Neurotransmitters in the visual system of the fruitfly, Drosophila melanogaster

Agata Kołodziejczyk

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ABSTRACT

Nearly all organisms on earth are sensitive to light. During evolution they

evolved biological clocks, which synchronize their activities to the environmental daily light rhythms. Light adaptation in the visual system is provided by fast neurotransmission and slower modulatory pathways. Fast neurotransmission is mostly driven by ion channel receptors and slow neuromodulation is provided by G-protein coupled receptors (GPCRs). The lamina is the first synaptic region (neuropil) localized beneath the fly compound eye, and is a well-studied part of the visual system in insects. Findings of circadian structural changes in synaptic contacts and other neuronal components in this region (Pyza 1998), have inspired us to analyze neurotransmitters and their receptors in the lamina of Drosophila melanogaster.

In paper I we used immunocytochemical methods combined with several transgenic Gal4 fly lines to reveal components of GABA, glutamate and acetylcholine neurotransmission in the lamina. We show evidence for acetylcholine signaling in monopolar neurons, including L4 neurons, and in boutons of the newly discovered cha-Tan neurons. By using a marker to the vesicular glutamate transporter (vGluT) we revealed staining in processes of amacrine cells surrounding cartridges in the lamina, and weak expression in large monopolar interneurons. The centrifugal cells, C2 and C3, express GABA and its metabotropic receptor, GABABR2. This receptor was also visualised on boutons of the above-mentioned cha-Tan neurons. GFP expression driven in rdl-Gal4 flies revealed monopolar L4 neurons and a new type of tangential neurons: rdl-Tan cells.

Paper II is a continuation of the neurotransmitter studies in the Drosophila optic lobe. We tried to combine the previous results with an analysis of a putative biological clock neurotransmitter: serotonin. Using markers for serotonin and its receptors, as well as clock and glial cells, we describe relations between lamina cells expressing serotonin and serotonin receptors. We found that a major type of lamina glial cells, epithelial glia, express a clock gene (timeless), a metabolic enzyme (Ebony) and the two types of serotonin receptors. Thus serotonergic neurons derived from the central brain and with processes adjacent to the lamina, may act in a paracrine fashion on epithelial glia cells to modulate lamina neuron activities.

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PAPERS

I. Kolodziejczyk A. and Nässel D.R. Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system.

II. Kolodziejczyk A. and Nässel D.R. Serotonin and two of its receptors in

the optic lobe and clock system of Drosophila melanogaster.

Additional paper (not included in this thesis): Enell L. Hamasaka Y. Kolodziejczyk A and Nässel D.R. 2007. GABA signaling components in Drosophila: Immunocytochemical localization of GABAB receptors in relation to the GABAA receptor subunit RDL and a vesicular GABA transporter. J Comp Neurol (in press)

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CONTENTS

ABSTRACT ……………………………………………………………………..3

PAPERS......……………………………………………………………………..4

INTRODUCTION ………………………………………………………………..7

The optic lobe of Drosophila melanogaster ….……………………….7

Retina …………..………………………………………...7

Lamina …………..………………………………………..8

Medulla………….……………………………………….10

Lobula……………………………………………………10

Lobula plate ……………………………………………..11

Neurotransmitters in the visual system.…………………………....11

Fast neural responses…………………………………….11

Modulatory responses …………………………………...11

The endogenous biological clock …………………………...............13

Peripheral circadian oscillators in the visual system ……13

Central pacemaker function in the visual system...……...14

AIMS OF THIS THESIS………………………………………………………….14

MATERIALS AND METHODS ………………………………………………….15

PAPER I ……………………………………………………………………….16

PAPER II……………………………………………………………………….18

CONCLUSIONS AND FUTURE PERSPECTIVES…………………………………..18

ACh, Glu, GABA and 5-HT circuits in the lamina….………………..18

Neurotransmitter circuits in relation to the peripheral clock………….18

Neurotransmitter circuits in relation to the central pacemaker………..19

AKNOWLEDGEMENTS………………………………………………………….19

BIBLIOGRAPHY………………………………………………………………...20

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INTRODUCTION The visual system of insects registers light pulses and transduces them into neuronal action potentials. Electrochemical signals are amplified and filtered via several interneuronal layers and get transmitted to higher visual centers in the brain, where complex signal processing occurs. The visual system continuously adjusts to exogenic light stimuli and the endogenous biological clock. Neuronal modulation enabling adjustments of visual sensitivity is performed by different kinds of neurotransmitters, neuromodulators and their receptors. The optic lobe of Drosophila melanogaster

