Distribution and modulatory roles of neuropeptides and ...352353/FULLTEXT01.pdf · 5 Distribution and modulatory roles of neuropeptides and neurotransmitters in the Drosophila brain

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

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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

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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.