Role of neurotransmitter GABA in the brain of the Madeira cockroach Rhyparobia maderae with focus on the circadian system Dissertation To obtain the academic degree of Doktor der Naturwissenschaften (Dr. rer. nat.) Presented by: Azar Massah University of Kassel - Department 10 Mathematics and natural sciences Institute of Biology - Department of Animal Physiology Kassel, February 2019 ii Examination Board: Prof. Dr. Monika Stengl Prof. Dr. Charlotte Helfrich-Förster Prof. Dr. Markus Maniak Prof. Dr. Georg Mayer Day of the oral exam: 6th of May 2019 iii Statutory Declaration I hereby declare that I have authored the present dissertation independently and without unauthorized help and not use any aids not specified in this dissertation. All passages that are taken literally or analogously from published or unpublished writings, I have identified as such. No part of this work has been used in any other doctoral or habilitation process. Kassel, February 2019 Azar Massah iv Dedication This thesis is dedicated: To my parents, for their endless love and support and for always respecting my life choices. To my brother and sisters, for their kindness, devotion, encouragement, and invaluable support. v “Education is not the learning of facts, but the training of the mind to think.” Albert Einstein vi Contribution Statements My contributions for each part will be stated clearly according to the “Allgemeine Bestimmungen für Promotionen an der Universität Kassel (AB- PromO) § 8 vom 18.05.16. Parts of this thesis have already been published and figures were reformatted to be consistent with the rest of the thesis. Exact wording is highlighted in blue in this doctoral thesis. Giese M, Gestrich J, Massah A, Peterle J, Wei H & Stengl M (2018) GABA- and serotonin-expressing neurons take part in inhibitory as well as excitatory input pathways to the circadian clock of the Madeira cockroach Rhyparobia maderae. Eur J Neurosci First published: 27 February 2018 DOI: 10.1111/ejn.13863. 3.1. Overview of GABA immunoreactivity in the brain of the Madeira cockroach R. maderae • All immunocytochemistry experiments were performed by the author. • Camera lucida reconstruction of figures 3.1 and 3.6 were performed by the author. • Documentation via light microscope and confocal laser scanning microscopy was performed by the author. • Analysis of the preparations and statistical analysis was performed by the author. • All figure plates were created by the author. • Images in figure 3.5 were taken from preparations of Bernhard Petri. These images were not used in any other publications. • The first version of the manuscript was written by the author; the final version was written together with Prof. Dr. Monika Stengl. 3.2. What is the role of GABA in the circadian system of R. maderae? • All immunocytochemistry experiments were performed by the author. • Documentation via light microscope and confocal laser scanning microscopy was performed by the author. • All figure plates were created by the author except figure 3.13 created by Dr. Achim Werckenthin. vii • The first version of the manuscript was written by the author; the final version was written together with Prof. Dr. Monika Stengl. 3.3. GABA as a possible photic entrainment pathway to the clock. • All immunocytochemistry experiments were performed by the author. • Documentation via light microscope and confocal laser scanning microscopy was performed by the author. • All figure plates were created by the author. • Analysis of the preparations and statistical analysis of figure 3.16 and 3.17 were performed by the author together with Thordis Arnold using ImageJ. • An ELISA system to measure GABA levels in brains of R. maderae was established by the author assisted by Dr. Thomas Schendzielorz. • Collection of samples of all ELISA experiments was performed by the author. • Statistical analysis of the ELISA experiments was performed by the author together with Dr. Thomas Schendzielorz. • The first version of the manuscript was written by the author; the final version was written together with Prof. Dr. Monika Stengl. 3.4. GABA could be involved in coupling pathways between the AMAE. • Backfill experiments in combination with multiple label immunocytochemistry were performed by the author, however, for statistical analysis of data other preparations from Melody Wintrebert (under the author’s guidance 2018) were used. • Documentation via confocal laser scanning microscopy was performed by the author. • All figure plates were created by the author. • The first version of the manuscript was written by the author; the final version was written together with Prof. Dr. Monika Stengl. 3.5. Output pathway of the clock and GABA 3.5.1. Characterization of the neurobiotin backfill from the first TG • All backfill experiments in combination with multiple label immunocytochemistry were performed by Anastasija Gajdasch under the author’s guidance (advanced internship 2018). • Documentation via confocal laser scanning microscopy was performed by the author. • All figure plates were created by the author. viii • For counting the backfilled cells, some of the backfilled-preparations done by Clarisse Leseigneur (under the author’s guidance 2016) were used. • 3D-Amira reconstruction of neurobiotin-labeled fibers and projection as well as neuropil reconstruction performed by the author assisted by Dr. Julia Gestrich. • The first version of the manuscript was written by the author; the final version was written together with Prof. Dr. Monika Stengl. 3.5.3. GABA and the ocelli • All backfill experiments in combination with multiple label immunocytochemistry were performed by Anastasija Gajdasch under the author’s guidance (advanced internship 2018). • Documentation via confocal laser scanning microscopy was performed by the author. • All figure plates were created by the author. • 3D-Amira reconstruction of neurobiotin-labeled fibers and projection, as well as neuropil reconstruction, were performed by the author and assisted by Dr. Julia Gestrich. • The first version of the manuscript was written by the author; the final version was written together with Prof. Dr. Monika Stengl. 