The complex network of neurons incorporated in the visual system has been extensively studied using Golgi impregnation (pioneering work of Cajal and Sánchez, 1915). Visual neurons have been described for Dipteran fly species since 1970s (e.g. Strausfeld, 1970; Strausfeld and Nässel, 1980; Fischbach and Dittrich, 1989). The optic lobe is divided into four neuropil layers (Fig.1). Under the retina, the following visual centers are distinguished: lamina, medulla, lobula and lobula plate. Throughout the whole optic lobe two major types of cells, columnar and tangential neurons, are ordered in a characteristic mosaic. The regular anatomical structure of the insect optic lobe has been first shown in Musca domestica (Fig.2).

Fig.1. General structure of the optic lobe in Drosophila melanogaster (modified from Fischbach and Dittrich, 1989). The retina contains 8 types of photoreceptors (R1-R8). R1-R6 send the light input to the large monopolars (L) in the lamina (La), while R7-R8 proceed to the medulla (Me). The proximal layers of the optic lobe consist of the lobula (Lo) and the lobula plate (Lp). The retina

Each compound eye of the fruitfly contains more than 700 hexagonal units called ommatidia (about 780 in females and about 730 in males; Wolff and Ready, 1993). Each ommatidium consists of a cuticular lens and a crystalline cone. Under the crystalline cone there are eight retinular neurons (photoreceptors) with photosensitive rhabdomers composed of densely packed microvilli, where phototransduction processes take place. Axons of the six photoreceptors from each ommatidium exit the

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retina and terminate in units named cartridges in the peripheral neuropile layer, lamina. Two other photoreceptors, R7 and R8, have their terminals in the next neuropil layer – the medulla (Trujillo-Cenóz 1965, Boschek, 1971) The photoreceptors express 6 different opsin genes (Salcedo et al. 1999): Rh1 -Rh6. Retinular cells R1-R6 express the major visual pigment, Rhodopsin1 (Rh1), which display spectral sensitivity with its peak in blue (O’Tousa et al. 1985). R1-R6s are the most efficient at low intensities of light (Heisenberg and Buchner 1977, Fischbach 1979). Two other kinds of photoreceptors, R7 and R8 dominate in phototactic behavior at higher light intensities. The majority of the R7s express Rh4 and in 30% Rh3 (Fortini and Rubin 1990). Both R7 and R8 have their spectral sensitivity in the UV range. R8s express Rh5 or Rh6 and absorb best blue or green-yellow light respectively (Bausenwein et al. 1992, Chou et al. 1996, 1999, Salcedo et al. 1999). Rh2 – a violet absorbing rhodopsine, is expressed in simple eyes (ocelli) located on the vertex of the head (Cowman et al. 1986). Better visual resolution in the compound eye is achieved by pigmented cells (also called screening pigment), which optically isolate each ommatidium from its neighbours. Lamina The most extensively studied part of the visual system, the lamina, consists of about 6000 neurons in Drosophila. In addition to the local lamina neurons (amacrine cells), there are retinal centripetal and centrifugal neurons connecting the lamina to the medulla (Cajal and Sanchez 1915, Strausfeld 1971). Most of the neurons compose ordered units called optical cartridges (Trujillo-Cenóz 1965). A single cartridge contains 12 different kinds of neurons listed in Table 1 and presented in Fig.2 and Fig.3. Neurons building a single cartridge have highly ordered spatial topology visualized in Fig.4. Additionally, there are in lamina wide field tangential cells (in Musca domestica: Tan1, Tan2 and LBO5HT – large bilateral optic lobe 5-HT immunoreactive neuron), which are neurons connecting the lamina to the medulla or protocerebrum. The final group of lamina cells are six types of glial cells. Some glial cells express clock genes and regulate circadian rhythms in the peripheral visual system, others are involved in several house-keeping functions such as neurotransmitter metabolism or nourishment of lamina neurons (Eule et al. 1997).

