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Chemical signalling in the Drosophila brain: GABA, short neuropeptide F and their receptors

Doctoral dissertation 2011 Lina E. Enell Department of Zoology Stockholm University 106 91 Stockholm ABSTRACT γ-aminobutyric acid (GABA) and short neuropeptide F (sNPF) are widespread signalling molecules in the brain of insects. In order to understand more about the signalling and to some extent start to unravel the functional roles of these two substances, this study has examined the locations of the transmitters and their receptors in the brain of the fruit fly Drosophila melanogaster using immunocytochemistry in combination with Gal4/UAS technique. The main focus is GABA and sNPF in feeding circuits and in the olfactory system. We found both GABA receptor types in neurons in many important areas of the Drosophila brain including the antennal lobe, mushroom body and the central body complex. The metabotropic GABAB receptor (GABABR) is expressed in a pattern similar to the ionotropic GABAAR, but some distribution differences can be distinguished (paper I). The insulin-producing cells contain only GABABR, whereas the GABAAR is localized on neighbouring neurons. We found that GABA regulates the production and release of insulin-like peptides via GABABRs (paper II). The roles of sNPFs in feeding and growth have previously been established, but the mechanisms behind this are unclear. We mapped the distribution of sNPF with antisera to the sNPF precursor and found the peptide in a large variety of interneurons, including the Kenyon cells of the mushroom bodies, as well as in olfactory sensory neurons that send axons to the antennal lobe (paper III). We also mapped the distribution of the sNPF receptor in larval tissues and found localization in six median neurosecretory cells that are not insulin-producing cells, in neuronal branches in the larval antennal lobe and in processes innervating the mushroom bodies (paper IV). In summary, we have studied two different signal substances in the Drosophila brain (GABA and sNPF) in some detail. We found that these substances and their receptors are widespread, that both sNPF and GABA act in very diverse systems and that they presumably play roles in feeding, metabolism and olfaction.

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Table of contents ______________________________________________________________ List of papers 3 1. Introduction 4 1.1. The nervous system of insects 4 1.1.1. Antennal lobe 5

1.1.2. Mushroom body 5 1.1.3. Other neurons and circuits of interest 6 1.2. Neurotransmitters and neuropeptides 6 1.2.1 Receptors 7 1.2.2. GABA and GABA receptors 8 1.2.2.1. Ionotropic GABA receptors 9 1.2.2.2. Metabotropic GABA receptors 10 1.2.3. Short neuropeptide F 11 1.2.3.1. Short neuropeptide receptor 12 1.2.4. Insulin, insulin-like peptides and insulin receptor 13 2. Aims of the thesis 15 3. Methods 16 3.1. The Gal4/UAS system and RNA interference 16 3.2. Fly stocks 17 3.3. Antisera, immunocytochemistry and antisera characterization 19 3.4. In situ hybridization 21 3.5. Quantification of immunofluorescence 21 3.6. Survival assays 21 3.7. Measurements of trehalose, lipids and growth 21 4. Results and discussion 22 4.1. Paper I 22 4.2. Paper II 23 4.3. Paper III 25 4.4. Paper IV 26 5. General discussion 27 6. Conclusions and future perspectives 29 7. References 31 8. Acknowledgements/tack 39

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LIST OF PAPERS This thesis is based on the following papers and will be referred to by their Roman numerals. ______________________________________________________________ I. Enell LE, Hamasaka Y, Kolodziejczyk A, Nässel DR. (2007) Gamma- Aminobutyric acid (GABA) signalling components in Drosophila: immunocytochemical localization of GABA(B) receptors in relation to the GABA(A) receptor subunit RDL and a vesicular GABA transporter. J Comp Neurol. 505:18-31. II. Enell LE, Kapan N, Söderberg JAE, Kahsai L, Nässel DR. (2010) Insulin signaling, lifespan and stress resistance are modulated by metabotropic GABA receptors on insulin producing cells in the brain of Drosophila. PLoS One. 30;5:e15780. III. Nässel DR, Enell LE, Santos JG, Wegener C, Johard HA. (2008) A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions. BMC Neurosci. 19;9:90. IV. Enell LE, Carlsson MA, Aumann D, Nässel DR. (2011) Distribution of the short neuropeptide F receptor and its ligands in the chemosensory and hormonal systems of larval Drosophila. (Manuscript) ______________________________________________________________ Publications not included in the thesis Root CM, Masuyama K, Green DS, Enell LE, Nässel DR, Lee CH, Wang JW. (2008) A presynaptic gain control mechanism fine-tunes olfactory behavior. Neuron 59:311-21. Johard HA, Enell LE, Gustafsson E, Trifilieff P, Veenstra JA, Nässel DR. (2008) Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: relations to extrinsic neurons expressing different neurotransmitters. J Comp Neurol. 507:1479-96. Nässel DR, Enell LE, Hamasaka Y, Johard H. (2005) Neurotransmitters and their receptors in circadian clock circuits of Drosophila. Pesticides 3:133-140.

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1. INTRODUCTION 1.1. The nervous system of insects The insect nervous system consists of a dorsal brain (Fig. 1) and a ventral nerve cord. The brain is divided into three lobes or neuromeres that are in fact three fused ganglia. The first neuromere is called protocerebrum and receives inputs from the compound eye. The mushroom body and central body complex are also part of the protocerebrum, as well as neurosecretory cells projecting to neurohemal organs of the corpora cardiaca and corpora allata. The deutocerebrum is the middle neuromere that receives inputs from the antennae and processes sensory information like odors, taste and tactile sensation. The antennal lobe is located in this region. The third neuromere is the tritocerebrum that contains neurons that connect to the labrum and anterior digestive canal. The subesophageal ganglion is located just below the brain and is primarily in control of the mouthparts and a relay for connections to the ventral nerve cord. The ventral nerve cord consists of segmentally arranged ganglia running along the midline of the thorax and abdomen. There are three thoracic ganglia and these are mainly in control of locomotion, whereas the abdominal ganglia control reproduction and other functions of the abdomen. In Drosophila, the abdominal and thoracic ganglia are fused into one thoracic-abdominal ganglion (Strausfeld, 1976).

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Figure 1. The adult Drosophila melanogaster brain. A. Frontal view. Labelled are the pars intercerebralis (PI), the antennal lobe (AL), the subesophageal ganglion (SOG), the lobes of the mushroom body (α-lo and β,γ-lo) and the compound eye (eye). The unlabelled arrows indicate parts of the protocerebrum. B. The calyx (Ca) of the mushroom body, protocerebral bridge (PB), the parts of the optic lobes: lamina (La), medulla (Me), lobular plate (Lo p) and lobula (Lo), lateral horn (l ho), lateral deutocerebrum (l deu) and the esophageal opening (oe) are labelled. Pictures modified from flybrain.org.

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1.1.1. Antennal lobe The olfactory pathway in insects starts in the antennae, where olfactory sensory neurons (OSNs) express one type of olfactory receptor (Or). Each OSN sends information about an odor to the antennal lobe. The antennal lobe is the insect homolog of the olfactory bulb in vertebrates and both are organized similarly in spherical packed neuropils called glomeruli. Adult Drosophila have 43-50 glomeruli and larvae only 21 (Vosshall and Stocker, 2007), compared to the over 1500 in mammals (Kosaka et al., 1998). This renders Drosophila a simple, but yet adequate model for olfaction studies. Each glomerulus in the antennal lobe receives information from one or sometimes two types of olfactory receptors (Vosshall and Stocker, 2007). OSNs synapse with local interneurons and projection neurons in the glomeruli. The local interneurons interconnect glomeruli and each local interneuron sends branches to almost all glomeruli (Stocker et al., 1997). Many local interneurons contain GABA, but also acetylcholine, tachykinins and other neuropeptides have been associated to these cells (Carlsson et al., 2010; Roy et al., 2007; Wilson and Laurent, 2005; Winther et al., 2003). Projection neurons send axons to higher protocerebral brains centres such as the mushroom bodies and lateral horn. 1.1.2. Mushroom body The mushroom bodies in insects have a role in olfactory memory and learning. They are composed of the thousands of Kenyon cells, the small but numerous intrinsic neurons of the mushroom bodies. Kenyon cells send their axons through the peduncle (or stalk) from which they bifurcate into a dorsal α-lobe and a medial β-lobe. Some axons do not bifurcate and form a medial γ-lobe. A third morphological subdivision deriving from the Kenyon cells can be distinguished, the α´- and β´-lobe (Crittenden et al., 1998). Just below the cell bodies of the Kenyon cells is the calyx, which is the input (dendritic) region of the mushroom body. The calyces receive information mainly from the antennal lobes (Vosshall and Stocker, 2007), but neurons of the optic lobes can in some insects send axons to the calyces (Strausfeld and Li, 1999). The mushroom body derives from four neuroblasts that give raise to all Kenyon cells and supporting glial cells. One neuroblast contributes to most, if not all, types of cells within the mushroom body and all are practically identical in structure and gene expression patterns (Ito et al., 1997). In other words, the mushroom body is composed of four identical clonal units.

