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Distribution and modulatory roles of neuropeptides and
neurotransmitters in the Drosophila brain
Lily Kahsai Tesfai
Department of Zoology
Stockholm University
Stockholm 2010
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© Lily Kahsai Tesfai, Stockholm 2010 Doctoral dissertation 2010 ISBN 978-91-7447-150-2 Printed in Sweden by universitetsservice US-AB, Stockholm, 2010
The background of the cover is a series of confocal images taken by Lily Kahsai Tesfai. Immunolbeling of 210yGAL4 (red) with serotonin antibody (green) showing from anterior to posterior, the ellipsoid body, fan-shaped body, protocerebral bridge and large neurosecretory cells as well as small interneurons in the adult Drosophila brain.
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Distribution and modulatory roles of neuropeptides and neurotransmitters in the Drosophila brain
Doctoral Dissertation 2010
Lily Kahsai Tesfai Department of Zoology Stockholm University
106 91 Stockholm
ABSTRACT The central complex is a prominent neuropil found in the middle of the insect brain. It is considered as a higher center for motor control and information processing. Multiple neuropeptides and neurotransmitters are produced in neurons of the central complex, however, distribution patterns and functional roles of signaling substances in this brain region are poorly known. Thus, this thesis focuses on the distribution of signaling substances and on modulatory roles of neuropeptides in the central complex of Drosophila.
Immunocytochemistry in combination with GAL4/UAS technique was used to visualize various signaling substances in the central complex. We revealed different central-complex neurons expressing the neuropeptides; Drosophila tachykinin (DTK), short neuropeptide F (sNPF), myoinhibitory peptide (MIP), allatostatin A, proctolin, SIFamide, neuropeptide F and FMRFamide. Subpopulations of DTK, sNPF and MIP-expressing neurons were found to co-localize a marker for acetylcholine. In addition, five metabotropic neurotransmitter receptors were found to be expressed in distinct patterns. Comparison of receptor/ligand distributions revealed a close match in most of the structures studied.
By using a video-tracking assay, peptidergic modulation of locomotor behavior was studied. Different DTK and sNPF-expressing neurons innervating the central complex were revealed to modulate spatial distribution, number of activity-rest phases and activity levels, suggesting circuit dependent modulation.
Furthermore, neurosecretory cells in the Drosophila brain that co-express three types of neuropeptides were shown to modulate stress responses to desiccation and starvation.
In summary, we have studied two different neuropeptides (DTK and sNPF) expressed in interneuronal circuits and neurosecretory cells of the Drosophila brain in more detail. We found that these neuropeptides display multiple actions as neuromodulators and circulating hormones, and that their actions depend on where they are released.
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TABLE OF CONTENTS
LIST OF PAPERS 7
INTRODUCTION 8
The central complex of Drosophila melanogaster 8
Biosynthesis and processing of neuropeptides and neurotransmitters 11
Neuropeptides and neurotransmitter receptors 12
Tachykinins related peptides (TKRPs) 13
Short neuropeptide F (sNPF) 15
Classical neurotransmitters 16
Acetylcholine 16 γ-aminobutyric acid 17 Glutamate 18
Biogenic amine neurotransmitters 18 Serotonin 18 Dopamine 19 Tyramine and octopamine 20
AIMS OF THE THESIS 21
METHODS 22
The GAL4/UAS system 22 Immunocytochemistry and antisera 23 Image analysis 25 Quantification of immunofluorescence 25 Locomotor behavior assay and data analysis 25 Conditional neuronal silencing experiment using UAS-shibire 26 Trikinetics locomotor activity 26 Assays of survival during starvation and desiccation 26 Measurement of water content and loss 27 RESULTS AND DISCUSSION 27 Neuropeptide and neurotransmitter signaling in the central complex: functions and distribution (paper I, II, III) 27 Peptidergic modulation of metabolic stress response (paper IV) 31 SUMMARY AND CONCLUSION 32
REFERENCES 33
ACKNOWLEDGEMENTS 44
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LIST OF PAPERS
This thesis is based on the following papers
Paper I Kahsai L, Martin JR, Winther ÅME. 2010. Neuropeptides in the
Drosophila central complex in modulation of locomotor behavior.
J Exp Biol 213(Pt 13):2256-2265.
Paper II Kahsai L and Winther ÅME. 2010. Chemical neuroanatomy of the
Drosophila central complex: distribution of multiple neuropeptides
in relation to neurotransmitters. J Comp Neurol. (In press).
Paper III Kahsai L, Nässel DR and Winther ÅME. 2010. Distribution of
metabotropic receptors of serotonin, dopamine, GABA and
glutamate in the central complex of Drosophila. (Manuscript)
Paper IV Kahsai L, Kapan N, Dircksen H, Winther ÅME, Nässel DR. 2010.
Metabolic Stress responses in Drosophila are modulated by brain
neurosecretory cells that produce multiple neuropeptides. PLoS
One 5(7):e11480.
Publication not includes in this thesis
Dircksen H, Tesfai LK*, Albus C, Nässel DR. 2008. Ion transport peptide splice forms in
central and peripheral neurons throughout postembryogenesis of Drosophila
melanogaster. J Comp Neurol 509(1):23-41.
* Tesfai LK is the same author as Kahsai L
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INTRODUCTION
The nervous system consists of two parts, the central nervous system (CNS) and the
peripheral nervous system (PNS). In insects, the CNS is composed of the brain and the
ventral nerve cord that is analogous to the vertebrate spinal chord. The insect brain is
made of three regions, the protocerebrum, deutocerebrum and tritocerebrum. The
protocerebrum receives inputs from the compound eyes and other organs, processes
information and relays signals. It contains centers, or neuropils, where synaptic
connections are made between axon terminals and dendrites. Neuropil structures such as
the central complex and the mushroom body are located in the protocerebrum. In
addition, the protocerebrum contains neurosecretory cells that project to the neurohemal
organs, the corpora cardiaca and corpora allata. The deutocerebrum receive inputs from
the antennae and process olfactory information. Neurons in the tritocerebrum controls the
mouthparts and innervate the digestive canal (Strausfeld, 1976)
Signal transmission between neurons in the nervous system is mainly performed
by different chemical substances such as neuropeptides and various types of
neurotransmitters. Neuropeptides and neurotransmitters are found in the whole animal
kingdom, starting from one of the simplest levels of organization, the hydra (phylum
Cnidaria) to one of the most complex systems, the mammals. These signaling substances
regulate different physiological processes such as locomotion, metabolism, homeostasis,
reproduction and growth in multicellular organisms.
The central complex of Drosophila melanogaster
More than sixty-five years ago Maxwell Power (1943) named a group of structures found
in the middle of the fruit fly brain as the central complex. The central complex, located
between the two peduncles of the mushroom body, consists of four interconnected
neuropil regions: the ellipsoid body (EB), the fan-shaped body (FB), the noduli (NO) and
the protocerebral bridge (PB). A comprehensive study, based on Golgi silver staining
analysis, of Hanesch and co-workers (1989) on the neuroarchitecture of the central
complex in Drosophila has shown at least 50 different neuronal types in this structure
(Hanesch, 1989).
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The doughnut-shaped EB is the most anterior substructure. The FB is the largest
substructure in the central complex, and is composed of 8 vertical segments and at least 6
horizontal layers. The two spherical NO are mirror images of each other and lie ventral to
the FB and posterior to the EB. The PB lies posterior to the FB and is composed of 16
segments, 8 segments in each brain hemisphere, (Figure 1A). Two paired accessory areas,
the lateral triangles (LTR) and the ventral bodies (VBO) are closely associated with the
central complex (Figure 1B). The general design of the central complex, also known as
central body in other insects, is well conserved. (Homberg, 2008; Loesel et al., 2002).