Cell type Symbol Localization Function Photoreceptors R1-R6

R7-R8

All of them are localized in the retina; R1-R6 terminate in lamina cartridges, R7-R8 terminate in medulla, their axons pass through the centers of cartridges

Phototransduction, depolarize under light pulses

Monopolar cells L1-L5 Located in lamina, terminate in medulla; L1 and L2 receive synapses from photoreceptors, their axons with extensive branching are located in the middle of cartridges

Amplify and filter signals from photoreceptors, regulate photoreceptor activity, hyperpolarize under light pulse, send output to the medulla

Centrifugal cells C2-C3 Neurons from medulla, terminate in the distal part of the lamina

Inhibitory neurons

Amacrines Am Local lamina neurons Wide-field regulation T-shape cell 1 T1 Local lamina neurons Send output to the medulla

Table 1. General characteristics of the lamina neurons that build a single cartridge.

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Fig.2. Several types of neurons in lamina of Musca domestica visualized by Golgi impregnation (modified from Strausfeld and Nässel, 1980). This can be compared to similar neurons in Drosophila (Fig.3).

Fig.3. Schematic composition of the lamina neurons in Drosophila melanogaster revealed by Golgi impregnation (modified from Meinertzhagen and O’Neil, 1991). Wide field amacrine neuron (Am) and wide field tangential neuron immunoreactive to serotonin (5-HT-IR Tan) are visualized on the left side. Several small field neurons found in the lamina and their projections to the medulla are displayed on the right side. 8 types of photoreceptors (R1-R8), 5 types of the large monopolar neurons (L1-L5), 2 types of centrifugal neurons (C2-C3), one T-shaped centripetal neuron T1 and one wide field tangential neuron (Tan).

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Fig.4. A spatial organization of the neurons within the cartridge (from Meinertzhagen and O’Neil, 1991). On the left side: synaptic contacts between selected lamina neurons are revealed and enlarged in boxes in the middle. A a tetrad synapse, B a feedback synapse from α-processes of the amacrine neurons, C a feedback synapse from L2 neuron, D a C2 or C3 cells input to the monopolar neurons. On the right side: an arrangement of the lamina neurons in cross-section of the lamina cartridge. The orientation of the cartridge model to the head of the fly is presented by the posterior (P) and ventral (V) directions, the orientation to the cartridge array is labeled by axes x and y. Profiles of the neurons are not in scale. Medulla Neurons of the lamina send axons into the second neuropil layer, the medulla, through the first optic chiasma. The medulla is topologically subdivided into 10 layers formed by multiple stratified sets of processes from various neurons. In 1989, Fischbach and Dittrich proposed that medulla layers reflect a structural division of organized functional pathways. It has been claimed (Strausfeld and Lee 1991), that in the fly there is a division between parallel pathways subserving colour-blind movement-sensitive and colour-processing visual processing, similar to the primates division for parvo- and magnocellular pathways. Lobula The third neuropil, the lobula, is subdivided into 6 layers (Lo1-Lo6). However little is known about functional pathways in the lobula. Anatomical studies (Strausfeld 1989), have constituted 3 classes of neurons: (1) Lcn cells – isomorphic lobula columnar neurons projecting to the descending neurons in the brain (Fischbach and Dittrich 1989); (2) Neurons of the dorsal eye zone (marginal zone) – probably

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involved in the analysis of the polarized light (Fortini and Rubin 1990); (3) Several types of the wide field neurons (Fischbach and Dittrich 1989). Lobula plate The lobula plate contains wide field motion-sensitive neurons and plays an important role in controlling compensatory motions of both the head and body in flight and during walking in response to turning stimuli (Hausen and Egelhaaf 1989). Using different technics like genetic dissection (Bausenwein et al. 1986), laser ablation (Geiger and Nässel 1982) and electrophysiological recordings (Gauck and Borst 1999), 4 directionally movement-sensitive layers have been identified in the lobula plate. Neurotransmitters in the visual system