Extrinsic cells associated with the mushroom bodies seem to be important for the functional role of the mushroom bodies. Two large dorsal paired medial neurons (DPM) are innervating the mushroom bodies and express the amnesiac gene. Mutations in amnesiac results in a defect memory consolidation (Feany and Quinn, 1995). The predicted peptides produced by the amnesiac gene have however not been found in Drosophila (Nässel and Winther, 2010). DPM neurons also seem to express acetylcholine (Yu et al., 2005) that might be the major transmitter of these cells. Six dopaminergic neurons that innervate the mushroom bodies have been shown to have a role in appetitive memory performance (Krashes et al., 2007). Food deprivation is normally a motivator for olfactory learning, and stimulation of the dopaminergic neurons proved to decrease olfactory learning

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in starved flies, whereas blocking them increased learning in fed flies. These cells express receptors to neuropeptide F (NPF) and were demonstrated to be under control of NPF (Krashes et al., 2007). 1.1.3. Other neurons and circuits of interest Gustatory and olfactory inputs signal about presence if food and about food quality. However, in order to monitor nutritional needs and maintain homeostasis, the organism utilizes internal cues. For example, there are 20 neurons in the subesophageal ganglion (SOG) that contain the neuropeptide gene hugin. The hugin expressing cells connect to higher brain circuits that regulate feeding behaviour. Branches of the hugin expressing cells are found in the SOG, in the protocerebrum where insulin-producing cells are located, in the ventral nerve cord, in the pharyngeal muscles and in the ring gland (Melcher and Pankratz, 2005). The ring gland is a larval endocrine organ involved in metabolism and growth and is producing ecdysteroids and juvenile hormone. It is composed of the corpus cardiacum, the corpus allatum and the prothoracid gland. Cells of the corpus cardiacum produce adipokinetic hormone (AKH), the insect equivalent to glucagon that raises circulating glucose levels (Kim and Rulifson, 2004). The insulin-producing cells (IPCs) in the pars intercerebralis project to the ring gland and several other sets of peptidergic neurosecretory cells have axon terminations there (Cao and Brown, 2001; Siegmund and Korge, 2001; Wegener et al., 2006). The central body complex is positioned between the peduncles of the mushroom body and is comprised of the ellipsoid body, fan shaped body, noduli and the protocerebral bridge and is believed to serve as integration centre for motor and sensory functions (Hanesch et al., 1989; Homberg, 2008). Flies with mutations in the central body have defective walking activity and learning behavior [see (Davis, 1996)]. 1.2. Neurotransmitters and neuropeptides Neurons are communicating with each other by chemical and occasionally electrical signalling. The chemical transmission is based on various types of substances such as neuropeptides or different kinds of neurotransmitters. Classical neurotransmitters include γ-aminobutyric acid (GABA), glutamate, acetylcholine (ACh) and biogenic amines such as serotonin, octopamine, histamine and dopamine. These are small molecules that can bind to and activate both ion channels and G-protein-coupled receptors (GPCRs) (Squire, 2003). Classical transmitters are synthesized and stored in small vesicles at the axon terminal. When the presynaptic membrane gets depolarized, calcium channels open that will activate calcium-sensitive proteins on the vesicles. These proteins (SNAREs) change shape, making the vesicles fuse with the presynaptic cell membrane and release their content into the synaptic cleft (Chen and Scheller, 2001).

Neuropeptides are by far the most structurally and functionally diverse signalling molecules and exist in all animals with a nervous system. They consist of chains of 3-200 amino acids that are produced in neuronal cell bodies and transported in vesicles to the presynaptic terminal. Transcription and translation of neuropeptides in the rough endoplasmic reticulum first

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produce precursor proteins (prepropeptides). The prepropeptides are thereafter transported to the Golgi apparatus where they are modified and packed into large dense-core vesicles. The vesicles are then transported along microtubules to the presynaptic terminal (Nässel. 2002).

There are only about ten classical neurotransmitters, whereas there is a huge diversity of neuropeptides. It is not really clear how many neuropeptides mammals possess, but the number is considerably higher than in invertebrates. With the entire Drosophila genome sequenced we know from database mining or peptidomic analysis that there are beyond 30 genes encoding neuropeptide precursors plus an additional seven genes encoding insulin-like peptide precursors (Hewes and Taghert, 2001; Nässel, 2009; Yew et al., 2009). Neuropeptides act on G-protein-coupled receptors and there are at least 44 putative peptide-activated GPCRs identified in Drosophila (Hewes and Taghert, 2001; Nässel, 2009). Neuropeptides can act as neuromodulators or as neurohormones. A neuromodulator in this sense means that it is released within or outside the synaptic cleft, mediating many different effects, normally by acting on G-protein-coupled receptors. A neurohormone on the other hand is released into the circulation and acts on targets far away from the release site. Neuropeptides are often colocalized with classical transmitters with the advantage that different functional messages can be signalled to the target cell. Classical transmitters are often released in response to smaller changes in Ca2+ levels, whereas peptides are released in response to much higher Ca2+ levels. Neuropeptides can often diffuse longer distances and therefore activate other target cells than the classical transmitter [reviewed in (Nässel, 2009)]. This thesis mainly focuses on GABA (a classical transmitter), as well as sNPF and insulin (both neuropeptides) and their receptors. 1.2.1. Receptors As mentioned earlier, classical transmitters (with few exceptions) act on both ion channel receptors (ionotropic receptors) and G-protein-coupled receptors (GPCRs or metabotropic receptors), whereas neuropeptides are believed to act on GPCRs and in some cases tyrosine kinase receptors (e. g. insulin-like peptides). Ion channels consists of proteins that form pores and regulate ion transport across the plasma membrane (Fig. 3A). When a transmitter (ligand) bind to an ion channel on the postsynaptic cell a conformational change of the receptor opens the channel and ions can pass and change the membrane potential within milliseconds. Ionotropic receptors are usually very selective to one or a few ions, such as K+, Cl-, Ca2+ or Na+, and the flow of ions can be inwardly or outwardly directed. Thus, a ligand can cause different responses of the postsynaptic cell depending on what type of receptor it activates. An excitatory receptor depolarizes the postsynaptic membrane, whereas an inhibitory receptor causes a hyperpolarization (Squire, 2003).

GPCRs are composed of a polypeptide chain that spans the membrane seven times (Fig 3B). The hydrophilic regions between the transmembrane domains form an extracellular N-terminal, three intracellular loops, three extracellular loops and an intracellular C-terminal. Binding of a ligand to a GPCR can be accomplished in various ways. Small ligands, like

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acetylcholine, bind to a pocket formed by a part of the extracellular regions of the polypeptide chain. Neuropeptides bind with higher affinity, since it involves both extracellular loops and transmembrane domains. Whereas binding of a ligand to an ion channel causes a fast response, GPCR mediated activation is a slightly slower process, but usually with a longer response as a result. The G protein complex is composed of three subunits, Gα, Gβ and Gγ. In an inactive form of G-proteins, the Gα binds to GDP (guanosine diphosphate). When a ligand binds to a GPCR, a change occurs in the orientation of the transmembrane domains that involves the second and the third intracellular loops. This conformational change results in an exchange of GDP for a GTP (guanosine triphosphate), and thereby activates the G-protein. Gβ and Gγ stay on the membrane, whereas the Gα- and GTP-complex is released into the cytoplasm where it can start a signalling cascade by influencing ion channels directly or by affecting the adenylate cyclase pathway. This pathway includes the conversion of ATP to cyclic AMP (cAMP) that functions as a second messenger. Second messengers can influence ion channels or affect nuclear or transcriptional activity. There are various kinds of Gα subunits, for example Gi (inhibitory), Gs (stimulatory) and Gq/0 (Vanden Broeck, 1996). The β/γ subunits can also vary in cellular responses they are causing upon GPCR activation. They can for example activate the phospholipase C pathway. Phospholipase C mediates the production of inositol 1,4,5-trisphosphate (IP3) and diacyl glycerol (DAG) through cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). IP3 diffuses through the cytosol and binds to and opens Ca2+ channels particularly on the endoplasmatic reticulum, thus increasing the cytosolic levels of calcium. Calcium and DAG can activate protein kinase C that phosphorylates other molecules. Another function of the β/γ subunits is to act on G-protein coupled inward rectifying potassium channels (GIRKs) (Squire, 2003). 1.2.2. GABA and GABA receptors Gamma-aminobutyric acid (GABA) is a major inhibitory transmitter in the brain of many animals (Fig. 2). It is produced by a large number of neurons in the central nervous system (CNS), but there is no GABA containing axons emerging to the periphery in Drosophila. In mammals, many conditions are caused by an imbalanced amount of GABA in the brain. Epilepsy, Parkinson’s disease, stress, sleep disorders, depression, addiction, pain, anxiety and Huntington’s disease are examples of disorders correlated with decreased GABA activity, whereas increased GABA levels can cause schizophrenia (Albin and Gilman, 1989; Benes and Berretta, 2001; Bettler et al., 2004). In insects, GABA has roles in olfaction memory and learning, vision and the circadian clock (Cayre et al., 1999; Hamasaka et al., 2005; Stopfer et al., 1997; Wilson and Laurent, 2005).

A major difference between GABA and glutamate (amino acid transmitters) on one hand and other transmitters on the other is that the former are derived from glucose metabolism. Synthesis of GABA occurs in cells with glutamic acid decarboxylase (GAD), a cytosolic enzyme that converts GABA from glutamate (Squire, 2003). GAD is activated by inorganic phosphatases and inactivated by ATP, aspartate and GABA. Increased GABA

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GABA is loaded into vesicles at the synapse by vesicular GABA transporters, vGAT (Owens and Kriegstein, 2002). These transport GABA against its concentration gradient by a vacuolar ATPase that exchange one H+ for one neutral amino acid [reviewed in (Gasnier, 2000)]. GABA is released into the synaptic cleft upon vesicle fusion with the membrane (described in more detail in section 1.2), and can then bind to receptors on the postsynaptic cell. Reuptake of GABA is achieved with different GABA transporters placed on both neurons and glia. There are also autoreceptors (GABABR) located on the presynaptic cell that serve as a feedback regulators (Squire, 2003).

Figure 2. Structure of gamma-aminobutyric acid, GABA. 1.2.2.1. Ionotropic GABA receptors There are two types of GABA receptors, the ionotropic (GABAAR) and metabotropic receptors (GABABR). The GABAAR is a pentameric subunit complex (Fig. 3A) that forms a pore in the membrane (Hosie et al., 1997). Each subunit has an extracellular N-terminus, four transmembrane regions and an extracellular C-terminal (Buckingham et al., 2005). When GABA binds to this type of receptor a conformational change will occur, the channel opens and ions (particularly Cl- ions) flow through down a concentration gradient. This causes hyperpolarization of the postsynaptic membrane (Darlison et al., 2005). Vertebrates have two major classes of ionotropic GABA receptors, GABAA and GABAC receptors. GABAA receptors are more widespread in the CNS than GABAC receptors and the latter are not antagonized by bicuculline as GABAA receptors are. Both are, however, blocked by the plant toxin picrotoxin (Hosie et al., 1997). Five subunits of the ionotropic GABA receptor have been identified in mammals: α, β, γ, δ and ρ. GABAA receptors are formed as heteromers of α, β and γ or δ subunits whereas GABAC receptors are composed of ρ subunits (Hosie et al., 1997). To date three subunits have been cloned and characterized in Drosophila; RDL (resistant to dieldrin) (Ffrench-Constant et al., 1991), GRD (GABA- and glycine-like receptor of Drosophila) (Harvey et al., 1994) and LCCH3 (ligand-gated chloride channel homologue 3) (Henderson et al., 1993). RDL can form functional homomultimers, whereas LCCH3 and GRD cannot. This suggests that these subunits form heteromers with RDL or other subunits (Hosie et al., 1997), giving rise to GABA receptors with different pharmacological and kinetic properties (Gisselmann et al., 2004). The most studied subunit in insects is RDL, since it is a suitable model of insect ionotropic GABA receptors. It is located throughout the entire nervous system

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in many different insect orders of both embryos and adults and has been found both at synapses as well as on neuronal cell bodies (Buckingham et al., 2005). 1.2.2.2. Metabotropic GABA receptors The last major neurotransmitter receptors to be cloned in mammals were the G-protein-coupled GABAB receptors (Kaupmann et al., 1997), and some years later they were cloned in Drosophila (Mezler et al., 2001). In accordance with other GPCRs, the GABAB receptor is composed of a single polypeptide that spans the membrane seven times (fig 3B). It has a large domain in the extracellular N-terminal called the venus flytrap module (VFTM) that is responsible for binding the ligand. The intracellular C-terminal activates G-proteins (Gi, see section 1.2.1) and contains a coiled-coil region, making it possible for the receptors to form dimers. As will be outlined below GABAB receptors must form dimers to be functional.