The EB in other insects is not circular as in Drosophila but has an arch like structure and
is called the lower division of the FB (Homberg, 2008)
Figure 1. (A) The four substructures of the central complex from posterior to anterior,
protocerebral bridge (pb), the fan-shaped body (fb), the nodule (no) and ellipsoid body
(eb). (B) Frontal view of the central complex substractures with two paired accessory
areas, lateral triangle (ltr) and ventral bodies (vbo). Schematic drawing taken from
(Hanesch, 1989).
Morphological studies in the fruit fly, Drosophila melanogaster;(Hanesch, 1989; Renn et
al., 1999; Young and Armstrong, 2010), in the house fly, Musca domestica (Strausfeld,
1976), and in the locust, Schistocerca gregaria, (Heinze and Homberg, 2008; Homberg,
1991; Williams, 1975) suggest that the neurons of the central complex can be divided
into three groups; tangential, columnar and pontine neurons (Fig. 2). Tangential neurons
typically arborize in a single central-complex substructure, and connect with various
regions outside the central complex. The majority of the tangential neurons are
A B
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considered to receive input from these different brain regions (Hanesch, 1989; Young and
Armstrong, 2010). Typical examples of tangential neurons are the ring (R) neurons of the
EB and the F-neurons of the FB. In dipterans, the R neurons form ring-like arborizations
around the EB-canal. Columnar neurons interconnect two or more different substructures,
for example, neurons that connect the FB-EB-NO. Most columnar neurons project to the
accessory areas or to the NO and are regarded as output pathways (Hanesch, 1989). The
third group consists of the pontine neurons which are intrinsic neurons of one
substructure and connect two regions of this substructure, right and left hemisphere or
dorsal and ventral parts.
Figure 2. Three examples of different types of central complex neurons. Schematic
drawing taken from (Hanesch, 1989).
The central complex receives input from many parts of the brain and is believed to be an
integration center for motor and sensory functions (Homberg, 2008). Investigations on
the functional role of the central complex in Drosophila began with behavioral analysis
of central complex structural mutants. These studies showed the importance of the central
complex in control of walking behaviors (Strauss and Heisenberg, 1993) and visual flight
control (Ilius et al., 1994). Later on, using GAL4 directed neuronal disruption or
combining mutational analysis with GAL4 directed rescue-experiments, other studies
reported the role of the central complex in coordination of locomotion and in regulation
of walking activity (Martin et al., 1999; Poeck et al., 2008; Strauss, 2002). Memory
behaviors such as visual pattern memory (Liu et al., 2006; Pan et al., 2009), spatial
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orientation memory (Neuser et al., 2008) and (Wu et al., 2007) have also been associated
with the central complex. The central complex has also been implicated in courtship
associated behaviors (Popov et al., 2003; 2004). Furthermore, in locusts and
grasshoppers, the central complex was shown to play a role in sky compass orientation
(Heinze and Homberg, 2007; Homberg, 2004; Vitzthum et al., 2002) and in sound
production (Wenzel et al., 2005).
Biosynthesis and processing of neuropeptides and neurotransmitters
Synthesis of neuropeptides occurs within the ribosomes in the neuronal cell body. Gene
transcription in the nucleus leads to the synthesis of a precursor protein, a pre-propeptide,
in the rough endoplasmic reticulum. The pre-propeptide is then transported to the Golgi
apparatus where it is further modified and packaged into large dense-core vesicles (100-
200 nm in diameter). The mature peptide in vesicles is then transported along the
microtubules by motor proteins towards the pre-synaptic terminal and vesicles are stored
in preparation for release. If an action potential reaches the pre-synaptic terminal, voltage
gated calcium channels are opened resulting in an influx of calcium ions. The rise of
cytosolic calcium triggers fusion between the vesicle membrane and the plasma
membrane of the pre-synaptic neuron and causes release of neuropeptides. The
neuropeptide then binds to a receptor on the post-synaptic neuron and causes a post-
synaptic effect which can either be an excitatory or inhibitory effect, but more common a
modulatory effect in the CNS (Nässel, 2002). Neuropeptides can function as
neuromodulators when released at a short distance, synaptically or non-synaptically, or
they can also act as neurohormones when released at a longer distance into the blood
stream (Nässel, 2009; O'Shea and Schaffer, 1985).
Until now, in Drosophila about 42 confirmed neuropeptide precursor genes have
been identified (Hewes and Taghert, 2001; Nässel, 2002; Nässel and Winther, 2010).
Neuropeptides in insects are found expressed in interneurons, neurosecretory neurons,
and in motor and sensory neurons, suggesting multifunctional roles of peptides. (See
review by Nässel, 2002; Nässel and Winther, 2010).
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Neuropeptides are sometimes found co-localized with classical neurotransmitters
within the same neuron termination. Classical neurotransmitters include acetylcholine,
glutamate, γ-aminobutyric acid (GABA) and biogenic amines (such as serotonin,
dopamine, octopamine, tyramine and histamine). In contrast to neuropeptides that are
synthesized in the cell bodies, neurotransmitters are synthesized and packaged into small
synaptic vesicles (50 nm in diameter) within the pre-synaptic terminal. One or more
biosynthetic enzymes are required for production of the mature neurotransmitter.
Moreover, removal of a neurotransmitter from the synaptic cleft (to terminate a signal) is
accomplished by uptake into the pre-synaptic terminal or into glial cells via vesicular
transporters whereas neuropeptides are removed from the synaptic cleft by enzymatic
degradation or diffusion (Nässel, 2002).
Neuropeptides and neurotransmitter receptors
Signal transduction most commonly occurs through two types of receptors; G-protein
coupled receptors (GPCRs) also known as metabotropic receptors and ligand-gated ion
channel receptors (LGICs). GPCRs are characterized by seven transmembrane (7TM)
protein domains that are connected by three extracellular and three intracellular loops.
The extracellular N-terminus domain can be glycosylated whereas the intracellular C-
terminus domain can be phosphorylated. GPCRs are directly coupled to heterotrimeric
GTP-binding (G) proteins that are composed of three subunits α, β, and γ. G proteins
exist in two functional states, in an inactive state when the α subunit is bound to GDP and
an active state when the α subunit is bound to GTP. Activation of a GPCR leads to
binding of the α subunit to GTP, the α-GTP then disassociates from the βγ subunits.
Depending on the signal transduction pathway, the Gα-GTP complex activates or inhibits
one of the effector enzymes; adenylyl cyclase, guanylyl cyclase, phospholipace C or
calcium channels; to generate second messenger such as cAMP, cGMP inositol 1,4,5-
triphosphate (IP3) and diacylglycerol and calcium respectively (Lodish et al., 1999).
LGICs are transmembrane proteins usually made of five subunits with an
extracellular ligand-binding site and an ion channel that selectively transmits small ions
such as sodium, potassium, calcium or chlorine. LGICs mediate fast post-synaptic
effects, usually lasting less than a millisecond. GPCRs on the other hand signal slower
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post-synaptic transmission lasting from seconds to hours (Lodish et al., 1999). If a
receptor is continually stimulated by a signaling substance, the post-synaptic neuron can
decrease its responsiveness for that ligand; this reaction is known as receptor
desensitization.
GPCRs in eukaryotes represent the largest group of cell surface receptors. In
Drosophila out of 13600 genes about 163 genes are coding for GPCRs which is about 2%
of the total genome. In the nematode Caenorhabditis elegans, GPCRs constitute about
6% of the total genome (Brody and Cravchik, 2000; Hauser et al., 2006; Hewes and
Taghert, 2001). Based on sequence similarity, GPCRs are classified into four families.