Functioning of the visual system is based on two main types of neuronal connections: electrical and chemical synapses. In addition, non-synaptic, paracrine transmission is common. Fast neurotransmission occurs via gap junctions and synapses with ionotropic receptors. Slow modulation of neurons and presynaptic modulation relies on pre- or postsynaptically located metabotropic G protein coupled receptors. Most visual processes are fast events relying on fast neurotransmission. However phenomena such as light adaptation and sensitivity levels are set by slow modulatory actions. The visual system is known to use several kinds of neurotransmitters, neuromodulators and their receptors. In the CNS in general there may be simple molecules (NO, CO), amino acids (glutamate, glycine, aspartate, gamma-butyric-acid – GABA), amines and their derivatives (dopamine, serotonin, octopamine, histamine, acetylcholine), purines and their derivatives (adenosine, ATP), lipid derivatives (arachidonic acid) and a big group of protein-like transmitters called neuropeptides (such as neuropeptide F, proctolin, tachykinins, pigment dispersing factor,PDF, and many others). Fast neural responses The fastest neurotransmission between neurons occurs in very thin clefts (2-4nm), called electric synapses or gap junctions. The depolarization is directly transferred from one neuron to another. In that way the neuronal signal is transported with the fastest speed – about 120 m/s! In the fly, lamina symmetric gap junctions are extensively present in R1-R6 axons (Ribi 1978), interneurons and glial cells (Saint Marie and Carlson 1985). Another kind of fast neurotransmission occurs in thicker clefts (30-50nm) called chemical synapses. The neuronal signal is transformed to a chemical one acting on a receptor that induces a cellular response. This process is slower because it requires release of neurotransmitters and activation of their receptors. The structural organization of a chemical synapses is much more complex, but functionally it is more plastic than the gap junction, because it can transmit different types of neuronal responses. For the fast neurotransmission, ionotropic receptors, which are ligand gated ion channels, are required. In Table 2, well studied ionotropic receptors in Drosophila melanogaster are listed.

Modulatory responses Metabotropic receptors (GPCRs) play a crucial role in neuronal signal modulation. They form seven helical transmembrane regions. After ligand binding

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Receptor Chemical structure Function Localization References GABAAR (gamma-amino-butyric acid receptor A)

Pentameric, 3 subunits known: -RDL (resistance to dieldrine) -GRD (GABA- and glycine-like receptor) -LCCH3 (ligand-gated chloride channel homologue3) different combinations of these subunits

Inhibitory receptor, permeable to chloride ions, hyperpolarize neuron

Central Nervous System (CNS), postsynaptic

Hosie et al. 1997

GluCl (L-glutamate-gated chloride channel)

Tetrameric, homomeric and heteromeric,

Similar to the GABAAR but activated by glutamate

CNS and muscles, postsynaptic

Raymond et al. 2005

idmGluR (ionotropic Dros. Mel. Glutamate receptor)

Tetrameric, 5 subunits are known: DGluRII A-E

Most of the fast excitatory transmission, Permeable to calcium, sodium and potassium cations

CNS and neuromuscular junctions (NMJ), postsynaptic

Featherstone et al. 2005

NMDAR (N-methyl D-aspartate receptor)

Heterotetrameric 5 subunits: NR1(glycine or D-serine binding domain) 4 kinds of NR2 subunits (A-D) (Zn2+ and glutamate or aspartate binding domains)

Open only when the neuron is depolarized. Then Mg2+ ions unblock the channel and Na2+, Ca2+ and K+ pass through

CNS, postsynaptic

Ultsch et al.1993

HisCl1 HisCl2 (histamine-gated chloride channel)

Multimeric, homomeric and heteromeric

Permeable to chloride ions, Hyperpolarize neuron

Tetraedric synapses in the lamina, postsynaptic

Zheng et al. 2002; Hardie 1987

nAChR (nicotinic acetylcholine receptor)

Pentameric 2xα (ligand binding domain),β,γ,δ

Excitatory, Permeable to Na+ and K+ ions

CNS, postsynaptic

Sawruk et al. 1990; Satelle et al. 2006

Table 2. The main neurotransmitter receptors involved in fast neuronal signaling in Drosophila.