3.5.4. GABA and neuroendocrine system of the Madeira cockroach. • All backfill experiments in combination with multiple label immunocytochemistry were performed by the author. • Documentation via confocal laser scanning microscopy was performed by the author. • All figure plates were created by the author. • The first version of the manuscript was written by the author; the final version was written together with Prof. Dr. Monika Stengl. ix Abbreviations a1-2 area 1-2 AFF Anterior fiber fan system AFP Anterior fiber plexus AKH Adipokinetic hormone ALA Accessory lamina ALAs Accessory laminae AMAE Accessory medullae AME Accessory medulla AMMC Antenno-mechanosensory and motor center ANes Anterior neurons AOC Anterior optic commissure AOTU Anterior optic tubercle aPDFME anterior PDFME B0 Maximum binding of antibody respectively tracer in ELISAs BB Blocking buffer BSA Bovine serum albumin BU Bulb C2-C3 - Centrifugal feedback neurons CA Calyxes CA Corpora allata cAMP Cyclic adenosine monophosphate CBL Lower division of the central body CBU Upper division of the central body CC Corpora cardiaca CE Compound eye cGMP Cyclic guanosine monophosphate CK2 Casein kinase 2 subunits CLK Clock CREB cAMP responsive element binding protein CRY Cryptochrome x CT Circadian time CWO Clockwork orange CX Central complex CYC Cycle DAB 3,3-diaminobenzidine tetrahydrochloride DAG Diacylglycerol DBT Doubletime DCV Dense core vesicle DD1 First day under constant condition DD2 Second day under constant conditions DE Deutocerebrum DFVNes Distal-frontoventral neurons DL Dorsal lobe dLA dorsal lamina cells Dm Distal medulla neuron DNs Dorsal neurons dPDFLA dorsal PDFLA DT Distal tract DUM dorsal unpaired median EC50 Half maximal effective concentration EDAC 1-Ethyl-3-(3-dimethylaminopropyl) carbodii-mid EDTA Ethylenediamine tetraacetic acid EGTA Ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′- tetra-acetic acid ELISA Enzyme-linked immunosorbent assay E-oscillator Evening oscillator E-oscillator Evening- oscillator FAB Antigen-binding fragment FaRPs FMRFamide related peptides GA Glutaraldehyde GABA γ-aminobutyric acid GaR Goat anti rabbit GluT Vesicular Glutamate Transporter xi GPCR Guanine nucleotide-binding protein coupled receptor HB Homogenization buffer HRP Horseradish peroxidase IB Incubation buffer ICA Inner layer of the calyces IgG Immunoglobulin G ILP Inferior lateral protocerebrum IP3 Inositol trisphosphate ir Immunoreactive ITP Ion transport peptide JH Juvenile hormone KLH Keyhole limpet hemocyanin L1-6 Lamina monopolar cells LA Lamina LAL Lateral accessory lobe lALT Lateral antennal lobe tract LAS AF Leica application suite – advanced fluorescence LC lateral cluster LD cycle Light/dark cycle LH Lateral horn l-LNvs Large ventral lateral neurons LMS Leucomyosuppressin LN Local neuron LNs Lateral neurons LNds - dorsal lateral neurons LNvs Ventral lateral neurons LO Lobula LOVT Lobula valley tract LPN Posterior-lateral neurons M Medulla layer MALDI-TOF MS matrix-assisted laser desorption/ionization – time of flight mass spectrometry mALT Medial antennal lobe tract MB Mushroom body xii MC Medulla commissural cell ME Medulla MFVNes Medial-frontoventral neurons Mi Medulla intrinsic neuron MIP Myoinhibitory peptide mlALT Mediolateral antennal lobe tract MLF Median fiber fan system MLFT Median fiber fan tract MNes Medial neurons M-oscillator Morning oscillator M-oscillator Morning- oscillator Mt Medulla tangential neuron NCC I Nervous corporis cardiaci I NCC II Nervous corporis cardiaci II NDS Normal donkey serum NGS Normal goat serum NPF Neuropeptide F NSB Non-specific binding O Ocellus OCA Outer layer of the calyces OCH1 First optic chiasm OL Optic lobe OSNs Olfactory sensory neurons p1-3 plexus 1-3 PB Phosphate buffer PDF Pigment-dispersing factor PDFLA PDF Lamina cells PDFME PDF medulla cells PDH Pigment-dispersing hormone PED Pedunculus PEDD Pedunculus divide PER Period PI Pars intercebralis xiii PIP2 Phosphatidylinositol 4,5-bisphosphate PKA Protein Kinase A PL Pars lateralis pLA proximal lamina Pm Proximal medulla neuron PMP Posterior median protocerebrum PN Projection neuron POC Posterior optic commissure POTU Posterior optic tubercle pPDFME Posterior PDFME PR Protocerebrum PRC Phase-response curve R Photoreceptor cell rMEd Dorsal soma rind of the medulla rMEv Ventral soma rind of the medulla RT Room temperature s-LNvs Small ventral lateral neurons SLP Superior lateral protocerebrum SMP Superior median protocerebrum sNPF short neuropeptide F SOG Subesophageal ganglion Ƭ period length TBS Tris buffered saline TIM Timeless Tm Transmedullary neurons TR Tritocerebrum TrX Triton X-100 VIP Vasoactive intestinal (poly)peptide vLA ventral lamina cells VMNes Ventro-median neurons VNes Ventral neurons vPDFLA ventral PDFLA VPNes Ventro-posterior neurons VUM neurons Ventral unpaired median neurons xiv WB Wash buffer ZT Zeitgeber time Δφ Phase shift xv Table of Contents Examination Board: .................................................................................................. ii Statutory Declaration ............................................................................................... iii Dedication................................................................................................................ iv Contribution Statements .......................................................................................... vi Abbreviations ........................................................................................................... ix Table of Contents ................................................................................................... xv Summary ............................................................................................................... xxi 1. Introduction ................................................................................................ 1 1.1. Biological clocks ........................................................................................ 1 1.2. Main components of the circadian system ................................................. 1 1.3. The circadian clock in Drosophila melanogaster ........................................ 3 Molecular basis of the circadian clock in Drosophila melanogaster ............ 3 Cellular basis of the circadian network in D. melanogaster ........................ 4 Morning and evening components in the activity rhythms .......................... 7 1.4. Circadian clock in the cockroach Rhyparobia maderae .............................. 7 Localization of the circadian clock in the cockroach Rhyparobia maderae . 7 Internal structure of the accessory medulla as circadian clock of the R. maderae ...................................................................................... 9 The AME is remarkably heterogeneous in neurochemistry ........................ 9 Light input phase-shifts the circadian driven locomotor activity .................12 Neuronal pathways of the AME ................................................................13 The accessory medullae are coupled via several coupling pathways ........13 Neuropeptide PDF as a key factor in the circadian timekeeping of the R. maderae .....................................................................................16 Photic entrainment pathways to the circadian clock: the function of GABA ................................................................................20 GABA signaling in insects .........................................................................22 The morning- and evening-oscillators model in cockroach R. maderae ....24 xvi 1.5. Circadian clock in mammals and comparison with insects ........................25 1.6. Neuroanatomy of the photoreceptive organs in insects .............................28 Structure of the external photoreceptors, the compound eye and the ocelli ............................................................................................28 Neuronal organization of the optic lobe neuropils .....................................29 1.7. Neural network with the thoracic ganglia and premotor area in the brain ..34 1.8. The neurosecretory system of insects ......................................................35 Neuropeptides and their