Presynaptic GABAB receptors can inhibit release of more transmitter by downregulating voltage-gated Ca2+ channels via Gβ/γ or inhibit adenylate cyclase via Gi that results in decreased cAMP levels and protein kinase A (PKA) activity (Kaupmann et al., 1998; Kubota et al., 2003). Postsynaptic GABABRs can affect the adenylate cyclase pathway and commonly, via Gβ/γ, act on G-protein-coupled inwardly rectifying K+ channels (GIRKs), that induce hyperpolarization (Kaupmann et al., 1998).

There are three subtypes of GABABRs in Drosophila (and presumably other insects), namely Dm-GABABR1-3 (Bettler et al., 2004). In mammals, however, only two subtypes has been identified, m-GABABR1-2. Dm-GABABR1 and Dm-GABABR2 are similar to the mammalian GABABR1 and R2. Dm-GABABR3 seem to be insect specific (Bettler et al., 2004) with a possible role in the circadian clock (Dahdal et al., 2010), and will be discussed in more detail below.

A functional GABABR requires formation of a dimer of GABABR1 and R2 subunits. The ligand binding part is situated in the GABABR1, with its venus flytrap module (Kaupmann et al., 1997), and the presumed ligand binding sequence is not conserved in GABABR2. The GABABR2 on the other hand activate G-proteins, increase GABABR1 agonist affinity (Pin et al., 2003) and assist GABABR1 to reach the cell surface. GABABR1 stays in the endoplasmatic reticulum (ER) after its biosynthesis due to an ER retention signal. This signal will be masked upon interaction of the coiled-coil regions of the two subunits, making GABABR1 reach the surface together with R2. GABABR1 with mutations in the ER retention signal can reach the surface alone, form homodimers, but is not able to activate G-proteins (Pin et al., 2004). GABABR2 with a mutation in the part of the intracellular loop that contact the G-protein can logically not couple to G-protein, whereas the same mutation in GABABR1 has no effect (Pin et al., 2003). GABABR1-deficient mice have no detectable pre- and postsynaptic responses to known GABABR agonists and the GABABR2 subunit is strongly downregulated in these mice (Bowery et al., 2002).

The third, insect specific GABABR3 subunit also has a coiled-coil region that might suggest that it form dimers (Mezler et al., 2001). Whether it forms heterodimers with other subunits or if it is acting as homodimer is not

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known, but the possibility that it forms dimers with GABABR1 or R2 is small, since the distribution of GABABR3 in embryos, observed with in situ hybridization is rather different from these subunits. GABABR1 and R2 however, seem to be co-localized in neurons both in insects and mammals (Mezler et al., 2001). The functional roles for GABAB receptors are not fully unraveled. One possible function seems to be a role in addiction. GABABR-deficient flies appear to be more tolerant to ethanol sedation than wildtype flies (Dzitoyeva et al., 2003) and nicotine-resistant flies have decreased levels of GABABR transcripts (Passador-Gurgel et al., 2007). Lower levels of GABABR1 also affect growth negatively and can be lethal (Dzitoyeva et al., 2005). The receptor also seems to be involved in the circadian clock (Hamasaka et al., 2005), as it is expressed in a subset of the clock cells (s-LNv) and responses to GABA is prevented when blocked with GABABR antagonist (but not with GABAAR antagonist). A recent study (Dahdal et al., 2010) suggest that also GABABR3 is expressed in the s-LNvs and helps generating 24 hr rhythms via GABA-mediated inhibition of acetylcholine-induced Ca2+ responses. GABABR3 RNA levels and the response to GABA were reduced in isolated larval LNvs (known to develop into adult s-LNvs) in Drosophila expressing GABABR3-RNAi specifically in the clock neurons. Furthermore, these flies showed a lengthened 24 hr rhythm. This is the first report on a possible function of the GABABR3 subunit (Dahdal et al., 2010). Figure 3. Schematic drawings of receptors. A. An ionotropic receptor is composed of five subunits and forms a pore in the membrane. B. A metabotropic receptor with an extracellular N-terminal, seven transmembrane domains, three extra- and three intracellular loops and an intracellular C-terminal. 1.2.3. Short neuropeptide F The mammalian neuropeptide Y (NPY) plays an important role in food-intake regulation, metabolism as well as in memory and learning [see (Lee et al., 2004)]. The invertebrate equivalent is named neuropeptide F (NPF) and the name refers to a phenylalanine (F) residue in the C-terminal instead of a tyrosine (Y) of the vertebrate peptide. NPF is involved in larval feeding, foraging and social behavior (Wu et al., 2003). The levels of NPFs are decreased upon the behavioral switch from the continuous feeding of the 3rd instar larva to the wandering stage when the larva search for a puparation site

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outside the food source (Wu et al., 2003). Whereas NPF seem to be a motivator for food intake, another peptide is regulating feeding rate and interact with insulin to regulate growth. This peptide, encoded by a different precursor gene, is called short neuropeptide F (sNPF). The snpf gene was first discovered in Drosophila (Vanden Broeck, 2001) and has up to date only been found in arthropods (Nässel and Wegener, 2011). As the name implies, its products are only 6-11 amino acids (Vanden Broeck, 2001), whereas NPF consist of 36 amino acids in Drosophila and are sometimes referred to as long NPF. This name is however misleading since the active peptide in some insects are even shorter than the sNPFs (Nässel and Wegener, 2011). The one snpf precursor gene gives rise to four sNPFs (sNPF1-4), whereas the npf precursor gene only produces one NPF in Drosophila (Lee et al., 2006; Mertens et al., 2002). The number of sNPF peptides varies in different insects and it has been suggested that the multiple forms of sNPFs have been generated by intragenic duplication, whereas NPFs are multiplied by gene duplication (Nässel and Wegener, 2011). Even though the functions of these peptides to some extent are similar and the names imply close relationship, sNPF does not seem to be related to NPF nor to the vertebrate NPY (Nässel and Wegener, 2011). The only “relationship” is the similarity in receptors these peptides activate (discussed more detailed in next section).

The expression of the snpf gene in the CNS occurs from late stage of the embryo and in the adult brain it is widely distributed (Lee et al., 2004). The four different forms of sNPFs in Drosophila vary to some extent in size and sequence. sNPF1 and sNPF2 have an RLRFamide sequence at the C-terminal, whereas sNPF3 and sNPF4 have an RLRWamide in the same position (Nässel, 2002).

One possible function of sNPF seems to be control of feeding and thus body size. Overexpression of the peptide results in bigger and heavier flies due to increased food consumption, whereas knockdown of the snpf gene produces smaller flies (Lee et al., 2004) and is believed to be caused by interaction with insulin signalling (Lee et al., 2009; Lee et al., 2008). Mass spectrometry and HPLC has shown presence of sNPFs in the hemolymph of Drosophila (Garczynski et al., 2006), which might suggest a possible neuroendocrine role. This is further supported by immunocytochemistry and in situ hybridization, showing localization in the neurohemal organs but not in the gut (Lee and Park, 2004). The peptide is expressed in a subset of the clock neurons, the LNd cells (Johard et al., 2009), indicating a role in the circadian rhythm. In Locusta migratoria, sNPF stimulates ovarian development (Cerstiaens et al., 1999) and might therefore have a role in reproduction in these animals. Since the peptide is so abundant in the brain, one would suspect many different functional roles, but these are still far from understood. 1.2.3.1. Short neuropeptide receptor As mentioned earlier, the mammalian NPY and its invertebrate homolog NPF are not related to sNPF, but the receptors of these peptides seem more related (Feng et al., 2003; Mertens et al., 2002; Reale et al., 2004). The mammalian NPY receptor, Y2, and the Drosophila sNPF receptor, sNPFR, show 33% identity and 49% homology (Mertens et al., 2002). As all neuropeptide receptors, the sNPFR is a G-protein-coupled receptor (Mertens