Receptor Chemical structure

Function Localization References

mAChR Muscarinic acetylcholine receptor

heptahelical Activates phosopholipase C to produce inositol 1,4,5-triphosphate

CNS, presynaptic

Millar et al. 1995; Honda et al. 2007

DmGluRA (Drosophila metabotropic glutamate receptor A)

Homodimeric (2 x heptahelical)

Inhibits Ca2+ channels and adenylate cyclase Regulates K+channels

CNS and NMJ, pre- and postsynaptic

Parmentier et al. 1996

GABABR Three subtypes: R1-R3

Heterodimeric (2 x heptahelical)

Slow inhibition due to the activation of potassium channels in the postsynaptic region Voltage-dependent inhibition of the calcium channels

CNS, pre- and postsynaptic

Bettler et al. 2004

d5-HT1B (Drosophila serotonergic receptor 1B)

heptahelical

Circadian processes and sleep

CNS, clock neurons, glia

Yuan et al. 2005

d5-HT2 (Drosophila serotonergic receptor 2)

heptahelical Higher order complex behaviors, possibly in clock control

CNS, glia

Nichols 2006

PDFR Groom-of-PDF (gop)

heptahelical Required for PDF neuronal effects on circadian phase

CNS Lear et al. 2005, Mertens 2005

Table 3. Characteristics of some well studied receptors involved in neuromodulation in the optic lobe of the fruitfly.

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they activate intracellular G-proteins which are responsible for several types of reactions, for example: modulation of specific ion channels or activation of intracellular enzymatic pathways. Table 3 presents characteristics for the main metabotropic receptors in Drosophila that may be expressed in the visual system. The endogenous biological clock

The biological clock has developed during evolution as the response to synchronize behavioral, metabolic and cellular rhythms of the organism with daily environmental changes. Drosophila posesses central pacemaker neurons in the brain (Fig.5), and autonomous and semiautonomous peripheral pacemakers in different parts of the body and the CNS. The latter are found for example in different sets of glial cells, antennal chemoreceptors and Malphigian tubules (Hall 1995, Stanewsky 2003). The biological clock is reset by environmental factors (mainly by light), which entrain the pacemaker neurons through specific neuronal input pathways. Clock cells possess 9 major clock genes (period, timeless, clock, cycle, doubletime, shaggy, vrille, par domain protein 1ε, casein kinase 2). Their cyclic expression is based on interlocking transcriptional and translational feedback loops and generate oscillations that drive output pathways and generate rhythmic behavior such as locomotor activity, feeding, mating, etc. (rewieved in Allada 2003).

Fig.5. Clock neurons in the adult brain of the Drosophila melanogaster (from Helfrich-Förster 2002). The main pacemaker neurons, large l-LNVs and small s-LNVs are localized in the accessory medulla (aMe) and project to the medulla, dorsal part of the brain and to the second hemisphere. Other clusters of clock neurons, DNs, PLs and LNds are localized in the dorsal region of the brain. Photoreceptors are labeled (R1-R6), the Hofbauer-Buchner eyelet localized close to the lamina (asterisk) is involved in circadian light entrainment. . Peripheral circadian oscillators in the visual system

In the visual system of the fly there are two kinds of peripheral clock-cells: the retinal photoreceptors and glia (especially in the lamina). Neurons in the lamina exhibit oscillations controlled by biological clock either by connections from the peripheral clock or via inputs from the central clock. Circadian rhythms are observed in a number of synapses (Pyza and Meinertzhagen 1993) and the vertical migration of screening pigment in the photoreceptor terminals (Pyza and Meinertzhagen 1997). Two kinds of monopolar cells, L1 and L2, undergo circadian modulation by daily

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changes in diameter of their axons (Pyza and Meinertzhagen 1995). Axons of these interneurons swell during the day and shrink by night. Other studies revealed morphological changes driven by the clock in nuclei and dendritic spines of the L2s (Gorska-Andrzejak et al. 2005); these are larger during the day. It is speculated that circadian regulation in lamina interneurons is provided by epithelial glial cells (Siwicki et al. 1988), which exhibit circadian changes in volume opposite to that of monopolars: they shrink during the day and enlarge by night (Pyza and Meinertzhagen 1995, 1996). Interestingly, injections of neurotransmitter and neuromodulator into the lamina region of Musca domestica induce different effects on monopolar cells. Glutamate and GABA decrease diameters of the monopolar neurons, while histamine and pigment dispersing factor (PDF) increase them. More details are presented in the table below from Pyza and Meinertzhagen (1996).