role in the circadian and neurosecretory system 36 Corazonin .................................................................................................37 Myoinhibitory peptide (MIP) ......................................................................37 Leucokinin ................................................................................................37 1.9. Aim of study ..............................................................................................38 2. Material and methods ...............................................................................40 2.1. Animals.....................................................................................................40 2.2. Immunocytochemistry ...............................................................................40 Dissection and fixation ..............................................................................40 Single staining ..........................................................................................41 Double labeling with antibodies raised in different species .......................42 Immunostainings with primary antibodies raised in same species.............42 Whole-mount immunohistochemistry ........................................................44 Quantification of GABA immunoreactivity .................................................45 Quantitative analysis of CLSM scans ........................................................45 Neuronal tracing (backfill) combined with immunostaining ........................46 General procedure ....................................................................................47 Single backfill from the optic stalk .............................................................47 Backfill from the antennal nerve ................................................................48 Backfills from the neurohaemal complex...................................................48 Backfill from the thoracic ganglia (TG) ......................................................49 Backfill from the ocelli ...............................................................................50 xvii Double backfill from the cut optic lobe and from the thoracic ganglia (TG) 50 Microscopy imaging and evaluation ..........................................................51 Antibody characterization .........................................................................52 2.3. Biochemical experiments ..........................................................................53 Animals.....................................................................................................53 Competitive ELISA ...................................................................................53 GABA measurement in the 12:12 LD cycle ...............................................54 Sample preparation ..................................................................................54 Coupling reaction ......................................................................................55 GABA measurement in the constants darkness ........................................57 GABA measurement in the short and long day photoperiods ....................57 Manufacturing of competitive ELISAs .......................................................57 Analytical Specificity (Cross reactivity) ......................................................58 Evaluation of the Data ..............................................................................58 Statistical analysis ....................................................................................59 3. Results .....................................................................................................60 3.1. Overview of GABA immunoreactivity in the brain of the Madeira cockroach R. maderae ......................................................60 Distribution of GABA-immunoreactivity in the midbrain .............................60 GABA-ir soma groups in the midbrain .......................................................60 GABA-ir neuropils in the protocerebrum ...................................................62 GABA-ir neuropils in the deutocerebrum and tritocerebrum ......................65 GABA-ir soma groups in the optic lobe .....................................................68 Glutamic acid decarboxylase (GAD) antibody is a reliable marker for GABAergic neurons .............................................................................71 3.2. What is the role of GABA in the circadian system of R. maderae? ............73 Six of seven soma groups next to the accessory medulla exhibited GABA-immunoreactivity.............................................................73 Three distinct GABA-ir tracts connect the AME to the medulla and/or to the lamina ..................................................................................75 xviii Output regions of PDF-ir fibers from the optic lobe revealed remarkable overlap with GABA-ir pattern, mainly in the lateral and median protocerebrum .............................................................................................78 GABA-ir cells express the circadian clock protein period ..........................80 Circadian clock protein PERIOD expressed in neuronal cells as well as glial ..........................................................................................81 3.3. GABA as a possible photic entrainment pathway to the clock ...................84 GABA-ir branches overlapped with termination sites of the short and long photoreceptor axons of the compound eyes ...............................84 GABA levels cycle in the distal tract ..........................................................86 Biphasic oscillation of GABA levels only in DD2 in optic lobe neuropils ....89 Light-duration (photoperiod) dependent increase of GABA levels .............89 Double-labelled 5-HT- and PDF-ir lamina cells (PDFLAs) connect the proximal lamina, accessory laminae and accessory medulla .............90 Triple-labelled 5-HT-, FMRFamide-, and PDF-ir lamina cells (PDFLAs) connect the proximal lamina, accessory laminae, and the accessory medulla ....................................................................................93 5-HT- and FMRFamide immunoreactivity colocalized in two VNes and one MNes.................................................................................96 GABA- and FMRFamide immunoreactivity colocalized in two VNes and one MNes.................................................................................99 One large PDF-ME neuron colocalized FMRFamide and MIP ..................99 Neurochemical profile of the accessory lamine ....................................... 102 3.4. GABA could be involved in coupling pathways between the AMAE ........ 104 At least two contralaterally projecting neurons belong to MCI (VNe) and MCII (VMNe) were GABA-ir ........................................... 104 3.5. Output pathway of the clock and GABA .................................................. 107 Characterization of the neurobiotin backfill from the first thoracic ganglion .................................................................................... 