These are: (1) The rhodopsin receptor family that are activated by a wide range of ligands
such as light, odorants, neurotransmitters, neuromodulators and neuropeptides. (2)
Secretin receptor family that are mostly receptors for numerous peptide hormones. (3)
Metabotropic glutamate receptor family, which include receptors for GABA, glutamate
and calcium ions. (4) Atypical metabotropic receptor family (Brody and Cravchik, 2000;
Hauser et al., 2006; Hewes and Taghert, 2001).
Tachykinin-related peptides
Tachykinin-like peptides are found in both vertebrates and invertebrates (Hökfelt et al.,
2000; Nässel, 2002). Both vertebrate and invertebrate tachykinins are suggested to be
multifunctional peptides involved in modulatory processes in the central and the
peripheral nervous system (Nässel, 2002; Nässel and Winther, 2010). The invertebrate
tachykinins are divided into two groups, one group share an identical C-terminus amino
acid sequence (FXGLMamide) as the vertebrate tachykinins. Invertebrate tachykinins of
this group have only been isolated from the salivary gland of two octopus species,
Eledone aldovrandi (Anastasi and Erspamer, 1962) and Octopus vulgaris (Kanda et al.,
2003), and from one mosquito, Aedes aegypti (Champagne and Ribeiro, 1994). The
second group, known as the tachykinin-related peptides (TKRPs), is only found in
invertebrates and contains the amino acid sequence (FX1GX2Ramide) at the C- terminus.
TKRPs have been isolated from the brain and gut of various invertebrate species
(reviewed in Nässel, 2002).
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In Drosophila, the TKRP encoding gene, (dtk) is composed of 289 amino acids
and encodes six different TKRPs (DTK-1-6) (Siviter et al., 2000). The dtk gene is
localized on the third chromosome and contains four exons. DTK-1-4 are encoded on
exon 2 while DTK-5 and DTK-6 (also designated TAP-5) are on exon 3 (Figure 3). Two
metabotropic tachykinin receptors have been identified in Drosophila; DTKR
(Drosophila TK receptor) (Li et al., 1991) and NKD (neurokinin receptor from
Drosophila) (Monnier et al., 1992). Recent report revealed that NKD is activated by
DTK-6 (Poels et al., 2009) whereas DTKR is suggested to be sensitive to DTK-1-5 (Birse
et al., 2006). Using immunocytochemistry and in situ hybridization techniques,
tachykinins in Drosophila have been detected in the optic lobes, antennal lobes, the
central complex, in the ventral ganglion and in endocrine cells of the midgut (Siviter et
al., 2000; Winther et al., 2003).
Figure 3. Organization of the Drosophila tachykinin gene (Taken from Siviter et al,
2000)
Studies have shown a possible role of TKRPs as cotransmmiters and neuromodulators
(see Nässel, 2002). For instance, TKRPs in Drosophila (Ignell et al., 2009) and
cockroaches (Nässel, 2002) have been found colocalized with the inhibitory
neurotransmitter GABA in local interneurons of the antennal lobe indicating the action of
the peptide as cotransmitter and/or neuromodulator in GABA signalling. Moreover,
TKRPs in crayfish were shown to modulate photoreceptors via presynaptic GABA
inhibition (Glantz et al., 2000). Several in vitro studies on TKRPs have also revealed
myostimilatory action of the peptide on gut muscles in Drosophila, regulation of AKH
release and induction of secretion in Malphigian tubules of moths and locusts (Nässel et
al., 1995). See also reviews (Van Loy, 2009). However, recently in vivo studies
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employing tachykinin deficient flies have shown that tachykinins have a role in
modulating locomotion (Winther et al., 2006) and olfactory processing (Ignell et al.,
2009; Winther et al., 2006; Winther and Ignell, 2010)
Short neuropeptide F (sNPF)
Peptides in invertebrates designated neuropeptide F are structurally related to the
vertebrate neuropeptide Y (NPY) (Brown et al., 1999; De Jong-Brink et al., 2001). The
name NPF and NPY refers to the amino acid phenylalanine (F) and tyrosine (Y) at the C-
terminal end, respectively. In Drosophila, distantly related or totally unrelated NPF-like
peptides known as short NPFs (sNPF) have been identified (De Loof et al., 2001; Nässel,
2002; Vanden Broeck, 2001). The sNPF gene in Drosophila encodes a precursor protein
which is predicted to consist of four different peptides (sNPF1-4) (Hewes and Taghert,
2001; Vanden Broeck, 2001). sNPF-1 and sNPF-2 contain RLRFa at their C- terminal
whereas sNPF-3 and sNPF-4 have RLRWa at the C-terminal end. So far, only one
metabotropic sNPF receptor (sNPF-R) has been identified (Feng et al., 2003; Mertens et
al., 2002; Reale et al., 2004).
sNPF is found widely expressed in numerous small interneurons in the CNS, in
the mushroom body, in the central complex, in chemosensory neurons of the antennal
lobe as well as in neurosecretory cells and in certain clock neurons(Johard et al., 2008;
Johard et al., 2009; Nässel et al., 2008). Studies have shown a role of sNPF in regulation
of feeding and growth via the insulin pathway (Lee et al., 2009; Lee et al., 2008; Lee et
al., 2004). In addition, a possible neuroendocrine function of sNPF has been reported
after the identification of sNPF1-3 in the hemolymph of Drosophila (Garczynski et al.,
2006) as well as sNPF immunoreactivity in gut endocrine cells (Veenstra et al., 2008).
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Classical neurotransmitters
Acetylcholine
Acetylcholine is the principal excitatory neurotransmitters in the insect CNS. It is
synthesized from the compounds choline and acetyl-CoA by the enzyme choline acetyl
transferase (ChAT). Immunoreactivity against ChAT in Drosophila has revealed an
abundant and widespread distribution in the nervous system including the calyces of the
mushroom bodies, antennal lobes, optic lobes, suboesophageal ganglia and in the central
complex (Buchner et al., 1986; Yasuyama and Salvaterra, 1999). By employing
Drosophila mutants for the enzyme that degrades acetylcholine (acetylcholinesterase) the
importance of acetylcholine in numerous behaviors, such as optomotor behavior and
courtship, was shown (Greenspan et al., 1980).
Acetylcholine receptors are divided into ionotrpic nicotinic acetylcholine
receptors (nAChRs) and metabotropic muscarinic acetylcholine receptors (mAChRs).
nAChRs are activated by nicotine and blocked by α-bungarotoxin, a snake poison,
whereas mAChR is activated by the mushroom alkaloid muscarine and blocked by
atropin. Five mAChR subtypes (M1-M5) has been identified in mammals (Eglen et al.,
2001). M1, M3 and M5 are coupled to the stimulatory G protein (Gq) thus, stimulating
phospholipase C whereas M2 and M4 receptors are coupled to the inhibitory G protein,
(Gi), inhibiting adenylyl cyclase. In Drosophila, only one mAchR gene (Dm1) with
similar paharmacological profile to the mammalian M3 and M1 subtypes has been
identified (Shapiro et al., 1989). mAchR in Drosophila is expressed in the whole nervous
system with strong expression in the antennal lobe glomeruli (Blake et al., 1993). The
Drosophila genome predicts 10 genes that express nAChRs (Sattelle et al., 2005). Of
these seven genes are encoding α subunits; Dα1 or ALS (alpha-like subunit), Dα2 or
SAD (second alpha-like subunit), Dα3-Dα7 and the remaining three genes are coding for
non α subunits, Dβ1-Dβ3. Using immunocytochemistry and in situ hybridization Dα1-
Dα4 and Dβ1 and Dβ2 have been shown to be expressed only in the nervous system
(Kopczynski et al., 1998; Lansdell and Millar, 2000). The expression pattern for the rest
of nAChR subunits is not yet described.