Central pacemaker function in the visual system

The circuits underlying modulatory effects on the lamina neurons from the main pacemaker neurons are not well studied. It has been shown that the main clock cells, LNv’s (ventral lateral neurons), are localized in the lateral brain close to the optic lobe (in a region called accessory medulla) and exhibit circadian morphological changes similar to monopolars in the lamina (Pyza and Meinertzhagen 1997b). LNv’s secrete the neuropeptide PDF, which is the only known clock output factor. PDF is responsible for modulation of rhythms in locomotor activity and for synchronizing endogenous rhythms in the visual system (PDF neurons send processes to the medulla and the contralateral clock neurons) (Helfrich-Förster 2007). Another transmitter, serotonin, is suspected to be involved in circadian modulation of the visual system (Pyza and Meinertzhagen 1996) and may be part of an output pathway from the central clock. Wide field serotonin neurons send processes to the vicinity of the lamina, but the targets of this neurotransmitter are not yet known.

AIMS OF THIS THESIS

Lack of knowledge about neurotransmitter circuits in the visual system of Drosophila melanogaster has inspired us to localize neurotransmitters and some of their receptors in the lamina of this animal. We were interested in relations of acetylcholine, glutamate, GABA and serotonin to circuits in the lamina and also relations of those neurotransmitters to peripheral clock cells (glial cells). Ultimately we want to unravel the signaling pathways from the central clock to the lamina neurons since some of these neurons are known to be under circadian control.

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MATERIALS AND METHODS

Our studies were based on immunocytochemical methods combined with Gal4-UAS technique to drive green fluorescent protein (GFP) or LacZ (encoding β-galactosidase) in specific neurons of experimental flies. In each experiment we used minimum 10 fly brains sectioned in frontal and horizontal planes. All flies were raised in 25°C and fed on standard Drosophila cornmeal. Fly stocks and antibodies used in the experiments together with immunocytochemical comments are listed in the table below (Table 5). Flies were decapitated in PBS-Tx (0.01M phosphate buffer saline with 0.5% TritonX-100, pH 7.4) and proboscises were removed to facilitate penetration of fixative. The tissues were usually fixed in 4%PFA or Zamboni fixative 2h on ice. In case of α-GABA, α-GAD and α-RDL staining, tissues were fixed 30 min in ice-cold Bouin. The fixed tissues were washed in 0.1M phosphate buffer (PB), pH 7.4 and incubated with 20% sucrose in 0.1M PB over night in 4°C. Cryostate sections 20-50µm thick were cut on Leitz Cryostat at -23°C. After washing in PBS-Tx the sections were incubated over night with primary antibodies at 4°C. The specimens were washed in PBS-Tx and incubated with fluorescent secondary antisera either over night in 4°C or 2h at RT. After washing in 0.01M PBS and 0.1M PB, the tissues were mounted in 20% glycerol in 0.1M PB. Specimens were imaged using 63x objective and 2-4x electronic zoom in a Zeiss LSM510 laser confocal microscope processed with its software. Confocal images were edited in Adobe Photoshop CS3 Extended version 10.0.

Marker for: Fly stock Antibody Additional comments Photoreceptors R1-R6 - α-Discs large 4%PFA 2h on ice L2 monopolar cells 21D-Gal4 - Crossed with UAS-GFP C3 neurons 5-6-8/CyO;TM2/TM6B-Gal4 - Crossed with UAS-GFP,

GFP signal increased by α-GFP antisera

Glial cells - α-Ebony 4%PFA 2h on ice Clock cells Tim-Gal4

Pdf-Gal4 α-PDH (pigment

dispersing hormone) Crossed with UAS-GFP, GFP signal

increased by α-GFP antisera

Vesicular acetylcholine transporter

- α-vAChT 4%PFA 2h on ice

Choline acetyltransferase

Cha-Gal4 α-ChaT 4%PFA 2h on ice

Vesicular glutamate transporter

OK371-Gal4 α-vGluT (4 types) Crossed with UAS-GFP, 4%PFA 2h on ice

Metabotropic glutamate receptor

- α-DmGluRA Zamboni 2h on ice

Ionotropic glutamate receptor

- NMDA1 Bouin 30min on ice

Gamma-aminobutyric acid

- α-GABA Bouin 30min on ice Zamboni 2h on ice

Glutamic acid decarboxylase

- α-GAD Bouin 30min on ice, Zamboni 2h on ice

GABA metabotropic receptor

- α-GABABR2 4%PFA 2h on ice

GABA ionotropic receptor

Rdl-Gal4 α-RDL 4%PFA 2h on ice Bouin 30min on ice Crossed with UAS-GFP, GFP signal