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et al., 2002). The only known ligands to sNPFR are sNPFs, since they are the only of tested peptides (including FMRFamides and other peptides with an RFamide C-terminus) that can activate the receptor when expressed in Xenopus oocytes or mammalian Chinese hamster ovary cells (Feng et al., 2003; Mertens et al., 2002). sNPFR transcript has been identified in CNS of embryos with in situ hybridization and in adult Drosophila with Northern blot (Feng et al., 2003), but its localization in the periphery is not at all established. RT-PCR have revealed presence of the receptor in the brain, gut, fat body, and Malpighian tubules of larvae and in whole bodies and ovaries of adults (Mertens et al., 2002). The sNPFR can activate inwardly rectifying K+ channels (GIRKs) (Reale et al., 2004) and Ca2+-dependent inward chloride currents (Mertens et al., 2002) when expressed in Xenopus oocytes. The G-proteins Gi and G0 are sensitive to pertussis toxin, and applying this to the oocytes decreases the activation of GIRK currents, which might indicate that Gi or G0 are involved in this process (Reale et al., 2004). Taken together, these results suggest that activation of the receptor is controlling neuronal excitability or induce hyperpolarization of the cell. This does not exclude, however, that sNPFR is activating other systems or function differently in vivo. 1.2.4. Insulin, insulin-like peptides and insulin receptor Insulin is the major hormone that regulates carbohydrate homeostasis, control fat and cellular uptake of amino acids. In humans, insulin is produced by β-cells of the pancreas and released into the circulation as a response to high sugar levels. Circulating insulin initiates uptake of glucose from the blood to store it as glycogen in the muscle and liver. The insulin propeptide includes a carboxyl A-chain and a terminal B-chain that is connected by a C-chain. The C-chain is cleaved off in the Golgi apparatus to produce the mature and functional insulin peptide. The presumed function of the C-chain is not completely established. The insulin receptor is a tyrosine-kinase and upon binding of insulin, the tyrosine kinase domains phosphorylate, which triggers a signalling cascade (Squire, 2003). Drosophila is a satisfactory model for insulin signalling because the pathway is well conserved between invertebrates and vertebrates. A simplified comparison of the insulin-signalling pathway in three different animals (C.elegans, Drosophila and mammals) is seen in Figure 4. Drosophila insulin-like peptides (DILPs), like mammalian insulin, regulate circulating sugar (glucose and trehalose) levels and store excess energy as glycogen and lipids [reviewed in (Teleman, 2010)]. Adipokinetic hormone, AKH, plays a role similar to mammalian glucagon and act as an antagonistic peptide to insulin-like peptide (Kim and Rulifson, 2004). Glucagon and AKH elevate circulating blood sugar and they function together with insulin to maintain glucose homeostasis. There are seven Insulin-like peptides in Drosophila, DILP1-7, but only one known receptor, dInR (Brogiolo et al., 2001). DILP1-5 are encoded on the 3rd chromosome and DILP6-7 on the X-chromosome, but all in different loci with their own promoter regions. All DILPs, except DILP6, have similar structure (Grönke et al., 2010; Teleman, 2010; Teleman et al., 2008), yet presumably diverse functions based on their dissimilar distribution patterns.

14

DILP 2, 3 and 5 are found in a specific cluster of the median neurosecretory cells, the insulin producing cells, IPCs. The IPCs send axons to the larval ring gland and aorta and adult corpora cardiaca and aorta where insulin is released into the hemolymph. DILP 2 is also expressed in the imaginal discs and salivary glands. DILP 4 and 5 are expressed in the midgut (Brogiolo et al., 2001; Broughton et al., 2005; Ikeya et al., 2002). DILP 6 is produced in fat bodies (Slaidina et al., 2009) and show sequence similarities to mammalian insulin-like growth factors, IGFs. DILP 7 is expressed in ten neurons in the abdominal ganglia, both during larval stages and in the adult fly (Brogiolo et al., 2001; Leevers, 2001; Miguel-Aliaga et al., 2008). One pair of DILP 7-expressing neurons in the ventral nerve cord sends axons that terminate on the MNCs in the protocerebrum in larvae (Miguel-Aliaga et al., 2008). Relaxin is an important reproductive hormone in mammals and DILP 7 is the assumed ortholog of relaxin due to its effect on female reproduction (Yang et al., 2008). DILP 1 has not yet been detected in tissues. Figure 4. The insulin-signalling pathway is well conserved among different animals. Picture modified from (Puig et al., 2003). Overexpression of any of the DILPs during development results in increased body size, whereas the same experiment in adult causes trehalosemia, but without altered body size (Ikeya et al., 2002). Growth is limited to the larval stages in Drosophila, since adult flies do not increase in size (Teleman, 2010). This means that genetic manipulation of insulin pathway components in adults only alter metabolism and stress responses, whereas also growth is affected in larvae. Deletion of the IPCs leads to decreased larval growth and also an altered lifespan is observed in adult flies; these flies live longer and are more resistant to metabolic stress. Increased

Mammals C. elegans Drosophila

Insulin Insulin DILPs

InR

IRS1-4

PI3K

PTEN

PDK1

Akt

FOXO mTOR

p27Kip1 4EBP

Growth

daf-2

age-1

PDK1

Akt

Daf-16

Lifespan

Daf-18

dInR

chico

dPI3K

dPDK1

dPTEN

dAkt

dTOR dFOXO

d4EBP

Growth

15

glucose levels in the circulation and more fat accumulation are also observed (Broughton et al., 2005; Ikeya et al., 2002). Upon larval fasting, DILP 2, 3 and 5 levels decrease (Ikeya et al., 2002; Teleman et al., 2008), but expression of DILP 6 and 7 is upregulated (Teleman et al., 2008). Knocking down only DILP 2 in IPCs produces flies that have higher trehalose levels, but lack the other changes in phenotypes, presumably due to a compensatory effect of the other DILPs (Broughton et al., 2008).

The Drosophila insulin receptor (dInR) is, like the mammalian InR, a receptor tyrosine kinase and shows homology with insulin receptors in other organisms. In fact, human insulin can activate the dInR with almost the same affinity (Nasonkin et al., 2002). 2. AIMS OF THE THESIS Little is known about GABA- and sNPF signalling and functions in Drosophila. The aim of this thesis is to learn more about sNPF and GABA signalling and to unravel some of their functional roles. Our main focus has been the role of sNPF and GABA in feeding circuits and in the olfactory system, including the mushroom bodies. We first decided to investigate the localizations of these transmitters and their other signaling components in the brain. For this we raised some new antisera and tested out various Gal4 drivers. The next step was to perform experimental work on sNPF and GABAB signalling by applying Gal4-UAS technology to cell specifically interfere with the peptide and receptor expression and then test behavior and physiology in various assays. PAPER I GABA is the major inhibitory transmitter in vertebrates as well as in invertebrates. The ionotropic receptor of GABA, GABAAR, used for fast, inhibitory action of GABA, has been extensively studied in insects. Little is however known about the metabotropic GABA receptor, GABABR, used for slow and modulatory actions of GABA. In paper I we therefore determined the distribution of GABABRs in relation to ionotropic GABA receptors and other markers for GABA-signalling in larval and adult Drosophila brains. This would give us an idea what circuits are involving GABA signalling via the GABABRs and facilitate future behavioral and physiological experiments. PAPER II Insulin-like peptides are important hormones regulating several physiological processes in an animal. Many studies regarding insulin have been carried out with main focus of the effects downstream of the insulin receptor. However, little is known about what regulates production and release of insulin. In mammals, insulin release depends on glucose/ATP via glucose transporters (GLUTs) (Zierler, 1999), whereas functional GLUTs or nutrient sensors have been less investigated in invertebrates. What are the factors controlling insulin release in Drosophila? We found that the GABABR is localized on the insulin producing cells, IPCs, and tested whether GABA might be one of the factors involved in the regulation of insulin. Previous studies have shown that insulin has roles in lifespan, growth, stress resistance, regulation of lipid and carbohydrate levels (Rulifson et al., 2002), so we tested these parameters after knocking down the GABABR in the IPCs.

16

PAPER III and IV The role of sNPF in feeding and growth has been established (Lee et al., 2008; Lee et al., 2004). To investigate the presence of sNPF signalling in feeding circuits and other relevant brain regions we mapped the distribution of sNPF and its receptor in the CNS and in the intestine of Drosophila in relation to different neuronal markers. In paper III we determined the localization of the mRNA (snpf) and the peptide precursor (sNPF) and asked whether the peptide has a global function or if its functions are dependent on the neurons releasing the peptide (i.e. multiple distributed functions). Paper IV focuses on the sNPF receptor in order to find out action sites for the peptide. Are the receptors postsynaptic on neurons involved in feeding? 3. METHODS 3.1 The Gal4/UAS system and RNA interference In all four studies of this thesis we have used immunocytochemical methods combined with the Gal4-UAS technique [developed by (Brand and Perrimon, 1993)]. This technique is based on two transgenic fly strains. One expresses Gal4, a yeast transcription factor (from Saccharomyces cerevisiae) that is not normally found in Drosophila. The gal4 gene is coupled to an endogenous Drosophila promoter of interest and is only expressed in those cells where the promoter usually is activated. The other fly stain contains a yeast-specific upstream activating sequence (UAS) coupled to a Drosophila gene of interest. When the two transgenic fly strains are crossed, Gal4 binds to UAS in the progeny and induce transcription of the gene it controls in specified cells only (Fig. 4). Figure 5. The UAS-Gal4 technique involves two parental transgenic fly stains (P), one bearing the yeast transcription activator protein Gal4 and the other the promoter region UAS (Upstream Activation Sequence) to which Gal4 bind to activate transcription. The parental flies are not affected, however, the progeny (F1) having both UAS and Gal4 will express the gene of interest (Gene A).

GAL4 Gene A UAS

Gene A UAS

A

X P

F1

17

We used this technique for two purposes. One was to drive expression of green fluorescent protein (GFP) in specific neurons of interest, often in combination with different antibodies. We also used this technique to cell-specifically downregulate the expression of different gene transcripts by RNA interference (RNAi). RNAi involves a double stranded RNA (dsRNA) with a complementary sequence to the target mRNA. The enzyme DICER cleaves the dsRNA into 20-25 nucleotide small interfering RNA (siRNA) that assembles to large complexes (RNA-induced silencing complexes, RISCs). The siRNA then guide the RISCs to the complementary, target mRNA and the catalytic component of the complex cleaves the target mRNA and prevents translation (Fire et al., 1998). 3.2. Fly stocks All transgenic strains of Drosophila melanogaster used in this thesis are listed in Table 1 and 2. In addition to the Gal4 and UAS strains, we used Oregon R and W1118 as wild type flies in all four studies. Some of the Gal4 and UAS strains were used in more than one study, whereas others were used in one only.

OK107-Gal4 was used in all four studies. It is expressed in the Kenyon cells of the mushroom bodies as well as in median neurosecretory cells (MNC) in the pars intercerebralis, including the insulin producing cells, IPCs. Except to localize the mushroom body neurons and MNCs, this strain was used in paper II to knock down the GABABR in these cells with UAS-GABABR-RNAi. UAS-GABABR-RNAi was also crossed with MB247-Gal4 that is expressed specifically in the intrinsic neurons of the mushroom body.

GH146-Gal4 and GH298-Gal4 were used in paper I to identify projection neurons and local interneurons of the antennal lobes, respectively. OK6-Gal4 for motorneurons and 21D-Gal4 for L2 type monopolar cells of the lamina were also used in paper I. Glutamic acid decarboxylase 1 (GAD1) is the biosynthetic enzyme for GABA, and thus a gad1-Gal4 strain was used in paper I, II and III to identify putative GABA-producing neurons.