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γ-aminobutyric acid
γ-Aminobutyric acid (GABA) is synthesized from glutamate through the biosynthetic
enzyme glutamic acid decarboxyalse (GAD). GABA is the major inhibitory
neurotransmitter in both vertebrates and insects. Immunocytochemical studies using
antibodies against GABA and GABA signaling components, such as GAD and vesicular
GABA transporter (vGAT), have shown GABA producing neurons in the nervous
system. GABA is expressed in major neuropil regions such as the anntenal lobe, the optic
lobe, the mushroom body calyces and in some substructures in the central complex.
(Enell et al., 2007; Fei et al., 2010; Hamasaka et al., 2005; Kolodziejczyk et al., 2008;
Okada et al., 2009).
In Drosophila, GABA activates two types of GABA receptors known as
ionotropic GABAA receptors and metabotropic GABAB receptors. The GABAA receptors
are ligand gated chloride channels that are blocked by picrotoxin. The GABAA receptors
can be formed by three subunits, the RDL (for resistance to dieldrin) (Buckingham et al.,
2005; French-Constant et al., 1993), LCCH3 (ligand-gated chloride channel homologue
3) (Zhang et al., 1995) and Grd (GABA and glycine-like receptor of Drosophila) (Harvey
et al., 1994). Immunocytochemical studies have localized RDL mostly in neuropil
structures such as the optic lobes, the antennal lobes, the mushroom bodies and in the
central complex in the adult Drosophila brain (Enell et al., 2007; Harrison et al., 1996).
LCCH3 was found mostly distributed in neuronal cell bodies in the adult nervous system
(Harrison et al., 1996). The distribution of Grd has not yet been mapped in Drosophila.
RDL has been shown to be significant for olfactory learning (Liu et al., 2009; Liu et al.,
2007).
Three types of GABAB receptors exist in Drosophila, GABABR1-3 (Mezler et al.,
2001). GABABR1 and GABABR2 hetrodimerize to mediate their synaptic transmission
via Go/Gi protein (Kaupmann et al., 1998; Mezler et al., 2001). Based on in situ
hybridization in the Drosophila embryo, GABABR1 and GABABR2 have been shown to
have similar expression patterns whereas the GABABR3 displayed a unique distribution
pattern and different pharmacology (Mezler et al., 2001). The GABABR protein is found
distributed in the adult nervous system, most abundantly in the mushroom body calyces,
central complex, optic lobe and antennal lobe (Enell et al., 2007; Hamasaka et al., 2005;
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Kolodziejczyk et al., 2008; Okada et al., 2009; Root et al., 2008). The distribution of
GABABR3 is not known in Drosophila. GABA signaling via GABAB receptors has been
shown to be involved in a wide variety of behaviors such as olfaction (Olsen and Wilson,
2008; Root et al., 2008; Wilson and Laurent, 2005), locomotion and ethanol tolerance
(Dzitoyeva et al., 2003), circadian behavior (Hamasaka et al., 2005) and in development
(Dzitoyeva et al., 2005).
Glutamate
Glutamate is known as one of the principal excitatory neurotransmitters in the vertebrate
brain and in insect motoneurons. Several studies have concentrated on its role in the
insect neuromuscular junction (Jan and Jan, 1976). However, more recent studies using
the vesicular glutamate transporter (vGluT) as a marker in Drosophila have localized
glutamate in sensory neurons, interneurons as well as in motoneurons in Drosophila
(Daniels et al., 2008; Mahr and Aberle, 2006).
Drosophila express both ionotropic and metabotropic glutamate receptors. Until
now, out of the 30 predicted ionotropic receptor types encoded in the Drosophila genome
only 9 have been cloned (Littleton and Ganetzky, 2000). Six of the nine cloned receptors
are expressed in NMJ (Qin et al., 2005) and the rest of the NMDA-like receptors are
found distributed in the Drosophila brain (Xia et al., 2005). The only functional
metabotropic receptor known in Drosophila is DmGluRA (Drosophila melanogaster
glutamate receptor A) (Parmentier et al., 1996). DmGluRA is expressed in most neuropil
regions except the lobes of the mushroom bodies (Devaud et al., 2008). In addition, it is
expressed in the neuromuscular junction where it modulates excitatory neurotransmission
(Bogdanik et al., 2004) and in certain clock circuits regulating circadian locomotor
behavior (Hamasaka et al., 2007).
Biogenic amine neurotransmitters
Serotonin
Serotonin (5-HT) is synthesized from the amino acid tryptophan by two enzymes:
tryptophan hydroxylase (TRH) and dopa decarboxylase (Ddc). By using an antibody
against 5-HT, the distribution of 5-HT in the developing and adult Drosophila has been
19
mapped (Nässel, 1988; Valles and White, 1988). 5-HT in Drosophila has been reported
to play an important role in aggression (Alekseyenko et al., 2010; Dierick and Greenspan,
2007; Johnson et al., 2009) and in learning and memory (Sitaraman et al., 2008).
Both ionotropic and metabotropic 5-HT receptors exist in mammals. However in
insects, only metabotropic 5-HT receptors have been identified (Tierney, 2001). In
Drosophila four types of 5-HT receptors that exhibit sequence similarity to the
mammalian 5-HT receptors are known; these are 5-HT1A, 5-HT1B (Saudou et al., 1992),
5-HT7 (Witz et al., 1990) and 5-HT2 receptors (Colas et al., 1995). 5-HT1A and 5-HT1B
belong to the same family of receptors and inhibit adenylate cyclase while stimulating
phospholipase C (Saudou et al., 1992) whereas 5-HT7 activates adenylate cyclase (Witz
et al., 1990). The transduction mechanism of the fourth receptor, 5-HT2, is not clearly
investigated, however, it is proposed that it is similar to the mammalian 5-HT2 receptor
that activates phospholipase C (Nichols, 2007). The distribution of 5-HT1A and 5-HT1B
in the brain of Drosophila has been partially mapped (Yuan et al., 2006; Yuan et al.,
2005). 5-HT1A is expressed in the mushroom body where it was shown to have a role in
sleep (Yuan et al., 2006). 5-HT1B is also expressed in the mushroom body and is
additionally found in certain clock neurons (Yuan et al., 2005). By using 5-HT1B mutant
flies a function of 5-HT1B in circadian behavior has been reported (Yuan et al., 2005). 5-
HT2 is expressed mainly in the EB of the central complex and in certain neurons in the
protocerebrum, and its role in circadian behavior has been shown (Nichols, 2007). In
addition 5-HT1A and 5-HT2 has been implicated in modulating aggressive behavior in
Drosophila (Johnson et al., 2009).
Dopamine
Dopamine is synthesized from the amino acid tyrosine into its precursor molecule L-
DOPA by the rate limiting enzyme tyrosine hydroxylase (TH), there after L-DOPA is
converted into dopamine by the enzyme dopa decarboxyalse (Ddc). Based on
immunocytochemical studies in the nervous system of Drosophila and in two other flies,
the distribution pattern of dopamine was found to match with that of TH (Friggi-Grelin et
al., 2003; Nässel and Elekes, 1992). In the Drosophila brain, using antibodies to TH and
GAL4 expression analysis, about 600 dopamine expressing neurons, organized in 15 cell
20
cluster have been reported to innervate all major neuropil structures (Friggi-Grelin et al.,
2003; Mao and Davis, 2009). Dopamine is involved in behaviors such as locomotion
(Pendleton et al., 2002), courtship (Liu et al., 2008; Neckameyer, 1998) sleep and arousal
(Andretic et al., 2005; Foltenyi et al., 2007; Kume et al., 2005).