107 GABA immunoreactivity in the lateral protocerebrum and the AMMC greatly overlapped with neurobiotin-labeled fibers backfilled from the thoracic ganglion ...................................................................... 112 xix GABA and the ocelli................................................................................ 113 GABA and neuroendocrine system of the Madeira cockroach ................ 116 4. Discussion .............................................................................................. 119 4.1. Methodological Considerations ............................................................... 119 Commercial GABA immunohistochemistry fulfilled all criteria for a good staining ...................................................................... 119 4.2. Widespread GABA labeling in different neuropils in the central brain suggest a crucial function of GABA in various physiological and behavioral processes in the cockroach R. maderae ................................................. 120 GABA possibly serves via three types of inhibitory circuits in the antennal lobe (feedforward-, feedback-, and lateral inhibition) ................ 121 GABA-mediated inhibition likely occurs in the calyces of the mushroom body ................................................................................ 123 Conservation of the pattern of GABA immunoreactivity in the central complex among insects ............................................................... 125 Feedback inhibition of GABA-ir centrifugal neuron on the lamina could adjust the light sensitivity of the photoreceptors ............................ 126 4.3. The role of GABA in the circadian timekeeping system of Madeira cockroach ................................................................................. 128 The possible roles of GABA-ir neurons in the circadian clock of R. maderae ......................................................................................... 128 Strong connection of the AME to the multipeptidergic layer 4 of the medulla via GABA-ir medial layer fiber tract .................................. 129 Could GABA-ir cells be circadian oscillators? ......................................... 131 4.4. GABA as a possible photic entrainment pathway to the clock ................. 132 Technical considerations ........................................................................ 132 The GABA-ir neurons in the optic lobe could transmit photic signals to the accessory medulla ............................................................ 134 GABA might receive light information from an extraretinal photoreceptive organ, the lamina organ .................................................. 137 Possible role of GABA and 5-HT in gating circadian clock inputs and outputs ............................................................................................. 138 xx Multiple-labeled medial neurons (MNes) are candidates for the ipsilateral/contralateral photic entrainment pathways to the circadian clock ....................................................................................................... 139 Triple-labeled large-sized PDF-immunoreactive(-ir) neuron colocalizing FMRFamide and MIP as an important labeled line .............. 141 Multipeptidergic accessory laminae might provide light input to the clock ............................................................................................ 143 4.5. Possible role of GABA in coupling pathways between the AMAE ........... 145 4.6. Output pathway and GABA ..................................................................... 146 ILP, SMP, and POTU are potential premotor areas that relay circadian information to locomotor centers in the thoracic ganglia .. 146 GABA might increase photosensitivity of the ocellar photoreceptors to provide entrainment at low light levels ........................ 149 GABA might be involved in the temporal control of the neurosecretory system via the circadian system ........................................ 151 Publication bibliography .........................................................................................154 Acknowledgments .................................................................................................178 xxi Summary Transplantation studies identified the accessory medulla (AME) at the ventro- median edge of the medulla as the circadian clock of the Madeira cockroach Rhyparobia maderae. About 250 neuropeptidergic neurons that innervate the AME are categorized into seven soma groups. Among them are pigment- dispersing factor-immunoreactive (PDF-ir) neurons that control circadian sleep-wake cycles. The main aim of the present dissertation was to characterize the distribution of γ-aminobutyric acid (GABA) in the circadian network. Previous studies showed that GABA-ir fibers in the distal tract (DT) connect the clock to the medulla, apparently from somata amongst the about 29 GABA-ir neurons adjacent to the AME. However, they never were assigned to the specific AME neurons and there were several open questions with regard to the GABA pattern in the circadian network. In the first part of my thesis I characterized the GABA immunoreactivity in the brain. I showed that except for anterior neurons, in all other AME soma groups some neurons expressed GABA. Next to GABA-ir fibers in the DT and in the anterior fiber fan system, I discovered a new GABA-ir tract which I called medial layer fiber tract (MLFT). The GABA-ir MLFT connected the AME to layer four of the medulla. Furthermore, I could show that the GABA-ir DT connects the AME to the medulla, only. I showed for the first time that the lamina organ, putative extraretinal photoreceptor, is also GABA-, but not histamine-ir. Regarding the optic lobe, neither photoreceptors nor lamina cells were GABA- ir. However, different types of GABA-ir medulla cells were found that were suggested to provide inhibitory feedback to output regions of the circadian clock. In second part of my thesis I performed multi-label immunocytochemistry to search for a possible role of GABA and serotonin (5-HT) in the light entrainment pathways. I showed that GABA immunoreactivity in the lamina and the second layer of the medulla overlapped with histaminergic terminals of the short- and long photoreceptor neurons, suggesting synaptic connections. In the accessory laminae which showed immunoreactivity to many neuropeptides histamine immunoreactivity was absent. Additionally, I xxii showed that GABA levels cycle in the optic lobes as well as in the DT day-time dependently. GABA-ir neurons next to the AME showed colocalization with FMRFamide. FMRFamide showed colocalization with 5-HT and MIP in some lamina and AME neurons. One large medium-sized PDF-ir cell colocalized PDF with MIP and FMRFamide. For the first time, I showed that PDF lamina cells that coexpress PDF and 5-HT projected via the anterior fiber fan from the lamina down to the AME, but not to the medulla, possibly relaying photic input to the clock. In the third part of my thesis I analyzed PDF-ir clock outputs to GABA-ir neurons contacting premotor areas, ocelli, or neurosecretory control centers. Backfill experiments