In paper III we used many Gal4 lines as markers; Cha-Gal4 (choline acetyltransferase) for cholinergic extrinsic neurons, th-Gal4 (tyrosine-hydroxylase) for dopamine, tdc-Gal4 (tyrosine decarboxylase) for octopamine and tyramine producing neurons, OK6-Gal4 for motorneurons, OK371-Gal4 (vesicular glutamate transporter) for glutamatergic neurons, npf-Gal4 for neurons expressing long neuropeptide F and snpf-Gal4 for short neuropeptide F. The latter was also used in paper IV. C929-Gal4 was used in paper III and IV to visualize large peptidergic neurons, since it is expressed in DIMM-positive cells. DIMM is required for differentiation of large peptidergic neurons and endocrine cells (Hewes et al., 2003).

In paper II and IV we used different markers for insulin-like peptides, Dilp2-Gal4 and Dilp3-Gal4. Dilp7-Gal4 in paper IV showed ten large cells in the ventral ganglia of the larvae. For overexpression of Dilp2, we used UAS-Dilp2 in paper III. Rdl-Gal4, UAS-Rdl-RNAi, GABABR-Gal4 and UAS-GABABR-RNAi for different types of GABA receptors were used in paper II. A new snfpr-Gal4 (unpublished) was tested in paper IV, but the expression pattern could not be confirmed when used together with sNPFR antisera.

18

To visualize Gal4-expression with green fluorescent protein, the Gal4 strains in all studies were crossed with transgenic flies expressing UAS-mcd8-GFP or UAS-GFP.S65t.

All flies have been fed standard Drosophila food and raised under 12h:12h light-dark conditions at 18°C or 25°C. Crosses of Gal4-UAS flies were made at 25°C. At least 10 larval or adult fly brains were used in each experiment.

Table 1. Gal4 strains used. Gal4 lines Description Reference Paper 21D L2 monopolar cells in the lamina of the

optic lobe (Gorska-Andrzejak et al., 2005)

I

c929 Large peptidergic neurons, endocrine cells

(Hewes et al., 2003) III, IV

Cha Choline acetyltransferase promoter-Gal4-GFP fusion

(Salvaterra and Kitamoto, 2001)

III

Dilp2 Insulin-like peptide 2

(Wu et al., 2005) II, IV

Dilp3 Insulin-like peptide 3

(Buch et al., 2008) II, IV

Dilp7 Insulin-like peptide 7

(Yang et al., 2008) IV

GABABR2 Metabotropic GABA receptor

(Root et al., 2008) II

gad1 GABAergic neurons

(Ng et al., 2002) I, II, III

GH146 Projection neurons in the antennal lobe

(Stocker et al., 1997) I

GH298 Local interneurons in the antennal lobe

(Stocker et al., 1997) I

Hug Hugin producing cells in the subesophageal ganglion

(Melcher and Pankratz, 2005)

IV

MB247 Intrinsic neurons of the mushroom bodies

(Aso et al., 2009) II

npf Neuropeptide F

(Wu et al., 2003) III

OK107 Mushroom body and median neurosecretory cells (MNC)

(Lee et al., 1999) I, II, III, IV

OK371 Vesicular glutamate transporter

(Mahr and Aberle, 2006) III

OK6 Motorneurons

(Aberle et al., 2002) I

Or83b Olfactory receptor neurons

(Larsson et al., 2004) IV

rdl GABAAR subunit

(Kolodziejczyk et al., 2008) II

snpf Short neuropeptide F

(Nässel et al., 2008) III, IV

Snpfr Short neuropeptide F receptor Unpublished

tdc Tyrosine decarboxylase

(Cole et al., 2005) III

th Tyrosine-hydroxylase

(Friggi-Grelin et al., 2003) III

19

Table 2. A list of UAS strains used. UAS-lines Description Reference Paper GABABR2-RNAi Metabotropic GABA receptor

(Root et al., 2008) II

rdl-RNAi Ionotropic GABA receptor subunit

(Liu et al., 2009) II

Irk3-RNAi Inwardly rectifying K+ channels

(Dietzl et al., 2007) II

Dilp2 Insulin-like peptide 2

(Wu et al., 2005) II

gfp.S65T Green fluorescent protein

I, II, III, IV

mcd8-gfp Green fluorescent protein

I, II, III, IV

3.3. Antisera, immunocytochemistry and antisera characterization All antisera used are listed in table 3 together with the fixative, dilution and reference where they were first used and characterized.

We designed antigens and had antisera produced for several proteins of interest. Three new antisera were thus used for the work of this thesis. In paper I we raised two rabbit polyclonal antisera against peptide sequences of each of the genes rdl and vgat. Anti-RDL was produced against a peptide sequence (CLHVSDVVADDLVLLGEE) of the C-terminal of the GABAA receptor subunit. The synthesized peptide was coupled to Limulus hemocyanin as a carrier. Anti-vGAT was produced against a peptide sequence (CQTARQQIPERKDYEQamide) of the N-terminal of the transporter and coupled to keyhole limpet hemocyanin. Both were injected into two rabbits each.

For detection of the sNPFR in paper IV, we produced four rabbit and four guinea pig polyclonal antisera against two different peptide sequences of the sNPFR gene. One was a peptide sequence of the N-terminal (CILADVAASDEDRSGGIIHNQ) and the other a portion of the C-terminal (ETDGDYLDSGDEQTVEVRC) of the receptor. The synthesized peptides were coupled to maleimid-coupled thyroglobulin.

All three types of antisera were characterized in similar fashion. Preimmune sera were collected prior to the first injection of the antigen. After three booster injections, the antisera were collected. The antisera were tested for specificity on CNS tissue by immunocytochemistry and Western blots with antisera, preimmune sera and antisera preabsorbed with the antigen. Preabsorbed antisera were prepared by incubation of 50 nmol/ml antigen in diluted antisera for 24 hours at 4°C. The procedure for Western blot is described in detail in paper I and IV.

To detect the products of the snpf gene, we tried three different antibodies (produced by Drs J. Veenstra, Bordeaux, France). The most frequently used was raised against 20 amino acids of the snpf precursor protein. The other two were raised against short sequences of the C-terminals of the peptides sNPF1 and 3 and would recognize sNPF1-2 or sNPF3-4 respectively. The antisera produced to sNPF1 are likely to cross-react also with other RFamides.

To amplify the GFP signal we applied a mouse monoclonal GFP-antibody or a rat monoclonal anti-cd8 in some specimens. Bouin´s fixative

20

weakens the GFP signal drastically and anti-GFP is not compatible with Bouin´s. Therefore, anti-cd8 proved to be a better choice in cases where Bouin´s fixation was required.

The procedure for immunocytochemistry is described in detail in material and methods in the papers, but briefly brains of 3rd instar larvae were fixed on ice for 2 hours in 4% paraformaldehyde (PFA) in 0,1M phosphate buffer (PB; pH 7.4) or in Bouin´s fixative for 30 minutes on ice. Adult flies were decapitated in PBS-TX and fixated for 4 hours in 4% PFA or 1 hour in Bouin´s fixative on ice. Larval brains were used as whole mounts, whereas adult brains were sectioned on a cryostat or used as whole mounts. The tissues were incubated in primary antiserum 12-72 hours (depending on the antiserum used) in 4°C, washed in PBS-TX (phosphate-buffered saline containing Triton X) and incubated with secondary antiserum conjugated to a florophore for 2 hours in room temperature or over night at 4°C. After washing in PBS-TX and PBS, the tissues were mounted in 80% glycerol in PBS. Specimens were imaged using a Zeiss LSM 510 microscope or a motorized Zeiss Axioplan 2 microscope with a CCD-camera. The images were processed with Zeiss LSM software and edited in Adobe Photoshop 7.0 or later version.

Table 3. Primary antisera used for immunocytochemistry. R, rabbit. GP, guinea pig. M, mouse. Antibody Host Dilution Fixative Recognize part of Reference GABABR R 1:16000 PFA C-terminus (Hamasaka et al., 2005)

RDL R 1:40000 Bouin´s GABAAR subunit This thesis (Paper I)

vGAT R 1:1000 PFA Vesicular GABA

transporter This thesis (Paper I)

GAD1 R 1:500-1000

PFA or Bouin´s

Glutamic acid decarboxylase

(Featherstone et al., 2000)

GABA R 1:1000 PFA γ-aminobutyric acid (Wilson and Laurent, 2005)

sNPFp R 1:4000 PFA sNPF precursor

This thesis (Paper III)

sNPFR R/GP 1:40000 Bouin´s N-terminus This thesis (Paper IV)

sNPFR R/GP 1:40000 Bouin´s T-terminus This thesis (Paper IV)

DILP2 R 1:1000 PFA A-chain (Cao and Brown, 2001)

DILP7 R 1:5000 PFA B-chain (Miguel-Aliaga et al., 2008)

Fas M 1:75 PFA Fasciclin-2 (Hummel et al., 2000)

CRZ R 1:2000 PFA Corazonin (Cantera et al., 1994)

GFP M 1:1000 PFA Green fluorescent protein

Invitrogen

CD8 Rat 1:100 Bouin´s

Membrane protein Invitrogen

21

3.4. In situ hybridization For distribution pattern of snpf transcript we performed in situ hybridization with a digoxigenin (Dig)-labeled snpf riboprobe on whole mount larval or adult brains. Probe production and in situ protocol is described detailed in paper III. Briefly, heads were fixed in 4% PFA and made permeable with proteinase K. After another postfixation in 4% PFA and prehybridization in hybridization buffer, the brains were incubated with the probes for 48 hours at 55°C. The probe was either hydrolyzed or used intact. The brain tissues were incubated in alkaline phosphatase (AP) tagged anti-Dig antiserum for 2 hours at room temperature. AP histochemistry was carried out in NBT/BCIP solution for about an hour, whereafter the tissues were mounted in 80% glycerol. 3.5. Quantification of immunofluorescence To determine the difference in insulin levels between starved and fed flies and between control flies and flies lacking the GABABR on insulin producing cells (paper II), we measured the intensity of immunofluorescence in these cells. This was performed in the confocal microscope with the same settings and exposure time for all specimens. The images were analysed and the intensity of immunofluorescence was quantified in each cell with ImageJ. 20-30 cells were analyzed for each genotype. 3.6. Survival assays We performed three different survival assays; lifespan under normal feeding conditions and during starvation or desiccation. To measure longevity under normal feeding conditions, male flies (4-6 days old) of different genotypes were counted each day for survival and transferred to new bottles with fresh food. Two replicates with at least 80 flies of each genotype per replicate were counted.