Three types of metabotropic dopamine receptors are known in Drosophila. Two
of these, DopR (also known as dDA1) (Kim et al., 2003) and DopR2 (also known as
DAMB) (Han et al., 1996) are orthologs of the mammalian D1 receptors. The third one,
D2R (also known as Dop2R) is the ortholog of the mammalian D2 receptors (Hearn et
al., 2002). The D1-like receptors are coupled to the stimulatory G protein (Gαs) and the
D2 like receptors are coupled to the inhibitory G protein (Gαi/o) thereby activating and
inhibiting adenylate cyclase, respectively. In addition a novel GPCR that is activated by
both dopamine and ecdysteroids has also been proposed as a fourth likely dopamine
receptor (Srivastava et al., 2005).
Tyramine and octopamine
Tyramine, a precursor to octopamine is synthesized by the enzyme tyrosine
decarboxyalse (TDC) from the amino acid tyrosine. Octopamine, an end product in the
synthesis of tyramine is synthesized from tyramine by the enzyme tyramine β-
hydroxylase (TBH). Immunocytochemical data has revealed about 100 octopamine
expressing neurons in the Drosophila brain (Busch et al., 2009; Monastirioti, 1999;
Sinakevitch and Strausfeld, 2006). Subsets of these neurons were shown to innervate the
central complex (Busch et al., 2009; Sinakevitch and Strausfeld, 2006). Octopamine in
Drosophila has been shown to be involved in several behaviors such as locomotion
(Yellman et al., 1997), ovulation and egg laying (Cole et al., 2005; Monastirioti, 2003),
ethanol tolerance (Scholz et al., 2000), appetitive reinforcement (Schwaerzel et al., 2003),
cocaine sensitization (Hardie et al., 2007) and aggression (Hoyer et al., 2008). Tyramine
has recently been implicated in locomotor activity (Saraswati et al., 2004) and cocaine
sensitization (Hardie et al., 2007).
Two GPCRs that are activated by both octopamine and tyramine has been cloned
in Drosophila. One receptor that has a preference to tyramine over octopamine is known
as tyramine/octopamine receptor (Octyr) (Arakawa et al., 1990). The second receptor
21
known as OAMB (octopamine receptor in the mushroom body) is activated by both
octopamine and tyramine but preferentially binds octopamine (Han et al., 1998). Both
receptors stimulate the production of intracellular calcium signal (Robb et al., 1994),
however Octyr and OAMB seem to exhibit opposite action towards adenylate cyclase,
OAMB being the activator and Octyr the inhibitor (Han et al., 1998; Saudou et al., 1990).
OAMB is found widely distributed in the mushroom body and in the central complex
(Han et al., 1998) in Drosophila. Reports have also revealed the expression of OAMB in
the thoracic and abdominal ganglion and in the reproductive regions (Lee et al., 2003). A
mutation in the OAMB gene in Drosophila resulted in female sterility due to defective
ovulation and egg laying (Lee et al., 2003).
AIMS OF THE THESIS
The main objective of this thesis is to understand the roles of brain neuropeptides by
using the central complex of Drosophila melanogaster as a model to study interneuronal
peptidergic circuits. Moreover, as an example of a different type of peptide expressing
cell we studied the roles of neuropeptides in brain neurosecretory cells.
Immunocytochemical studies have shown that various types of neuropeptides are
expressed in the central complex of different insects. However, the neuromodulatory
roles of these neuropeptides in the central complex are not known. Thus, we investigated
functional roles of two neuropeptides, DTK and sNPF in the central complex of
Drosophila. The central complex has been shown to be involved in the regulation of
locomotion and therefore, we examined the functions of DTK and sNPF in aspects of
locomotor behavior in Drosophila (paper I). In paper II, the objective was to provide a
detailed map of the distribution of neuropeptides in relation to other signaling substances,
as well as to describe the chemical neuroanatomy of the central complex of Drosophila.
Since signaling substances mediate their action through specific receptors, I wanted to
explore the correlation between neurotransmitters and GPCRs in the central complex
(paper III). In our final project (paper IV), I was interested in studying additional
22
functions of neuropeptides in the nervous system. Thus, we examined the hormonal roles
of DTK and sNPF in metabolic stress responses in Drosophila.
METHODS
The GAL4/UAS system
In all four studies we used the GAL4-UAS technique (developed by (Brand, 1993 #819).
This technique was used in combination with immunocytochemistry, RNA interference
(RNAi) and the temperature sensitive allele of shibire (shits1). The GAL4/UAS technique
involves two transgenic fly lines. One fly line express a yeast Gal4 gene, encoding for the
transcriptional activator protein GAL4, cloned downstream of an endogenous Drosophila
promoter. The second fly line express a yeast-specific regulatory sequence known as
upstream activating sequence (UAS) fused to a gene of interest. When these two
transgenic flies are crossed, the GAL4 protein (activator protein) binds to the UAS (target
sequence) and induces transcription of the gene of interest in the progeny of the cross.
We exploited the GAL4/UAS technique to drive the expression of green
fluorescent protein in order to visualize specific neurons and then label these neurons
with different antibodies. We also used the GAL4 to direct the expression of UAS-RNAi
into specific cells and thereby degrading the mRNA of the target gene. The RNAi
pathway in the cell is first triggered by the presence of double stranded RNA (dsRNA)
whose sequence is complementary to the target mRNA. The dsRNA is cleaved by the
enzyme DICER into small interfering RNA (siRNA) (21-23 nucleotides). The siRNAs
are then assembled into multiprotein complexes known as RNA-induced silencing
complexes (RISCs). The siRNAs binds to the complementary target mRNA and directs
gene silencing. The catalytic component of the RISC complex cleave the target mRNA
and prevent translation. The GAL4/UAS technique was also used to drive the expression
of a UAS-shits1 transgene in specific neurons to block synaptic transmission (Kitamoto,
2001). Shits1 encodes a temperature-sensitive mutation of dynamin that is essential for
synaptic vesicle recycling. Dynamin is inactivated at a restrictive temperature (>29°C)
23
resulting in a rapid and reversible inhibition of synaptic transmission in the specific
neurons where GAL4 is expressed.
Immunocytochemistry and antisera
Adult Drosophila heads aged 4-6 days old were dissected in 0.01M phosphate -buffered
saline with 0.5% Triton X-100, pH 7.2 (PBS-Tx) and fixed in ice-cold 4%
paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 (PB) for 4 hours. Whole-
mounted or cryostat sectioned adult Drosophila heads were used for
immunocytochemistry. Whole mount tissues were incubated in primary antisera for 72
hrs while sections were incubated between 24-48 hrs at 4°C. See Table 1 for primary
antisera and their references used in all four papers. For detection of primary antisera
Cy3-tagged goat anti-rabbit or goat anti-mouse antisera or Cy2-tagged goat anti-mouse or
goat anti rabbit antisera (Jackson Immuno Reasearch) were used. For each experiment at
least 5-10 adult brains were analyzed.