increased by α-GFP antisera

Serotonin - α-5-HT 4%PFA 2h on ice Serotonin receptor 1B w;;5HT1B-Gal4/TM3.SB - Crossed with UAS-GFP, GFP signal

increased by α-GFP antisera Serotonin receptor 2 5HT2DroGal4;UASLacSZ - α-β-Gal antisera were used to

visualize expression of the receptor

Table.5. Fly strains and antibodies used to reveal neurotransmitters, their receptors and cells in the lamina.

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PAPER I Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system The lamina of Drosophila melanogaster is a very well investigated part of the visual system in this insect, however, neurotransmitter signaling in this synaptic neuropil is still enigmatic, even with respect to the classical transmitters. Here we used immunocytochemical techniques and genetically modified flies to reveal the distribution of acetylcholine, glutamate and GABA signaling components in the most peripheral neuropil of the fly optic lobe, the lamina. Two new types of tangential lamina neurons were discovered: cha-Tan and rdl-Tan neurons. We provide evidence that L4 monopolar cells are cholinergic neurons and suggest, that acetylcholine signaling is utilized by two tangential pathways: distally in lamina via cha-Tan processes, and proximally via L4’s collateral branching that couples neighbouring cartridges together. Glutamate signaling was visualized by using vGluT (vesicular glutamate transporter) markers. A strong signal was obtained in alpha-processes of the amacrine cells surrounding cartridges. We were not able to confirm glutamate neurotransmission in L1 and L2 cells using specific vGluT antibodies, but this can be caused by too low levels of neurotransmitter in vesicles in this cells. Previous studies showed that centrifugal C2 and C3 neurons contain GABA. We presented new data, showing that these GABA-ergic neurons express metabotropic GABA receptors in their terminations but not ionotropic ones. Additionally, we detected metabotropic GABABR2 receptors in the cha-Tan neurons. Our results are schematically summarized in the diagrams below (Fig.6).

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Fig.6. Diagrams that summarize the lamina neurons visualised in our studies. A The general diagram with schematic arrangement of the neurons of interest in the present study. photoreceptors (R) are localized in retina (Re). In the lamina (La), monopolar interneurons L2, L4, and amacrine (Am), tangential (Cha-Tan, rdl-Tan) and centrifugal (C2, C3) neurons are shown. B Coloured diagram showing neurons possibly involved in acetylcholine signaling in the lamina: strong expression of the vAChT was observed in L4s collaterals and in cha-Tan neurons. Weak staining with the α-Cha antibodies in L2 neurons was also detected. C Possibly glutamatergic neurons in the lamina: amacrines and L2s. D GABA-ergic neurons, C2 and C3 have GABABR2 autoreceptors on the presynaptic side, while cha-Tan cells seem to respond to GABA by expressing GABABR2 receptor on the postsynaptic side of their boutons (GABABRs expression labelled by black dots).

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PAPER II Serotonin and two of its receptors in the optic lobe and clock system of Drosophila melanogaster Circadian changes described in the morphology of monopolar neurons and glial cells in the peripheral neuropil of the fruitfly inspired us to study the distribution of serotonin signaling components. This monoamine is suggested to be one of the main modulatory factors in the lamina that may serve as an output from the central circadian clock. Using immunocytochemical markers for serotonin and the epithelial glial cells, together with genetically modified flies with markers for two types of 5-HT receptors: 5HT1B and 5HT2, we visualized part of the serotonin circuitry in the optic lobe of the fruitfly. We found that both serotonin receptors are expressed in epithelial glia, together with the clock gene timeless. This suggests that clock-derived modulation in the lamina may be mediated by serotonin released in a paracrine fashion, and that the primary targets are glial cells. 5HT1B is weakly expressed in PDF-containing LNvs, what suggests that the main pacemakers may also be regulated by serotonergic neurons. CONCLUSIONS AND FUTURE PERSPECTIVES ACh, Glu, GABA and 5-HT circuits in the lamina