Survival during starvation was measured by placing individual flies in small bottles containing 0,5% aqueous agarose, making water accessible but not food. Dead flies were counted every 12 hours until all flies were dead. Three replicates with at least 40 flies per genotype in each replicate were counted. Male flies, 4-6 days old, were used.

Survival during desiccation was carried out similar to the starvation assay, with the difference that the bottles were empty with no access to either food or water and the flies were counted each hour.

The flies were kept in a 25°C incubator with 12:12 light:dark conditions and controlled humidity. 3.7. Measurements of trehalose, lipids and growth The trehalose levels were measured in normally fed flies as well as after 5 or 12 hours of starvation in different genotypes according to (Isabel et al., 2005). 4-8 days old male flies were kept in tubes with different food conditions and their wet weight was measured. The flies were then incubated in ethanol and sonicated. Yellow anthrone that turns green when it binds to sugar was added, and the color intensity was measured in an ELISA plate reader against a standard curve.

22

Levels of lipids were measured in whole bodies of different genotypes after 0, 12 and 24 hours of starvation. Wet weight of male flies was determined and thereafter dry weight, by placing the flies in 65ºC for 24 hours. Lipids were then extracted in dry flies by diethyl ether. When the ether was removed and the flies had been exposed to another drying, the lean dry weight was obtained. The total lipid content was considered as the difference between dry weight and lean dry weight. To investigate growth rate, we weighed either third instar larvae or 3-6 days old male or female flies. The length of the pupae was measured in a Zeiss Axioplan 2 microscope with a CCD-camera and analyzed in AxioVision 4.6.3.0. 4. RESULTS AND DISCUSSION 4.1. PAPER I We mapped the distribution of the metabotropic GABA receptor (GABABR) in Drosophila larvae and adults and analyzed the relations to known structures in the brain, to the ionotropic GABA receptor (GABAAR) and other markers for GABA signaling. GABA and both types of GABA receptors are abundant in the entire CNS of Drosophila and are present in the antennal lobe, mushroom bodies, parts of the central body complex and optic lobes. The GABAAR subunit RDL and GABABR2 have similar distribution in the Drosophila brain, but yet distinct patterns of the two can be observed in many brain regions.

We used an antiserum against the GABABR2 subunit, but this immunolabeling probably reflects the distribution of functional metabotropic GABA receptors since the two subunits (R1 and R2) have to dimerize to function (Kaupmann et al., 1997; Pin et al., 2003). Unfortunately, anti-GABABR2 only labeled neuronal fibers and commonly not cell bodies, making it difficult to directly identify specific neurons. However, double labeling with GABABR2-Gal4 driven GFP together with anti-GABABR2 revealed overlapping distribution patterns and we thus have a reliable marker for both GABABR positive cell bodies and fibers.

Vesicular GABA transporters are supposed to be located in the synaptic terminations of GABAergic neurons and staining with anti-vGAT probably corresponds to these sites. The antiserum to the GABAAR subunit RDL probably labels most GABAAR action sites. This antiserum also did not stain cell bodies, but labelled fiber branches that are most likely to be on the postsynaptic side of the neuron. The GABABR can be both pre- and/or postsynaptic, and that is reflected in the immunolabeling with anti-GABABR2. In the antennal lobe, we found GABAergic local interneurons expressing GABABR but not RDL. This could suggest that the metabotropic receptors are both pre- and postsynaptic and probably function as autoreceptors at these locations. Another location where GABABR-IR fibers are presynaptic is in cells of the lamina in the optic lobe. These are most likely the GABAergic centrifugal C2 neurons, and thus show presynaptic GABAB receptors also in this region. Another study (Kolodziejczyk et al., 2008) has shown GABABR2 immunoreactivity (IR) in C2 and C3 neurons (both GABAergic), but not the GABAAR subunit RDL. A later study revealed that the olfactory sensory neurons (OSNs) express the GABABR (Root et al., 2011). This presynaptic

23

GABABR was shown to modulate sensitivity in OSN to specific odors in a gain control mechanism. Other regions where the two receptor types differed were in the mushroom bodies, the ellipsoid body in the central body complex and in some of the clock neurons. In the peduncles and lobes of the mushroom body we only observed RDL-IR and not GABABR-IR. We found RDL and vGAT immunostaining in the entire ellipsoid body, whereas anti-GABABR2 only stained the outer layer. The protocerebral bridge and the fan-shaped body were weakly stained with all three antibodies. We found GABABR immunoreactivity in a subset of clock neurons where there where was no RDL staining. These results agree with a previous study (Hamasaka et al., 2005) that showed that GABA application decreased intracellular calcium levels in one group of clock neurons. Furthermore, this response was blocked by the GABAB-R antagonist CGP54626 and not by the GABAA-R antagonist picrotoxin. 4.2. PAPER II Insulin and insulin-like peptides (ILPs) are important hormones that are involved in many physiological processes. The ILP signaling pathway is well conserved among mammals and invertebrates, and consequently makes Drosophila a good model organism to understand functions and signalling mechanisms. Little is actually known about regulation of the production and release of insulin-like peptides in insects. Mammalian insulin is released when glucose enter the β-cells in the islets of Langerhans in the pancreas via glucose transporters (GLUT2), but such transporters have not been characterized in insects. When investigating GABA signalling with immunocytochemistry in combination with UAS-GAL4, we found that the metabotropic GABABRs are expressed in IPCs in the brain, whereas the ionotropic GABAARs (RDL) are not. This made us suspect that GABA is somehow affecting production and/or release of Drosophila insulin-like peptides (DILPs) via GABABRs. It was clear that most, possibly all, of the IPCs express GABABRs. In close vicinity to the IPCs are some other neurons containing GABABRs, but not DILPs. These cells are most likely other median neurosecretory cells (MNCs). We also tried to reveal the identity of GABAergic inputs on IPCs. This proved to be difficult because of the numerous neurons containing GABA, making tracing of individual neurons almost impossible. However, both GABA and GAD1 immunoreactivity displayed branching in close association with the IPCs. We show that none of the IPCs are GABAergic and therefore the GABABRs located in these sites are postsynaptic.

The mechanism behind the regulation of IPCs by GABA was further investigated by knocking down the expression of GABABRs in the IPCs by RNAi. Since GABA is inhibitory, we hypothesized that knocking down the GABA receptor would lead to increased production and/or release of DILPs and in consequence the flies would display phenotypes of increased DILP signalling. Insulin has reported effects on lifespan, stress resistance and growth (Broughton et al., 2005; Ikeya et al., 2002), so we tested these parameters in flies with diminished GABABR expression on the IPCs. Indeed lifespan and stress resistance were affected in the same manner as when

24

insulin is overexpressed. Normally fed Dilp2-Gal4 x UAS-GABABR-RNAi flies had a significantly reduced lifespan compared to controls, although the effect was not so drastic. When the same flies where tested in stress resistance assays (starvation and desiccation), these flies showed decreased resistance compared to the controls. Overexpression of DILP2 in IPCs produced the same phenotype, which would further support the idea that GABA is inhibiting insulin release or production. Also one Dilp3-Gal4 (for insulin-like peptide 3) and one OK107-Gal4 strain that include the mushroom bodies as well as most IPCs crossed with the same GABABR-RNAi gave the same phenotype: decreased survival at metabolic stress conditions. To make sure that the mushroom body is not involved in this process, we used a mushroom body-specific GAL4 line (MB247-Gal4) crossed with GABABR-RNAi. These flies did not display significantly altered survival at metabolic stress compared to their controls, and thus confirming that mushroom bodies are not involved.

We have found that GABA regulates insulin via its metabotropic GABABR, but not via the ionotropic GABAAR. The GABAAR subunit RDL-Gal4 expression was found in the neighboring cells, but not on the IPCs. This result was further supported by the lack of effect of starvation and desiccation on survival when knocking down RDL in IPCs.

Irk3 is a putative subunit of a G-protein-coupled inwardly rectifying K+ channel, GIRK, likely to be downstream of the GABABR (Kaupmann et al., 1998). Knocking down this subunit in IPCs decreased the survival at starvation, the same phenotype as when knocking down GABABRs.

We also measured relative DILP fluorescence in starved and fed flies lacking the GABABR on the IPCs and compared them to controls. The results indicate that DILP storage is increased and release is decreased in starved control flies as shown previously (Geminard et al., 2009), as DILP immunoreactivity is more intense than in fed control flies. Fed transgenic flies displayed even higher immunofluorescence compared to controls, indicating increased DILP production or reduced release. The highest relative immunofluorescence was seen in starved flies with diminished GABABR on IPCs. Taken together, these results indicate that GABA via GABABRs inhibits DILP signalling from IPCs. The fact that even fed transgenic flies had higher immunofluorescence than starved controls suggests that GABA is affecting both production and release. More release of DILPs is perhaps compensated by more production. Another study (Broughton et al., 2010) has shown that restricted diet can affect the different DILPs in various ways, one can be upregulated whereas another is downregulated. This variability might be masked in our assay, since the antiserum against DILP 2 used is most likely to crossreact also with DILP 3 and 5, and the immunofluorescence we see could be a total level of all three DILPs.

Since DILPs are known to regulate growth during development (Broughton et al., 2005; Ikeya et al., 2002), we determined weights of third instar larvae and length of pupae and adult flies lacking GABABR on IPCs. We could not see an effect on either weight or length. This might support the idea that GABA is released as a response to stress and is not affecting insulin signalling under normal conditions (also noted in weak affect on healthy lifespan).

GABA decreases insulin release via both GABAA and GABABRs in the pancreas in mammals in response to glucose (Dong et al., 2006). The

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Drosophila fat body has nutrient sensors, but the IPCs in the brain do not seem to. Thus many questions remain. How do GABAergic neurons sense nutrients? What is the identity of GABAergic neurons with outputs on IPCs? We know from immunocytochemistry that the IPCs are not GABAergic and thus we know that GABA is released from other neurons and that the receptors are postsynaptic to these cells. 4.3. PAPER III We mapped the distribution of sNPF (both expression of mRNA with in situ hybridization and the peptide precursor and peptides with immunocytochemistry) in larvae and adult Drosophila and found expression in a large number of diverse neurons. The widespread distribution of sNPF in a considerable variety of interneurons as well as in olfactory sensory neurons (OSNs) of the antennae indicates multiple roles as neuromodulator and probably cotransmitters.