24
Antibody Host Dilution Immunogen Source/characterization
Ast-A Rabbit polyclonal 1:5000 APSGAQRLYGFGLa (Vitzthum et al., 1996a)
FMRFa Rabbit polyclonal 1:1000 GAQATTTQDGSVEQDQPPG (Chin et al., 1990)
MIP Rabbit polyclonal 1:4000 GWQDLQGGWa (Predel et al., 2001)
Proctolin Rabbit polyclonal 1:1000 RYLPT (Taylor et al., 2004)
SIFa Rabbit polyclonal 1:4000 AYRKPPFNGSIFa (Terhzaz et al., 2007)
sNPF Rabbit polyclonal 1:4000 DPSLPQMRRTAYDDLLEREL (Johard et al., 2008)
ITP Rabbit polyclonal 1:1000 GGGDEEEKFNQ (Ring et al., 1998)
TK Rabbit polyclonal 1:2000 APSGFLGVRa (Winther and Nassel, 2001)
ChAT Mouse monoclonal 1:500 Choline acetyltransferase
(recombinant)
University of Iowa Hybridoma
Bank, Iowa City, IA
TH Mouse monoclonal 1:250 Full sequence of rat TH Immunostar, Hudson, WI
5-HT Mouse monoclonal 1:50 5-HT DAKO, Glostrup, Denmark
DopR Mouse polyclonal 1:200 (Kim et al., 2003)
GABABR2 Rabbit polyclonal 1:16000 CLNDDIVRLSAPPVRREMPS (Hamasaka et al., 2005)
DmGluRA Mouse monoclonal 1:10 (Eroglu et al., 2002; Panneels et
al., 2003)
nc-82 Mouse monoclonal 1:10 Drosophila head homogenate University of Iowa Hybridoma
Bank, Iowa City, IA
Table 1. Primary antisera used for immunocytochemistry (Paper I-IV)
25
Image analysis
Specimens were imaged on a Zeiss laser scanning microscope (LSM 510 META, Zeiss)
based on Axiovert S100 microscope (Jena, Germany). An argon2/488nm and HeNe
543nm laser were used for scanning inages. The images were obtained at an optical
section thickness of 0.5-2 µm and 20x, 40x and 63x oil immersion objective lenses were
used.The images were processed with Zeiss LSM software and edited for contrast and
brightness in Adobe Photoshop CS3 Extended version 10.0.
Quantification of immunofluorescence
Immunocytochemistry and imaging of specimens were carried out as described above.
Immunofluorescence was quantified in single optical sections in set regions of interest
(ROI) using ImageJ.v142. (http://rsb.info.nih.gov/ij). The data were analyzed using Prism
v4.0 (GraphPad, CA, USA). Three-five brains per genotype and experiment were
measured.
Locomotor behavior assay and data analysis
For paper I, spontaneous walking activity of female flies aged 3-4 days was quantified
using a video-tracking paradigm described by Martin (2004). Single flies were recorded
simultaneously at 5Hz sampling rate using the EthoVision software (Noldus, The
Netherlands) for 7 hours. Experiments were performed at 25°C in 60% relative humidity.
A total of 20-24 female flies were analyzed for each genotype. Analyses of data were
done according to Martin (2004). Four parameters were extracted from EthoVision
software: frequency of passages through a virtual zone located at the centre of the arena
(2 cm in diameter), total distance moved (mm), mean walking speed (mm/sec) and
number of episodes of activity and inactivity (start/stop). A fly was considered to be
active if it had an activity level of or above 4 mm/sec and considered inactive if it had an
activity level below 2 mm/sec. In order to account for differences in overall activity, the
frequency of passages in the central zone was normalized to the duration of movements
(sec). Statistical analysis were made using analysis of variance (ANOVA) and Tukey’s
post hoc test; Statistica version 4 (StatSoft, Inc.).
26
Conditional neuronal silencing experiment using UAS-shibire
Adult female flies aged 3-4 days were analyzed for total distance moved (mm), start/stop,
mean walking speed (mm/sec) and frequency in zone/sec using the video-tracking
paradigm as described above. To silence specific neuronal circuits UAS-shits1 was used at
restrictive temperature (30°C) (Kitamoto, 2001). Prior to monitoring the locomotor
activity, control flies and flies expressing shits1 were pre-warmed at 30°C for 1 hour and
then video-recorded at 30°C for 5 hours. All flies for this experiment were raised at 19 °C
during the entire development. A total of 24 female flies were analyzed for each
genotype.
Trikinetics locomotor activity
In paper IV, we measured the activity of starved flies. Male flies aged 4-6 days were
analyzed using Trikinetics activity monitor (Trikinetics, Brandeis CA USA). Single flies
were placed in individual 5 mm monitoring glass tubes filled in one end with 2 cm of
0.5% aqueous agarose. Tubes were placed in Trikinetic monitoring racks in an incubator
at 25o C with a light-dark cycle of 12:12h. All monitoring of activity started 3h after onset
of starvation (recordings started at 5.5h after lights on) and activity data (crossings of an
infrared beam) were collected by the Trikintics computer software every 15 minutes.
Data were collected for 40 h and activity records from a minimum of 64 individual flies
for each genotype were pooled to obtain average activity levels. The data were analyzed
using Microsoft Excel Assays of survival during starvation and desiccation
Starvation experiments were performed according to the protocol of Lee and Park (2004).
In brief, male flies, aged 4-6 days, were anesthetized on ice and then placed individually
in 2 ml cotton-capped glass vials containing 500 µl of 0.5% aqueous agarose. All vials
were placed in an incubator with 12:12 light:dark conditions at 25 °C. The vials were
checked for dead flies every 12 hours until no living flies were left. For desiccation
experiments the same protocol was followed, except that the vials were empty and the
vials were checked every hour until there were no living flies.
27
Measurement of water content and loss
Total water content of desiccated and non-desiccated male flies was determined. Flies
were weighed before and after drying the flies. Two groups of flies of each genotype
(experimental and controls) were weighed: one group with fed flies (0 hour desiccation)
and the second group after 16 hours of desiccation without food and water. Groups of 5
male flies were weighed (on a Sartorius, Göttingen, Germany) after anesthetizing them
on ice (wet weight), and then dried at 60°C for 24 hours. Dried flies were weighed after
reaching room temperature (dry weight). Thereafter the water content of each genotype
was calculated by subtracting the wet weight from the dry weight.Since we had to use
ded dry weight it was necessary to use separate flies for 0h and 16h.
RESULTS AND DISCUSSION
Neuropeptide and neurotransmitter signaling in the central complex: functions and
distribution (paper I, II, III)
In paper I, we investigated the roles of two neuropeptides, DTK and sNPF, that were
found to be abundantly expressed in the FB. By using immunocytochemistry in
combination with different FB-GAL4 lines, we first identified three types of TK-
expressing neurons: columnar, tangential and pontine neurons. In addition, we identified
sNPF-expressing tangential neurons that project to the FB. Then, using RNAi, we down-
regulated the levels of DTK and sNPF in these neurons and measured the locomotor
activity of the flies using the video-tracking locomotor behavior assay. We revealed the
involvement of DTK-expressing tangential and columnar neurons in modulation of
spatial orientation and activity-rest phases. On the other hand, sNPF-expressing
tangential neurons appeared to regulate in the fine-tuning of mean walking speed and
distance walked.
Furthermore, by employing UAS-shits1 to block synaptic transmission in these
neurons we confirmed that tangential and columnar DTK-expressing neurons are
involved in modulation of spatial distribution and activity phases. Although shi has been
reported to perturb behaviors when expressed in peptidergic neurons, it is not known if or
28
how shi-dependant vesicle endocytosis affects peptide signaling (Wülbeck et al., 2009).
Certainly it will block the effect of any co-localized classical transmitter. In paper II, we
were interested in investigating the relation between neuropeptides and other
neurotransmitters in the central complex of Drosophila since studies in mammals (see
review by (Burnstock, 2004; Hökfelt et al., 2000) and insects (see review by (Nässel,
2002) have suggested that neuropeptides mainly act as co-transmitters. Therefore, we
compared the distribution patterns of DTK, in relation to different neurotransmitters.