form the thoracic ganglia, the ocellar nerve, and the neurohaemal organs were combined with immunohistochemistry using anti- PDF and anti-GABA antisera. I showed that large second-order as well as 2 small multimodal ocellar neurons projected to the optic lobe and innervated the AME. The projections of the labeled ocellar neurons in the posterior optic tubercles, posterior optic commissure, lobula valley tract, and in the AME greatly overlapped with PDF and GABA immunoreactivity. I hypothesized that GABA might increase the photosensitivity of the ocellar photoreceptors to provide entrainment at low light levels to the AME. Interestingly, none of the neurosecretory cells in the brains were GABA-ir. However, the GABA-ir fibers were in close vicinity to backfilled fibers of neurosecretory cells in the SMP and SLP where PDF-ir fibers projections were present. Finally, evidence was provided that several regions in the brain including inferior lateral protocerebrum, superior median protocerebrum, and posterior optic tubercles which GABA- and PDF-ir are potential premotor areas that relay circadian information to locomotor centers in the thoracic ganglia. Introduction 1 1. Introduction 1.1. Biological clocks The periodic environment of the earth, such as daily rhythmic fluctuations of ambient light, temperature, humidity and other factors has affected life on earth. Different organisms from bacteria to humans, have adapted to these external rhythms by the evolution of endogenous biological clocks which enable them to predict serial circumstances and regulate behavior, physiology, metabolism and a wide range of other activities. The most remarkable periodic quality of the earth is the 24-hour cycle of the day and night generated by the planet’s rotation. Many physical, mental and behavioral patterns in living organisms follow these daily predictable cycles, which arise from their body’s response to the light and darkness. Circadian rhythm is the most common rhythm. Biological clocks adjusting to 24-hour rhythms are called circadian clocks. The word “Circadian” literally means “about a day “, comes from the Latin circa- “about” and -dies “day”. However, diversity of biological activity rhythms is essential for many important processes in living organisms. Ultradian cycles are slower than 24 hours. Examples for ultradian cycles are respiration, heartbeat and sleep patterns. Infradian rhythms are longer than 24 hours; for example, in the monthly menstrual cycle, migration in birds, hibernation in the animal and germination and photosynthesis processes in the plants. Among all endogenous clocks with different periods the circadian clocks were studied best. 1.2. Main components of the circadian system Across phyla, all circadian clocks are organized in three main parts: circadian oscillator, input and output pathways of the clock (Figure 1.1). The endogenous pacemaker generates circadian rhythms with a period length of approximately 24hours. Since this endogenous period is not accurately 24hours, the circadian clock is required to be synchronized with the environment (Pittendrigh 1957). This process by which the internal clock is synchronized by the external cues is called entrainment. Introduction 2 The external time cues are named Zeitgebers (from German words time giver). Different Zeitgebers such as light-dark cycle, temperature, social cues, food availability, etc. supply the entraining input signals to the circadian clock and synchronize it via time-dependent delays and advances. Eventually, the circadian oscillator via output pathways influences rhythms in various effectors such as locomotor control centers, the endocrine system, or neuronal circuits involved in reproduction and food consumption (Helfrich-förster et al. 1998; Homberg et al. 2003a; Roenneberg and Merrow 2005). Additionally, circadian clocks are described by several fundamental features. First of all, the circadian clock is endogenous and self-sustained. This means that, in the absence of the external cues or under constant conditions (i.e. constant darkness) the circadian rhythm persists with a period of about 24 hours. The period of the endogenous rhythm under constant conditions is called the free-running period = tau (Greek letter Ƭ). The period of the circadian free-running cycle is species-specific. For example, in humans it is longer than 24 hours (~24.30 h) and in night-active animals shorter than 24 hours. An average period in the male cockroach Rhyparobia maderae is ~23.72 h (Page and Block 1980). Another universal aspect of the circadian clock is called temperature compensation. It means that the period of the circadian clock remains constant in a wide range of temperatures. This property is even conserved in poikilotherm organisms such as insects, for which their internal temperature significantly varies (Bodenstein et al. 2012; François et al. 2012). Figure 1.1 A schematic drawing of the principal components of the circadian timekeeping system. External Zeitgebers such as light-dark cycles via photoreceptors and entrainment input pathways synchronize the circadian pacemaker. The circadian oscillator generates the circadian rhythm which in turn regulates the timing of rhythms in many effectors. Introduction 3 1.3. The circadian clock in Drosophila melanogaster Molecular basis of the circadian clock in Drosophila melanogaster Molecular mechanisms of the circadian clock were studied best in the fruit fly D. melanogaster. The mechanism of the circadian oscillations is based upon transcriptional/posttranscriptional autoregulatory feedback networks. These networks are formed by a set of genes– the clock genes. In 1971, the first clock gene was discovered. Owing to the effect of this gene on the circadian period length, it was named period (per) (Konopka and Benzer 1971). However, how per contributed to circadian timekeeping was not clear until the next clock gene, timeless was discovered (Sehgal et al. 1994). Both clock genes are the main component of the first discovered negative feedback loop in which the protein product of these genes inhibits its own transcription (Figure 1.2). Later, another dozen clock genes were identified by genetic screens. These genes include clock (clk) (Allada et al. 1998), cycle (cyc) (Rutila et al. 1998), Doubletime (dbt) (Kloss et al. 1998; Price et al. 1998), cryptochrome (cry) (Stanewsky et al. 1998), shaggy (sgg) (Martinek et al. 2001), casein kinase 2 (CK2) subunits (Akten et al. 2003). In the central core feedback loop, between ZT4 to ZT18 (midday) (Zeitgeber time, LD 12:12) transcription factor CLK and its heterodimer partner, transcription factor CYC, bind to the E-box element in the promoters of the per and tim and initiate their transcription. From about ZT12 on, the proteins PER and TIM1 begin to accumulate in the cytoplasm (Kloss et al. 1998; Kloss et al. 2001; Price et al. 1998). Simultaneously, the progressive phosphorylation of the CLOCK proteins begins by the PER:TIM1:DBT complex (Lee et al. 1998; Bae et al. 2001; Menet et al. 2010). This phosphorylation and additional posttranslational modifications are responsible for the generation of phase delays in the molecular circadian clock. Since