We found that acetylcholine may be one of the major neurotransmitters in the lamina. Markers for cholinergic signaling are located in cell bodies of the main columnar lamina neurons, the monopolar cells, and in two tangential networks: distally in cha-Tan projections and proximally in collaterals of L4 neurons. Glutamate signaling components were detected in α-processes of the amacrine neurons that surround the cartridges. GABA, a major inhibitory neurotransmitter, is expressed in the centrifugal C2 and C3 neurons. Metabotropic GABAB receptors are expressed by the same neurons and additionally in boutons of cha-Tan neurons. This suggests that GABA-ergic neurons may inhibit cholinergic signaling in tangential processes in the distal part of the lamina. Processes of serotonin-immunoreactive tangential neurons (LBO5HT) do not contact any cells in the peripheral part of the Drosophila visual system. However two kinds of receptors for this neurotransmitter are extensively expressed in the epithelial glia of the lamina, what suggests that serotonin release occurs in paracrine fashion. Neurotransmitter circuits in relation to the peripheral clock The epithelial glia in the lamina express clock genes and is claimed to function as peripheral clock cells. Among the investigated neurotransmitters we found evience for possible action of serotonin on these glial cells. The rest of the neurotransmitters may also interact with the glial cells, but we were not able to visualize any receptors for acetylcholine, glutamate and GABA on glial cells. Antibody staining against nicotinic and muscarinic acetylcholine receptors and improved immunolabeling for glutamatergic receptors is required to better understand the chemical circuitry in the fly lamina and further interactions between neurons and glial cells.

For the future it would be of interest to interfere with serotonin receptor expression in glial cells in the lamina and to analyze the effects on circadian and light-induced changes in lamina circuitry and and locomotor behavior of the flies.

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Neurotransmitter circuits in relation to the central pacemaker The main pacemaker neurons, the LNv’s, release PDF to the distal parts of the medulla but it is not clear, what the targets are for these neurons in the visual system. Mapping the expression of PDF-receptors would bring light on the role of the central clock output in the visual system of the fly. Weak expression of the 5-HTR1B in the main pacemaker cells (LNvs) suggest, that serotonin neurons may regulate activity of the central clock through specific input pathways. These serotonergic inputs, which may be part of a photic pathway, remain to be identified in Drosophila and other flies. It is possible that the large serotonin immunoreactive neurons (LBO5HT) are part of an output from the central pacemakers involved in circadian regulation of the peripheral visual system. One important link in supporting this idea would be to reveal the connections between the PDF-expressing central pacemakers (LNvs) and the LBO5HT neurons. It is possible that these neurons interact with each other in the accessory medulla where both types have branches. Another part of my future studies involves the role of GABA and its metabotropic receptors in the inputs to the central pacemakers, LNvs. We have started behavioral assays on transgenic flies with the GABABR1 and GABABR2 receptors knocked down by cell-specific RNA interference (by Gal4-UAS technique). Our preliminary data show significant changes in locomotor activity of flies with diminished levels of these receptors, especially GABABR1, in the LNv neurons. During constant darkness these experimental flies display strongly reduced circadian rhythmicity compared to controls. Knocking down the GABABR1 expression in all clock neurons resulted in a much weaker effect, and when interfering with GABABR1 in all neurons in the CNS there seemed to be no detectable influence on the circadian activity. Similar results have been shown when interfering with the metabotropic glutamate receptor (Hamasaka et al., 2007). In both cases this may be explained by these metabotropic receptors being located both pre- and postsynaptically and a global knockdown would not be readily detectable.

AKNOWLEDGEMENTS I will write shortly otherwise it would be a long list: thank you Dick, for all time devoted me during the last six months, especially for teaching how to process microscopic images in professional way (combining scientific message of the image together with its natural beauty). Thank you all, who helped me in any way, even by smile or tasty banana. Thank you all flies that sacrificed life for this studies. Thank you Paul for all help, patience and support. And thank you, not-yet-born Baby, for giving me strong motivation and optimism in described above scientific adventure.

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