Interestingly we revealed a population of DTK expressing neurons co-localizing with a
marker for the excitatory neurotransmitter acetylcholine. In paper I, we observed
locomotor phenotypes when shi was expressed in putative peptidergic neurons. Even
though, we at present do not know if these DTK/acetylcholine producing neurons are
involved in modulation of locomotor behavior, we may speculate that they do. Based on
their location in the superior median protocerebrum in the brain, comparing the labeling
patterns in Paper I and II, this is not unlikely. Thus, the locomotor phenotypes induced by
shi might be accomplished by interfering with the recycling of synaptic vesicles
containing acetylcholine.
When using the same GAL4 that was used to knock-down sNPF levels by RNAi,
to drive the expression of UAS-shits1, flies displayed different locomotor phenotypes than
when down regulating the peptide level. One possibility is that shi is interfering with
receptor internalization rather than with peptide release. This has recently been suggested
to occur when expressing shits1 in PDF expressing clock neurons in Drosophila (Wülbeck
et al., 2009). Unfortunately, the distribution of sNPF receptor is not known,
In paper II, we examined the distribution patterns of neuropeptides in the central
complex. By combining enhancer trap central complex-GAL4 drivers and
immunocytochemistry, we described the distribution patterns of eight different
neuropeptides in the FB. These were: DTK, sNPF, MIP, proctolin, FMRFa, allatostatin
A, SIFa and NPF. Three of these peptides, MIP, dFMRFa and SIFa were for the first time
described in the central complex of Drosophila. Each of the peptides displayed distinct
distribution patterns in different layers of the FB, suggesting different functional roles of
these peptides in the central complex. For example, DTK and sNPF are found abundantly
29
expressed in almost all layers of the FB whereas others such as proctolin, FMRFa,
allatostatin A and SIFa were found to have more restricted distributions.
In paper II we used a NPF-GAL4 (Wen et al., 2005) to visualize the expression of
this peptide in the central complex. Double labeling, combining NPF antibody and the
NPF-GAL4, has shown that the GAL4 drives expression in several additional layers of
the FB and in the EB, regions that are not labeled by the antibody (Krashes et al., 2009).
This additional labeling has been suggested to be post-synaptic areas and that the
antibody recognizes pre-synaptic varicosities only (Krashes et al., 2009). Here, we
confirmed this using the neuronal markers, neuronal synaptobrevin (nSyb)-GFP (Ito et
al., 1998) and Drosophila Down syndrome adhesion molecule (Dscam)-GFP (Wang et
al., 2004) (Fig. 4). UAS-nSyb-GFP is expressed in pre-synaptic sites, where axon
terminals are localized, whereas (Dscam)-GFP is used to visualize post-synaptic sites (Ito
et al., 1998; Wang et al., 2004).
30
Figure 4. Pre-synaptic and post-synaptic expression of dNPF-GAL4. Immunolabeling of
dNPF-GAL4-UAS-nSyb and dNPF-GAL4-UAS-Dscam with a synaptic marker nc82
antibody on whole-mounted adult Drosophila brains. (A) Strong GFP expression of
dNPF-GAL4-UAS-nSyb reveals pre-synaptic sites in a dorsal layer of the FB (arrow).
Weak labeling could also been seen in central layers. (arrowhead) (B) Labeling of dNPF-
GAL4-UAS-Dscam shows post-synaptic regions in central layers of the FB (arrow),
weak labeling was observed in a dorsal layer (arrowhead). Images are projections of
confocal sections. D, dorsal; C, central; V, ventral. Scale bars 20 µm. The dNPF-GAL4
fly line was kindly provided by Scott Waddell, University of Massachusetts Medical
School, UAS-nSyb-gfp and UAS-Dscam-gfp were obtained from the Bloomington
Drosophila Stockcenter.
Furthermore, in paper II, using markers to demonstrate signaling with acetylcholine,
serotonin, dopamine, tyramine and octopamine in the central complex we have shown the
distribution of these transmitters in the central complex. In paper III, we investigated the
relation between some of these neurotransmitters and their metabotropic receptors. Thus
we choose to explore the distribution pattern of five GPCRs, two serotonin (5-HT)
receptors, 5-HT1B and 5HT-7; dopamine receptor, DopR; GABA receptor, GABABR
31
and glutamate receptor, DmGluRA. We found all receptors expressed in the EB. DopR,
GABAB and DmGluRA were found expressed in the FB. GABABR and DmGluRA were
localized to the PB. Strong expression of DopR and DmGluRA was detected in the NO.
We found all GPCRs to match well with their ligands in most of the central complex
structures.
Peptidergic modulation of metabolic stress response (paper IV)
We identified five pairs of large neurosecretory cells, designated ipc1 and ipc2a
neurons that co-express three neuropeptides, DTK, sNPF and ITP, in the protocerebrum
of the adult Drosophila brain. ITP was first identified by Audsley and colleagues as a
peptide stimulating ion and water reabsorption in the locust (Audsley et al., 1992).
Recently, its distribution in the larval, pupal and adult Drosophila brain has been
described (Dircksen et al., 2008). This study revealed 4 pairs of ITP-immunoreactive
neurons in the larval brain and 7 pairs in the adult brain, designated as ipc1-4.
Tachykinin-related peptides in moths and locusts have been suggested to stimulate
secretion in Malphigian tubules (Johard et al., 2003; Skaer et al., 2002) and AKH release
in locusts (Nässel et al., 1995). They have also been associated with feeding in locusts
(Lange, 2001; Winther and Nassel, 2001). Moreover, the DTK receptor DTKR, is
expressed in cells of the Malpighian tubules (Birse et al., 2006). sNPF is known to be
involved in regulation of feeding and growth via insulin-like peptides in Drosophila (Lee
et al., 2009; Lee et al., 2008; Lee et al., 2004). Hence, based on the previous findings of
the roles of these three neuropeptides, we were interested in examining if DTK and/or
sNPF are involved in water reabsorption and/or starvation in Drosophila. We
demonstrate signaling with also wanted to determine whether sNPF and DTK are linked
to the AKH/insulin pathways since we found axonal terminations containing these
peptides in neurohemal organs adjacent to AKH and insulin producing cells. To answer
these questions, we down-regulated the levels of DTK and sNPF in ipc1 and 2a by using
RNAi and monitored fly survival under desiccation and starvation. Both desiccated and
starved flies with decreased amount of peptides in ipc1 and 2a exhibited shorter life span
32
than control flies. Next, by comparing whole body water content in desiccated flies to
none-desiccated flies, we revealed decreased water retention in DTK-knockdown flies.
Studies have revealed that ablation of AKH cells in starved flies leads to increased
locomotor activity in search of food (Isabel et al., 2005; Lee and Park, 2004). Thus, we
monitored locomotor activity of DTK-knockdown flies under starvation using Trikinetics
locomotor activity monitors. The transgenic and the controls flies displayed normal
locomotor activity until the experimental flies started to perish after 18h of starvation,
suggesting that DTK might not directly affect AKH in Drosophila. The effects of DTK
and sNPF knockdown in the ipc-1 and 2 neurons produced stress phenotypes opposite to
those seen after knockdown of insulin production/signaling. Since sNPF has been shown
to stimulate insulin production (Lee et al., 2008; Lee et al., 2004) we can suggest that the
ipc-1 and 2 neurons do not regulate insulin signaling. The overall findings indicate that
the circuits of DTK, sNPF and ITP expressing neurons (ipc1 and ipc2a) are not directly
linked to AKH or insulin signaling but target other pathways to regulate responses to
nutritional stress (desiccation and starvation). Perhaps, mapping receptor distribution of
DTK, sNPF and ITP in the future would reveal the targets for these hormonal peptides. In
summary, we have localized clusters of cells expressing three neuropeptides with axonal
terminations in neurohemal organs, and shown that two of these peptides regulate
nutritional-stress response in Drosophila.