the molecular process of the circadian clock theoretically takes less than 24 hours, these delays are essential for the generation of the period length. The levels of PER and TIM peak at the late night (ZT 22/23). Thereafter, they move back to the nucleus and inhibit their own transcription by interfering with CLK/CYC (Stanewsky 2002). At ZT0 and the beginning of the light phase, photo-activation of the blue-light photoreceptor Cryptochrome 1 (CRY1) results in the degradation of TIM1 and thereby the dissociation from PER Introduction 4 (Hunter-Ensor et al. 1996; Tomioka and Matsumoto 2010). Ultimately, PER is also targeted for degradation (around ZT4) which in turn leads to initiation of another cycle by binding heterodimer CLK-CYC to the E-box element of the per and tim again. Cellular basis of the circadian network in D. melanogaster The circadian clock of D. melanogaster was identified in the 1983 and was localized in each side of the brain (Konopka et al. 1983). Circadian pacemaker neurons were labeled by immunochemical staining against the clock proteins. This led to the identification of 150 pacemaker neurons per hemisphere which express clock genes (Figure 1.2). According to their anatomical location, they were divided into 7 major soma groups. Four groups are located laterally between the optic lobe and the median protocerebrum and they are called lateral neurons (LNs). They are further classified by their size and position into large ventro-lateral neurons (L-LNvs), small ventro-lateral neurons (S-LNvs), dorso-lateral neurons (LNds) and posterior-lateral neurons (LPNs). The other soma groups are located dorsally in the dorsal protocerebrum (DN1-3) (Siwicki et al. 1988; Zerr et al. 1990; Kaneko and Hall 2000; Nitabach and Taghert 2008; Tomioka and Matsumoto 2010; Peschel and Helfrich-förster 2011). The neuropeptidergic profile of these soma groups was studied using immunochemical methods. Among them, the functions of the neuropeptide pigment-dispersing factor (PDF), the insect homolog of the crustacean pigment-dispersing hormone (PDH) were studied best (Rao and Riehm 1988; Homberg et al. 1991a; Nässel 1991). In the crustaceans, PDH controls the circadian dispersion and translocation of the pigments of the retinal chromatophors in the compound eyes (Kleijn and van Herp 1995). Later, PDF was identified as an important anatomical marker for pacemaker neurons in several insects species (Homberg et al. 2003b; Helfrich-förster 2004; Helfrich- förster et al. 2000; Renn et al. 1999). In the fruitfly Drosophila eight clock neurons including four s-LNv and four l-LNv showed PDF immunoreactivity. PDF is required for normal behavioral circadian rhythm (Yao and Shafer 2014; Helfrich-förster 2014). Introduction 5 Both PDF-ir s-LNv and l-LNv project to the pacemaker centers- accessory medulla (AME) (Helfrich‐Förster et al. 2007). PDF-ir s-LNv also project to the dorsal brain area where the other clock neurons- DN1-DN3- are located. Since PDF-ir expressing l-LNv innervate the medulla neuropil and also via posterior optic commissure (POC) project to the contralateral side (Figure 1.2). Therefore, PDF was introduced to be a coupling factor as well. There is considerable evidence that PDF is essential for normal circadian behavior in Drosophila (Hermann-Luibl et al. 2014). Apparently, PDF-ir s-LNvs are the leading circadian pacemaker group under normal conditions (12:12 LD cycle) and constant darkness (Hermann-Luibl et al. 2014). Moreover, genetic ablation of PDF result in weakening of entrained rhythms and impairing of the free- running behavior (Renn et al. 1999; Stoleru et al. 2005; Rieger et al. 2006; Picot et al. 2007). It was shown that PDF acts via G protein-coupled receptors (GPRCs) (Hyun et al. 2005; Lear et al. 2005; Mertens et al. 2005). PDF increases cyclic adenosine monophosphate (cAMP) in PDFR-expressing cells (Shafer et al. 2008). Subsequent studies have shown that PDF also enhances Figure 1.2. General overview of the clock gene- expressing neurons and their arborization in Drosophila brain. Photoreceptor cells (R1-8), Hofbauer-Buchner (H-B), accessory medulla (aME), dorsal neurons (DN1-3), posterior-lateral neurons (LPN), dorso-lateral neurons (LNd), small ventro-lateral neuron (s-LNv), large ventro-lateral neuron (l-LNv). (Helfrich‐Förster et al. 2007). Introduction 6 the stability of the PER and TIM proteins via protein kinase A (PKA) regulation (Li et al. 2014; Seluzicki et al. 2014). The ion transport peptide (ITP) is another important neuropeptide which is expressed in two clock neurons, one of the l-LNv that is called 5th l -LNv and one of the LNd (Figure 1.3, (Dircksen et al. 2008). Both ITP- expressing neurons also express neuropeptide F (NPF) (Hermann et al. 2012; Lee et al. 2006). Further experiments have identified other neuropeptides in the clock neurons, e.g. short Neuropeptide F (sNPF) in some of the LNs (Johard et al. 2009), IPNamide (IPNa) in the two of the DN1 cells (Shafer et al. 2006), vesicular glutamate transporter (GluT) which indicates the presence of the glutamate and choline-acetyltransferase (Cha) which indicates the presence of the acetylcholine (Johard et al. 2009). The function of these neuropeptides is not fully understood. Figure 1.3. Neurochemistry of the clock neurons in one Drosophila brain hemisphere. Accessory medulla (AME), dorsal neurons (DN1-3), posterior-lateral neurons (LPN), dorso- lateral neurons (LNd), small ventro-lateral neuron (sLNv), large ventro-lateral neuron (lLNv), Pigment-dispersing factor (PDF, cyan), ion transport peptide (ITP, purple), neuropeptide F (NPF, orange), short neuropeptide F (sNPF, yellow), IPNamide (IPNa, magenta), choline- acetyltransferase (Cha, green), glutamate transporter (GluT, black). (Andreas Arendt dissertation, Modified after Hermann-Luibl and Helfrich-Förster 2015. Introduction 7 Apparently, none of the clock neurons in Drosophila expressed the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA). However, studies showed that the LNvs receive GABAergic inputs (Parisky et al. 2008). These input neurons are assumed to be involved in the regulation of the circadian activity. Moreover, PDF-expressing s-LNvs and l-LNvs in adult Drosophila also expressed GABA receptors (Parisky et al. 2008; Gmeiner et al. 2013). It was shown that GABAergic input to the LNvs effect the sleep homeostasis in Drosophila. It is assumed that sleep-promoting GABAergic neurons are able to suppress wakefulness by acting on their receptors on the s- and l-LNvs (Parisky et al. 2008). This finding is consistent with findings in humans in which GABAergic inputs to the wake-promoting centers drive daily sleep cycles (Roth 2007). Morning and evening components in the activity rhythms In many insects, locomotor activities show two peaks in one circadian cycle (Wiedenmann 1977b; Wiedenmann 1983; Koehler and Fleissner 1978). Therefore, it was proposed that two circadian oscillators control two activity peaks (Daan and Pittendrigh 1976). According to the dual oscillator model in Drosophila, there are two distinct oscillators which control morning and evening activity peaks of animals (Pittendrigh and Daan 1976; Grima et al. 2004; Stoleru et al. 2004). It appears that for the morning oscillator (M- oscillator) the PDF-expressing s-LNvs as well as CRY-positive DN1 and for the evening oscillator (E-oscillator) the ITP-expressing clock neuron are essential (Grima et al. 2004; Rieger et al. 2006). These two different peptidergic oscillators however are coupled to different extent in order to generate various outputs (Guo et al. 2014; Yao and Shafer 2014). 