First we investigated whether the peptides are likely to be released into the circulation and act as hormonal peptides. In order to do so we looked for Large cells that Episodically release Amidated Peptides, LEAP neurons, by using a c929-Gal4 line as a marker. These large cells express the transcription factor DIMM together with PHM which is an enzyme required to α-amidate neuropeptides (Park et al., 2008). We found no coexpression of c929-Gal4 driven GFP and anti-sNPF precursor in the larva, indicating that sNPF is not released as a hormone but instead signals locally and act as cotransmitter or neuromodulator. We did however find sNPF immunoreactivity in three pairs of large cells (LNCs) that also contained corazonin that have axons terminating in the anterior aorta and near the AKH-producing cells in the ring gland. We found punctuate sNPF immunoreactivity close to the AKH-cells, which could suggest that sNPF regulates AKH production, but we cannot eliminate the possibility that they are released into the circulation. The same sNPF/corazonin-immunolabelled cells arborize close to insulin producing MNCs. Thus it remains open whether these LNCs regulate AKH or DILP producing neurons (or none).

We also investigated whether sNPFs may be released as cotransmitters with some of the classical neurotransmitters, since this is often the case for small peptides in mammals. We screened for colocalization with GABA, glutamate, acetylcholine, dopamine and octopamine/tyrosine and found sNPF to be present in some neurons containing GABA, glutamate and acetylcholine. However, most of the sNPF-IR neurons did not contain any of the tested classical transmitters.

In the antennae we found sNPF-IR in a set of olfactory sensory neurons (OSNs) and their terminations in the antennal lobe. In the OSNs sNPF is colocalized with choline acetyltransferase, Cha-Gal4, that is the enzyme for synthesis of acetylcholine and is an indicator for cholinergic neurons. The projection neurons of the antennal lobe did not express sNPF, but Cha. A description of the detailed expression of sNPF in OSNs has been carried out (Carlsson et al., 2010). In this study, sNPF was found to be expressed in a subpopulation of OSNs in both the antennae and maxillary palps and their axons were detected in thirteen glomeruli of 50.

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One pair of dorsal median (DP) neurons in the first abdominal neuromere expressed both Cha and DILP7 together with sNPF. These neurons have branches in the vicinity of the DILP producing MNC dendrites in the pars intercerebralis.

In a previous study we found snpf and sNPF to be expressed in the majority of the mushroom body intrinsic Kenyon cells both in larvae and adults (Johard et al., 2008). The intrinsic neurons of the mushroom bodies are part of the central circuitry for olfactory learning and memory and we investigated whether sNPF was expressed together with any other transmitters in these cells. However, none of the tested transmitters labeled the Kenyon cells.

4.4. PAPER IV Short neuropeptide F seems to be involved in feeding, growth and regulation of insulin-like peptides, but other functions and sites of actions are not entirely established. In order to understand more about sNPF signalling, the sNPF receptor (sNPFR) needs to be mapped. The distribution of the receptor has not been revealed, except in Drosophila embryos using in situ hybridization (Feng et al., 2003). We therefore produced antisera against sNPFR, mapped the distribution in larvae and compared to its peptide ligands and other known markers, especially markers associated with feeding. The receptor was found in roughly 100 cells in the larval brain and ventral nerve cord. These cells do not produce sNPF, but branches of sNPF cells seem to contact fibers of sNPFR expressing neurons. It has been claimed that sNPFR is located on the IPCs (Lee et al., 2008), but our antisera did not label these cells. Instead, we found sNPFR immunoreactivity in three neighbouring MNCs in each hemisphere. These cells are included in OK107-Gal4 and c929-Gal4 and may produce diuretic hormone, DH31 and/or DH44 (see Park et al., 2008). Further sNPFR immunoreactivity was seen in AKH-producing cells in the ring gland and in 8-10 cells in the intestine.

We found fibers of both sNPF and sNPFR expressing neurons in the glomeruli of the larval antennal lobes. sNPF-IR in neuronal processes in glomeruli colocalized with Or83b-Gal4 driven GFP and are therefore likely to derive from OSNs similar to adult flies (Carlsson et al., 2010). There was no colocalization of Or83b-Gal4 and anti-sNPFR, but the antiserum labelled processes in the larval antennal lobe (LAL). This suggests that the sNPFR is not presynaptic in the larval OSNs, as it is in adult OSNs (Root et al., 2011). These authors also showed that sNPFR is upregulated presynaptically on the OSNs upon starvation and insulin proved to be the regulating factor. When the flies are fed and the level of insulin-like peptides in the circulation is high, the sNPFR is downregulated in OSNs. Upon starvation, and lower levels of circulating insulin-like peptides, there is an upregulation of sNPFRs (Root et al., 2011). If this is a generally occurring phenomenon in Drosophila, perhaps the levels of sNPFR in OSNs of the larvae are quite low. The larvae hatch in the food and they are continuous feeders submerged in food. This would result in continuously high insulin levels in feeding larvae and perhaps as a consequence lower levels of sNPFRs in OSNs (and maybe elsewhere in the brain). It could also be that larvae do not express sNPFR in the same pattern as adults do. Perhaps sNPF is not as important in feeding behaviours in larvae as it is in adults. That would for example explain why the Hugin

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producing cells (Bader et al., 2007; Meng et al., 2002) express sNPF in adults, but not in larvae. We had substantial difficulties with double labeling with receptor antiserum and Gal4 driven GFP. Bouin´s fixative was required to obtain labelling with the antisera to sNPFR and this harsh treatment repressed the GFP signal. One way to get around weak GFP signal is to use anti-GFP, but this antiserum required PFA as fixative and is not compatible with Bouin´s. When we finally thought about using anti-cd8 to increase the green signal in our specimens, the different sNPFR antisera had already passed their expiration date and did not function any longer. We also tried to apply the antisera to adult specimens, but the staining was not satisfactory. In cryosections or paraffin sections sometimes a few cells labelled weakly, but no staining was observed in the majority of the specimens. Whole mount specimens did not work at all for adults. We also tested a new sNPFR promoter Gal4 line to visualize sNPFR expressing neurons (kindly provided by Dr K. Yu, Korea Research Institute of Bioscience and Biotechnology, Korea). However, even after considerable efforts, we could not confirm that this Gal4 line was accurate in visualizing the sNPFR. The expression pattern of the antiserum against sNPFR and the Gal4 line did not match at all and therefore we did not use the Gal4 line in paper IV. The antiserum seemed to be reliable, since we designed two sequences specific for the sNPFR N-terminal and C-terminal respectively and injected into four rabbits and four guinea pigs. All antisera marked more or less the same cells and Western blot revealed a band at predicted size that was not visible when applying preimmune sera or antisera preincubated with the peptide used for immunization. 5. GENERAL DISCUSSION We wanted to learn more about sNPF and GABA signalling in the CNS of Drosophila and to unravel some of the functional roles of these molecules. This study has mainly focused on the role of sNPF and GABA in feeding and the olfactory system, including the mushroom bodies. First we mapped the distribution of these two transmitters and their receptors and compared their localization with other signalling components in the brain. For this we raised some new antisera and tried out various Gal4 drivers.

Regulated food intake is essential for survival and healthy life. For food search external signals, such as taste and olfaction, are needed. Internal signals are required to maintain homeostasis to initiate hunger and to signal satiety. Both sNPF and GABA seem to have roles in feeding and olfaction and presumably also in olfactory memory and learning. We found the GABABR to be localized on the IPCs in the pars intercerebralis and interfered with GABA receptors specifically in these cells. Our results indicate that GABA regulates production and/or release of DILPs via GABABR. The sNPFs have also been suggested regulators of DILP production in Drosophila (Lee et al., 2008), since overexpression of sNPFRs in the IPCs resulted in 10% heavier flies, and knocking down sNPFR produced smaller flies. Knocking down sNPFs in sensory neurons downregulated Akt in the fat body, dFOXO was translocalized to the nucleus, and the translational inhibitor 4E-BP was upregulated. All of these substrates are known to be signals downstream of

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the insulin receptor. Furthermore, sNPF knockdown reduced cell size, elevated circulating glucose levels and extended lifespan in adult flies (Lee et al., 2008), indicating that DILP signalling is reduced in these flies. Taken together, these data suggest that both sNPFR and GABABR are expressed on the adult IPCs. Earlier serotonin has been implicated in DILP regulation (Kaplan et al., 2008). It is not surprising that several factors are involved in the regulation of the DILPs since they are very important hormones. Interestingly, insulin has been shown to regulate the sNPFR levels on a subset of OSNs (Root et al., 2011).

It was stated that sNPFR immunoreactivity (IR) is displayed on the IPCs also in the larval brain (Lee et al., 2008). However, the images are somewhat ambiguous and the sNPFR-IR neurons could just as well be cells adjacent to the IPCs. We did not observe sNPFR-IR on the IPCs in the larval MNCs, but sNPFR-IR neurons were localized nearby IPCs in the same cluster. We suggest that these cells coexpress Diuretic hormone 31 or 44 (DH31 or DH44) or possibly myosuppressin (Park et al., 2008). It is possible that sNPFR is not expressed on the IPCs in larval brains, but the expression pattern might change during metamorphosis and be expressed on IPCs in adults. The GABAAR subunit, RDL, was also not detected in the IPCs, but in adjacent neurons in the same cluster and in close vicinity. Thus, we suggest that GABAAR and sNPFR are expressed in the same neurosecretory cells in the larva (but this needs to be confirmed).

We have not been able to determine the identity of the neurons containing GABA and sNPF that innervate the IPCs. Both transmitters are very abundant in the brain and individual neurons are difficult to trace. Two abdominal neurons containing DILP7 known to arborize close to the IPCs (Miguel-Aliaga et al., 2008) also contain sNPF and are likely candidates for DILP regulation at least in the larva.

