................................................................ 26

Buffers and Solutions................................................................................ 26

Fly rearing and fly stocks.......................................................................... 28

Molecular cloning and plasmid construction............................................ 29

Cell culture and transfection ..................................................................... 30

Ratiometric calcium imaging in HEK293 cells ........................................ 30

RNA isolation, reverse transcription, RT-PCR and Real-time quantitative

[35S] GTPS binding assay ...................................................................... 32

Larval chemotaxis assay ........................................................................... 33

Electroantennogram (EAG) ...................................................................... 34

Error! Reference source not found.

Antennal single sensillum recording......................................................... 35

Labellar single sensillum tip recording..................................................... 36

Odorant application system in EAG and single sensillum recording ....... 37

Whole mount fluorescent antibody staining of Drosophila larvae........... 38

SDS gel electrophoresis and Western-blotting ......................................... 38

cAMP assay .............................................................................................. 38

Different G subunits are expressed in Drosophila antenna. ........................... 39

RT-PCR analysis of G subunits in Drosophila antenna ......................... 39

Western-blot analysis showed that different classes of G are expressed in

the antenna ................................................................................................................ 40

Immunohistochemical analysis of the expression patterns of different G

proteins in Drosophila antenna................................................................................. 41

Real-time quantitative PCR analysis of Gs and Gq expression in

Drosophila antenna and head.................................................................................... 43

Functional study of G subunit in Drosophila olfactory signal transduction

Overexpression study of different G subunits and G-protein specific

protein toxins in Drosophila ORNs .......................................................................... 44

Dose-response curves of EAG responses from CTX and control flies..... 45

CTX blocks the olfactory response of ORNs in Drosophila .................... 47

CTX does not act as a non-specific toxin on cells .................................... 49

Functional study of OR and G protein interactivity in the recombinant

HEK293 expression system ...................................................................................... 49

Overexpression of a GTPase deficient Gs mutant in Drosophila antenna

leads to a change in response dynamics.................................................................... 52

Odorant exposure caused Gs redistribution in antenna ........................... 53

Generation of a Gs knockout or knockdown fly ..................................... 54

Secondary messengers and other components in the ORNs signal transduction

Error! Reference source not found.

The cAMP pathway is involved in odorant signaling in fly olfactory

Effect of MIA on Drosophila odorant receptors....................................... 61

Functional study of Ih channels in Drosophila odorant receptor neurons 63

Gs and Gq are expressed in larva olfactory receptor neurons ............... 65

CTX larvae have normal chemotaxis behavior......................................... 66

Expression of AcGq3 in larval olfactory receptor neurons causes olfactory

The role of Gs in the olfactory signal transduction pathway .......................... 67

Larval olfactory system..................................................................................... 73

Other signaling components in the olfactory signal transduction pathway ...... 74