SUMMARY AND CONCLUSION
Data presented in this thesis has shown multiple actions of neuropeptides by
exploring the roles of two types of peptide expressing neurons; interneurons of the central
complex and neurosecretory cells in the Drosophila brain. The peptides DTK and sNPF,
expressed in interneurons and in neurosecretory cells, were shown to modulate different
aspects of locomotor behavior and metabolic stress responses, respectively.
This study has also explored the co-localization of neuropeptides with other
neuropeptides and signaling substances. It is interesting to note that out of two classical
neurotransmitters and four biogenic amines examined, only a marker for acetylcholine
was found co-localized with DTK, sNPF and MIP in neuronal cell bodies innervating the
central complex. Coexpression of dFMRFa and a GABA marker was also detected in a
33
pair of neurons that may innervate the EB. These findings suggests that some GABAergic
and cholinergic neurons utilize neuropeptides as co-transmitters Certainly, we cannot
exclude the possibility that some of the GAL4 lines used to identify transmitter
expression may not be complete in their expression, thus additional co-localization of
neuropeptides and the signaling substances examined may be present. Co-localization of
two different neuropeptides in neurons of the insect CNS is not uncommon (see Nässel,
2006; Nässel, 2010), but here we for the first time demonstrated the co-expression of
three different neuropeptides, DTK, sNPF and ITP, in specific neurosecretory cells (in
total five neurosecretory cells in two clusters). Additionally, we studied the distribution
of metabotropic neurotransmitter receptors in relation to their ligands to explore possible
sites of transmitter action in the central complex. It is apparent that different serotonin
and dopamine receptor types display differential distributions in central complex circuits,
suggesting that each of these monoamines may activate different sets of follower neurons
whose GPCRs couple to different transduction systems. For glutamate and GABA we
only explored metabotropic receptors and thus presence of ionotropic receptors for these
neurotransmitters will further increase signaling complexity. This kind of receptor
distribution studies can serve to guide future exploration of the functional roles of these
signaling substances, just as the neuropeptide expression in the central complex guided us
to investigate peptidergic modulation of locomotor function.
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ACKNOWLEDGEMENTS
This thesis would not have been possible without the support and assistance of so many
great people. It’s a pity that I can not mention everybody but I sincerely wish to express
my gratitude to each and everyone who helped me in anyways all through the years.
My special thanks go to:
Åsa Winther, my supervisor for your continous support and flexibility you have given me
during these four years. I very much appreciate our communication which was always
clear and to the point. I am also grateful for giving me the opportunity to go to France
and learn new things. Dick Nässel my co-supervisor, thank you so much for always being
available whenever I knocked at your door, your advice is always valuable. I am also
thankful for helping me choose my next destination. I would also like to extend my
gratitude to our collaborator Jean-Rene Martin, CNRS-Gif-Sur-Yvette for introducing me
to the world of video tracking the paranoid and hyperactive flies. My gratitude also goes
to Heinrich Dircksen for the first year of my PhD time, I had a lot to learn. I also thank
you for your help in the lab whenever something was broken. Bertil Borg, thank you for
reading my manuscripts, for preparing Glögg in Christmas times and of course for the
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stock of beer upstairs. Rafael Cantera, thanks for the enthusiastic talks about new
experiments in Uruguay and Spain. Annete, thank you for always being helpful and for
the delicious cakes. Minna, thanks for helping me with KLARA, printers and copies. Siw
and Berit thank you for being cooperative with all paper works. My special thanks also
go to people in the other divisions of the Zoology department for for your friendly
gesture and the occasional social activities.
Agata and Neval, my wonderful roommates; Agata, i don’t know where to begin... you
always make me smile. Thank you, thank you so much for so many things but most of all
for being such a fun, energetic person and for “Oops! I did it” moments. Thank you
Neval for our cooperation on the PLoS One paper. It was always fun to discover common
things between Eritrea and Turkey. Lina, thanks for giving me advice on problems with
immunocytochemistry in the beginning and for being pleasant. Jeannette, thank you for
always being ready to help, to either translate or provide information almost about
everything. Micke, thank you for our cooperation about the nitric oxide story, though it’s
not yet finished. Johannes, my workaholic buddy thanks for the late lunch chit chats and
badminton even if it was in the end of my PhD. time. Jiangnan, thank you for letting me
see your nice 5-HT preps and I will not forget the different ways of speaking Chinese,
Japanese and Korean. Kristin another energetic person in the lab, it is nice meeting you
though it was in the end of my time. Yi Ta, thanks for the sweets from china. I am also
thankful to former members in the department; Kerstin, thanks for the” after PhD
courses”, i would not have remembered about AEA and stuff. Erik for the talks about
fishing and the help with the students. Ryan and Helena for your help in the lab.
My stay in Sweden is unforgetable because of the magnificent people i met through the
years. Gernot Reissmann, i don’t know where to start... I thank you for your continuous
love, kindness and encouragement all through these years. Thanks, for showing me the
Vienna life style and for the other side of the story. I am so lucky that i met you. My
gratitude also goes to Ronald and Martin for your hospitality. Lina Issa, thank you for all
the fun we had in the weekends. I am grateful to you for introducing me to so many
wonderful people like “The Duvboers”. Alessandra, Ferreira, Chicky, Sanches, Isabel and
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Karin, thanks for all the nice parties. My gratitude also goes to the people i met in France
for making me feel at home, most of all François and Meena for showing me Paris with a
French style.
All my beloved friends in Eritrea, who are all over around the world today, for always
being dependable. I cherish our friendship that dates back to Elementary school and
Asmara University. Semira, Winta, Solomon and Amir, thank you for being there with
me in the time of exams, classes and fun. Members of the “Hadegang”; Nebiyu Juppe,
John, Sami, Nathnael, Wael, Henok, Sewit, Sinit, Mello, Eyob Betty, thanks for the
evenings in DZ and the Massawa trips. Rahel, Semhar, Almaz, Jack and Elsa I am
grateful for the times we had here in Sweden, I will not forget our coffee celebrations,
wedding and all the nice times.
All my life I have been blessed with lots of families whereever i go. I don’t know what i
would have done with out my family. My family in Sweden, Daniel, Thige and the three
musketeers, I thank you for always checking up on me, it was so important that you are
here in Sweden. Jonny, thanks to you i am here today. I thank you for your support and
dedication. Soliana, thank you for all the good times, keep your free spirit. My warmest
gratitude also goes to Zemui and Abrehet for always being helpful and for all the
delicious lunches and dinners I had at your house.
My family in the US, Amoy Hidega, Amoy Tsehainesh, Baba Sibhat, Baba Weldezgi,
Baba Solomon and all my lovely cousins; I thank you for your contributions to my life.
Ada and Hirut, it means a world to me that you are here for my dissertation.
My family in Eritrea, Alemseged and Abrehet my heart is always with you. Alemseged,
my hero, thank you for being the amazing person that you are. Natsenet, I know you will
not read this but maybe one day you will and know that you have not been forgotten.
Timnit, Simret, Selam, Bereket and Hiben, you bring a smile to my face, it’s a fortune to
have you in my life not only as my siblings but also as my best buddies. Finally, Baba
and Mama thank you for providing me with the proper education and encouraging me to
pursue my interest. I am grateful for your love and support you have given me through
out all my life. I am who i am today because of you.