We found sNPF and GABABR2 in the antennal OSNs of Drosophila and we expected to find sNPFR-IR axon terminals from the OSNs in the larval antennal lobe, since this was detected in the adult (Root et al., 2011). However, we could not confirm this, but cannot entirely rule out the possibility that sNPFR is present in low levels in OSNs in larvae. Perhaps the sNPFR antiserum did not penetrate the tissue well enough because of the deep location of the larval antennal lobe. Another possibility is that the expression of the receptor changes during metamorphosis. The levels of sNPFR in adult OSNs increase as a result of starvation and lower levels of circulating insulin (Root et al., 2011). The larvae in our experiments are fed and therefore the levels of sNPFR might be lower and not detectable with the antisera.

The mushroom bodies are the main brain centres for learning and olfactory memory. We found intense GABABR staining in the calyces, but not in the peduncle and lobes. This is expected, since extrinsic GABAergic neurons terminate in the calyx (Keene and Waddell, 2007; Yasuyama et al., 2002). However, RDL has previously been shown to be present in the mushroom body lobes and peduncles (Cayre et al., 1999; Strambi et al., 1998). We also found that the intrinsic Kenyon cells of the mushroom bodies contain sNPF. The Kenyon cells do not express the receptor to sNPF, but we found sNPFR-IR neuron processes innervating the lobes of the mushroom bodies in larvae. We assume that sNPF probably acts as a cotransmitter in the intrinsic neurons of the mushroom bodies, but that another substance will

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be identified as a primary transmitter. There is no evidence that Kenyon cells contain GABA, neither in the cell bodies nor axons or dendrites. We can probably also exclude glutamate, monoamines and acetylcholine as transmitters in the same cells. These transmitters, however, seem to be used by extrinsic neurons innervating the mushroom body. It is of interest to determine the neurotransmitters of the Kenyon cells lacking sNPF and also to screen for an additional classical transmitter in the sNPF-expressing cells. 6. CONCLUSIONS AND FUTURE PERSPECTIVES In paper I we found that the ionotropic and metabotropic GABA receptors have similar distribution in the Drosophila CNS apart from a few regions. Parts of the mushroom bodies and ellipsoid body contain only GABAAR. A fraction of the clock neurons only contain GABABR as well as some neurons in the optic lobe and in the olfactory receptor neurons (Root et al., 2008) where the receptor presumably acts presynaptically. In paper II we found another location where the two receptor types differ, namely in the median neurosecretory cells in the pars intercerebralis where GABABRs are located on the insulin producing cells and GABAARs are located on adjacent neurons in the same cell cluster. We also found that GABA is regulating production and/or release of insulin-like peptides via GABABRs and not via GABAARs.

The functional roles for the metabotropic GABABR need to be further investigated. Drug addiction is a relevant condition to study. In mammals, GABABR agonists can reduce cravings for different drugs, e.g. cocaine, heroine, alcohol and nicotine. This occurs in the brain region involved in reward and reinforcement, where GABABRs are blocking the increase in dopamine release that otherwise would be induced by the drug [see (Bettler et al., 2003)]. A role for GABABRs in alcohol tolerance and nicotine-resistance in Drosophila has been suggested (Dzitoyeva et al., 2003; Passador-Gurgel et al., 2007), but the mechanisms remain unclear. Other addictive drugs would also be interesting to test in Drosophila. The functional roles of GABABRs in locomotion behaviors would be of interest since the receptor has been displayed in the central body complex. The relevance of GABABRs in circadian rhythm regulation also needs further investigation.

Paper III and IV focus on the locations of short neuropeptide F and its receptor. We found a widespread distribution of sNPF in many interneurons and in a subset of antennal olfactory sensory neurons, suggesting multiple roles as cotransmitters or neuromodulators. The receptor distribution in the larvae indicates roles in feeding and olfaction as we found the receptor in some cells of the midgut, in some neurosecretory cells and in the larval antennal lobe.

The finding of sNPFs in the Kenyon cells of the mushroom bodies is exciting, since it is the first and so far only neurotransmitter found in these cells. We believe that sNPF acts as a cotransmitter in these cells, and therefore it would be of interest to determine the classical or primary transmitter used by the Kenyon cells. In addition, we only found sNPF expression in a subset of the Kenyon cells. The transmitter utilized by the remaining cells is still unknown. It would also be of interest to study the role of sNPF in olfactory associative learning.

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The sNPFs have been shown to stimulate ovarian development in locusts (Cerstiaens et al., 1999) and therefore it would be of interest to investigate if this is also the case in Drosophila and investigate the roles of the peptide in reproduction.

Based on distributional studies of the peptide, the next step would be to study the function of sNPF in locomotor behaviors, hormonal release and in the circadian clock. The role of sNPF in locomotor behaviors has been investigated previously (Kahsai et al., 2010), but more experiments are needed. Another aspect is to further study the modulation of chemosensory inputs to the antennal lobe. Both sNPF and sNPFR have been found in the adult OSNs (Carlsson et al., 2010; Root et al., 2011) and the levels of sNPFR were increased in starved flies due to decreased levels of insulin in the circulation (Root et al., 2011). However, we did not find sNPFR in the axons of the larval OSNs. Perhaps this is because the larvae are continuous feeders and have constant high levels of circulating insulin. Therefore it would be interesting to starve larvae to see if sNPFR is upregulated in the OSNs. It is also possible that larvae do not possess sNPFRs in the OSNs and that the expression starts at a later stage during development. This is our suggestion for the lack of sNPFR in the larval IPCs, since the receptor most likely is present in the adult IPCs (Lee et al., 2009). We only studied third instar larvae, but perhaps a mapping throughout all development stages is necessary.

We have started to understand a little more about GABA and sNPF signalling in the brain of Drosophila, but further experiments on the role of the two substances are needed to unravel further functions of these substances and mechanisms of signalling.

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8. ACKNOWLEDGEMENT/TACK Finally I am here! There are so many people that made this work possible and that I would like to thank! First of all I would like to thank my supervisor Dick Nässel. You helped me to keep my motivation during these years, with your incredible ability to always say the right things at the right moment. Thank you for always keeping your door open, for your advices and expertise. My co-supervisor Åsa Winther, you have been a great help in the lab. I would not have been able to cross flies if it wasn’t for you. You have also been a good friend outside the lab. Thank you for trying to keep me fit by dragging me to the gym. My roomies deserve a special thanks. Jeannette ”Miss Google” Söderberg, thank you for all the laughters and tears that we have shared and for all the good talks about everything and nothing. Your curiosity and interest in other people is admirable and I learned a lot about myself thanks to you. Jiangnan Lou, thank you for interesting discussions and for teaching me about proper chinese food. My former roomies: Yasutaka Hamasaka, thank you for teaching me about immunocytochemistry and Ryan Birse for not giving up on my attempts to make that %&#€%”&# Gal4-line (I am sorry to tell you that I gave up on it when you left). Kerstin Mehnert and Ellan Gustafsson, I really miss you guys! I would also like to thank people at the department for making the environment pleasant to be and work in. Agata Kolodziejczyk, you are such a lovable and crazy person, thank you for cheering me up. Always! Neval Kapan, thank you for good cooperation and interesting conversations. Micke Carlsson, sorry that I didn’t always understand your skånska and forced you to speak stockholmska. Kristin Eriksson, you are such a ”frisk fläkt” at the department, keep up the good work. Bertil Borg, thank you for reading and commenting manuscripts and for introducing me to the weird world of Albert Engström. Count me in for the yearly glögg in the kitchen. Erik Hoffmann, you do a good job down in the basement, but we miss you upstairs. Heinrich Dircksen, thank you for valuable discussions and for always repairing broken apparatus in the lab. Yi Ta Shao, thank you for the sake and for the nice party after your lic. Good luck with your thesis! Rafael Cantera, you made the lectures about neurology interesting and perhaps I would not have continued in the field if it wasn’t for you. Minna Miettinen, thank you for always helping out with a smile on your face. Anette Lorents, Berit Strand and Siw Gustafsson, tack för trevliga diskussioner och korsordslösningar i lunchrummet och för all hjälp med allt runtomkring forskningen. Erland Dannerlid, tack för dina små teckningar, för dina underfundiga kommentarer och för hjälpen till rummet på Tjärnö (du vet vad jag menar). I would also like to thank former people at the department: Lily “Miss Ouri” Kahsai, you are always so nice and I wish you all the best! Helena Johard, my travel buddy. Whatever happens in Zakopane stays in Zakopane... Farideh Rezaei, thanks a lot for taking such good care of me when I was new and lost in the lab. Johannes ”Bert” Strauss, you somehow managed to make the lab more interesting with your fun and quirky ideas. Ana Beramendi and Helena Elofsson, ni var i slutfasen när jag började, men välkomnade mig och lärde mig mycket om hur det är att vara doktorand.

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To all other colleagues at Zootis, present and former, thanks for helping out in various ways. Ulf, thanks for all your help with the computers. Lus-Lina och Fjärils-Lina, tack för att jag fick bli Flug-Lina. ;) Jessica, Aleksandra, Marina, Marianne, Helena, Anna, Magne and everyone involved in doktorandrådet for standing up for our rights. Maria Lissåker, I forgive you for calling me juvenile, and I am sorry about your shoes... Sören Nylin, thank you for always listening to us PhD students and taking us seriously. “Biologbrudarna”, Ida von Scheele, Åsa Andersson, Yvonne Sundström, Agnes Karlson, Julia Borg och Anna Davidsson, tack för att ni gjorde pluggåren sååå mycket roligare och för att ni fortfarande gör åren så mycket roligare. There is also a world outside the walls of the university. I would like to thank all my family and friends for always being there for me. Min extrafamilj, Birgitta, Chrille och Peter, tack för att ni har låtit mig bli en del av er härliga familj! Tack till alla i min egen familj. Mormor och Teta, ni är underbara! Sara ”Kajan”, ”sis”, ”Inga”, du är den bästa syster och vän som går att få! Tack för att du alltid lyssnar, stöttar och finns där. Love u!! Mamma och pappa, tack för att ni alltid har stöttat och trott på mig. Ni ställer upp i vått och torrt, och för det är jag evigt tacksam! Tack för att ni finns! Bud, dig har jag så mycket att tacka för. Du har fått utstå mycket under dessa år, men du har stått vid min sida och väglett mig i rätt riktning. Jag älskar dig! Parp. Slutligen vill jag tacka min älskade dotter Lea, min lilla solstråle, du är en sån häftig liten människa. Du har öppnat mina ögon och fått mig att uppskatta det enkla och vackra i livet.