1.4. Circadian clock in the cockroach Rhyparobia maderae Localization of the circadian clock in the cockroach Rhyparobia maderae In spite of the fact that Drosophila has been well established as a powerful model with which to study the molecular basis of the circadian clock, a couple of hemimetabol insects, such as crickets or cockroaches, have also become Introduction 8 useful models for investigating the physiology of circadian timekeeping. Due to showing a clear circadian rhythm in the locomotor activity, as well as their large body size, the cockroach R. maderae (Figure 1.4) has been subjected to many surgical and physiological manipulations in the field of chronobiology. The cockroach R. maderae was the first animal model in which lesion and transplantation experiments could localize the internal clock in a specific region of the brain (Nishiitsutsuji-Uwo and Pittendrigh 1968). Removal of the optic lobe resulted in arrhythmicity in the locomotor activity of the cockroach. Further experimentation showed that the transplantation of the optic lobe from another cockroach could restore the circadian activity rhythm in a cockroach whose optic lobes were surgically removed (Page 1982). Later on, the location of the internal clock was further narrowed down to the region between the medulla and the lobula neuropils (Roberts 1974; Sokolove 1975). Immunostainings with an antibody against PDH have identified neurons which could function as pacemaker neurons in R. maderae (Homberg et al. 1991b; Nässel et al. 1991; Stengl and Homberg 1994). These PDH-ir neurons are located at the ventromedial edge of the medulla and innervate a small neuropil which is called the accessory medulla (AME). Additional experimentation showed that the cockroaches whose optic lobes were cut off and received an AME transplant in the antennal lobe were able to restore circadian rhythmicity in their locomotor activity. When followed by PDF staining, it turned out that PDF- ir fibers from the AME transplant regenerated to the superior lateral Figure 1.4. Dorsal view of the cockroach Rhyparobia maderae (Werckenthin 2014). Introduction 9 protocerebrum (SLP) and superior median protocerebrum (SMP) (Reischig and Stengl 2003a, 2003b). These two areas are original targets of the AME. Altogether, studies indicated that the AME and PDF-ir neurons are the location of the circadian clock. Internal structure of the accessory medulla as circadian clock of the R. maderae The AME is remarkably heterogeneous in neurochemistry Unlike other optic lobe neuropils which possess retinotopic organization (see 1.6.1), the AME shows a glomerular organization. It consists of dense glomeruli which are encapsulated in a coarse interglomerular neuropil. In its outer most layer, a loose neuropil shell surrounds the AME. The anterior region of the medulla merges with the shell region of the AME (Figure 1.5 A, (Reischig and Stengl 1996). The glomerular neuropil consists of glomeruli within different sizes. There are two large frontal and dorsal glomeruli and several small glomeruli located at the proximal and distal side of the AME (Reischig and Stengl 1996; Thordis Arnold 2016). The gap between the glomeruli is packed with the interglomerular neuropil. Figure 1.5. Three-dimension reconstruction of the accessory medulla (AME) (A) and the AME with associated soma groups (B). A) The AME structure is subdivided into glomerular neuropil, interglomerular and shell regions. B) The somata near the AME are classified into medial neurons (MNe), distal frontoventral neuron (DFVNe), medial frontoventral neuron (MFVNe), ventral neuron (VNe), ventroposterior neuron (VPNe), ventromedial neuron (VMNe) and anterior neuron (ANe). The latter are not shown. Distal tract (DT). Scale bar 50µm in B. (Figure A by Thomas Reischig and B from Soehler et al. 2011 modified after Reischig and Stengl 2003b. Introduction 10 Nearly 240 neurons are located in close proximity to the AME. They project to the AME. Based on different criteria such as morphology, the size of the soma, position and the heterochromatin content and size of the nucleus; neurons next to the AME are assigned to seven soma groups (Reischig and Stengl 1996; Reischig 2003; Soehler et al. 2008; Stengl et al. 2015b). These groups are: medial neurons (MNes), distal frontoventral neurons (DFVNes), medial frontoventral neurons (MFVNes), ventral neurons (VNes), ventroposterior neurons (VPNes), ventromedial neurons (VMNes), and anterior neurons (ANes) (Figure 1.5 B). The neurochemical profile of the AME soma groups was identified to some extent by means of immunochemical methods and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) analysis. Many neuropeptides and neurotransmitters such as GABA (Petri et al. 2002), myoinhibitory peptide (MIP) (Schulze et al. 2012; Schulze et al. 2013), leucokinin (Hofer and Homberg 2006b, 2006a; Petri et al. 1995; Lara Fricke 2016), gastrin, allatostatin, allatotropin (Petri et al. 1995), baratin (Nässel et al. 2000; Soehler et al. 2011), corazonin, serotonin (Petri et al. 1995) orcokinin (Hofer and Homberg 2006b), ITP (unpublished data by Thordis Arnold), FMRFamide related peptides (FaRPs; including short Neuropeptide F (sNPF) and leucomyosuppressin (LMS) (Söhler et al. 2007; Soehler et al. 2008) and PDF (Soehler et al. 2011) were detected in the AME soma groups. Some of these neuropeptides and neurotransmitters are assigned to different AME soma groups (Table 1.1), however, for some of them this information is still unknown. Other neurotransmitters such as histamine and acetylcholine have not been detected in any AME soma groups. However, it appears that they play an important role in the circadian timekeeping system of the R. maderae. Histamine is the neurotransmitter of the photoreceptors in R. maderae (Roberts 1965; Loesel and Homberg 1999). Injection of histamine induced light-like PRC indicating that it is involved in photic entrainment pathway (Arendt et al. 2017). Calcium (Ca2+) imaging experiments showed that the AME neurons in the primary cell culture responded to histamine and acetylcholine indicating that these cells possess the receptors for both neurotransmitters (Baz et al. 2013; Schulze et al. 2013). Moreover, behavioral experimentation Introduction 11 showed that application of acetylcholine resulted in all delay monophasic PRC, indicating that cholinergic neurons may be part of the light entrainment pathway (Stengl and Arendt 2016). Table 1.1. Neurochemical profile of the AME soma groups A N e D FV N e M FV N e M N e VM N e VN e VP N e Total number of cells 1.5±1 29±10 49±7 56±12 35±5 24±5 36±9 Allatotropin - 19- 31 (withVNe) - - Allatostatin* 16-21 Baratin 2-? 4 Corazonin - - - 1 - - - FaRPs 1.5±1 8.9±4.1 - 4.3±0.5 - 10.6±2.2 2.8±1.0 GABA* - 25 Gastrin* 2-10 ITP Leucokinin - 9.5±1.24 - - - 3.8 ± 0.98 - LMS - - - - - 1-3 - MIPs - 5.8±1.4 7.5±1.9 3.9±1.1 4.5±1.3 5.2±1.4 2.1±0.6 Orcokinin - 10.6±3.7 - 2.4±0.8 3.3±0.5 7.8 ±0.1 3.9±1.6 PDF - 3.6±0.5 - - - 8±1.4 - Serotonin* 18-25 sNPF 1.17±1.21 - - - - 1.67±1.51 - - : zero cells were counted. * : neuropeptide or neurotransmitter are not assigned into soma groups. ? : the maximum number of cells has not been clear. Values are given as arithmetic means ± standard deviation (SD). Medial neurons (MNe), distal frontoventral neuron (DFVNe), medial frontoventral neuron (MFVNe), ventral neuron (VNe), ventroposterior neuron (VPNe), ventro-median neuron (VMNe) and anterior neuron (ANe) Double-label and multiple-label immunostainings showed that neuropeptides are often colocalized with other neuropeptides or neurotransmitters in the AME neurons (Petri et al. 1995; Hofer and Homberg 2006a, 2006b; Soehler et al. 2011; Schulze et al. 2012; Arendt et al. 2017; Schendzielorz and Stengl 2014). Introduction 12 The co-occurrence of transmitter is a common incident in insect and mammals (Homberg et al. 1991a; Homberg et al. 1987; Homberg et al. 1990; Albers et al. 1991; Moore-Ede and Sulzman 1982). It was suggested that co-release of transmitters could increase the possibilities of the neuronal interaction in the nervous system (Homberg 1994b). In the insect circadian system, co- localization might indicate similar cooperative effects of transmitter interactions or multiple roles in synaptic transmission which are required for proper functioning of the AME as a master clock. Light input phase-shifts the circadian driven locomotor activity As described above, any Zeitgeber with similar period is able to entrain an endogenous clock. To determine whether a specific environmental cue or any kind of external stimulus functions as Zeitgeber is able to provide an input signal to the circadian clock, a phase response curve (PRC) can be obtained (Figure 1.6). The PRC is a graph representing the relationship between a certain stimulus e.g. light pulse, and the change of phase of an output rhythm of the circadian clock at specific circadian times (CTs). The CT is measured in constant conditions, when the animal´s activity is driven by its endogenous circadian clock, thus, expressing its free-running endogenous period which is either shorter or longer than 24 hrs. Accordingly, one circadian hour is not 60 min, but the endogenous period divided through 24. Circadian time from CT00 to CT12 encompasses the subjective day, CT12 to CT24 is the subjective night of the animal. For nocturnal animals the onset of activity under constant conditions is defined as CT12 (= the beginning of the subjective night) and for diurnal animals the beginning of locomotor activity is CT00 (= the beginning of the subjective day). When, under CT conditions, the animal is exposed to a light pulse, or injection of a neuroactive substance that is an input into its circadian clock, a phase shift of the beginning of its circadian locomotor activity rhythm occurs. Phase advance means that its locomotor activity starts earlier, phase delay means it starts later than predicted by its endogenous period. The amplitude of the phase shift (∆φ) is plotted on the Y-axis against the circadian time on the X-axis of the PRC (Figure 1.6). In all animal species, light- dependent PRCs resemble each other. Light pulses at dusk (early night) induced a phase delay in locomotor activity. In contrast, light pulses at dawn Introduction 13 (late night) induced a phase advance (Wiedenmann 1977a; Page and Barrett 1989; Golombek and Rosenstein 2010). A PRC that shows both phase delay and advance–such as the PRC of a light pulse- is named biphasic PRC. PRCs with only a delay or an advance are termed monophasic PRC (Golombek and Rosenstein 2010). Neuronal pathways of the AME The accessory medullae are coupled via several coupling pathways Figure 1.6. Phase response curve (PRC) after application of light pulses (white squares). Change in the running activity of the animal (black bars) in constant darkness after light exposure at different times of the day result in phase shifts (actograms above). The light- induced biphasic PRC is plotted below the actograms. A light pulse at the early subjective night leads to phase delay (-∆φ) (B, C) and a light pulse at the late subjective night leads to phase advance (∆φ) (D, E). A light pulse at the middle of the subjective day causes no phase shift (A). Figure from Moore-Ede and Sulzman 1982. Introduction 14 In order to generate a synchronized rhythmic output, the two bilaterally symmetric internal clocks must be coupled. Neuronal tracing experiments from the cut optic stalk showed that up to 50 neurons project to the contralateral optic lobe, at least some of them directly connecting both accessory medullae (AMAE) via either the anterior (AOC) or the posterior optic commissures (POC). Since these neurons are located near the medulla neuropil, they were called the medulla commissural cells (MC) and based on their position and size were assigned to one of four groups (MCI, II, III and VI) (see Table 1.2, (Reischig and Stengl 2002; Reischig et al. 2004). The MC I includes maximum of five VNes; MC II includes up to 35 VMNes; MC III consists of maximum of four posterior cells near the AME and approximately two MNes belong to the MC IV. The MC neurons connect both optic lobes via seven commissures (tracts). However, only three commissures directly connect both AMAE (Figure 1.7). Two tracts travel through the anterior optic commissure (AOC) and the third tract passes through the posterior optic commissure (POC) (Reischig and Stengl 2002). Both POC and AOC fuse and enter the contralateral optic lobe as the lobula valley tract (LOVT). In the contralateral optic lobe, two fiber systems can be distinguished. One fiber system includes branches which run over the anterior face of the medulla (=anterior fiber fan) and innervate the proximal lamina and the accessory laminae (ALA, plural ALAE). The ALAE form small neuropils at the proximal line of the lamina (Reischig and Stengl 2002). The other fiber system has arborizations in the middle layer (= medial layer) fiber system of the medulla. MC I neurons contribute to the anterior fiber fan system and middle layer fiber system, respectively. They are VNes with 4 aPDFMEs, (see part 1.4.5.). The largest aPDFME contains only PDF and projects both via the POC as well as the AOC. The other 3 medium-sized aPDFMEs project via the AOC. They express FMRFamide and orcokinine as well (Soehler et al. 2011). In contrast, MC II neurons which are VMNes and most likely all MCIII neurons which are posterior AME neurons, only project via POC to the contralateral optic lobe (Reischig and Stengl 2002; Reischig et al. 2004; Soehler et al. 2011). Electrophysiological experiments revealed that the MC I neurons are light-insensitive or very little light-responsive, while MC II neurons strongly react to changes in light intensity and are polarization- sensitive (Loesel and Homberg 2001a). This finding was consistent with the Introduction 15 earlier hypothesis in which it was assumed that both AMAE are connected via two functionally different pathways (Page 1983). The neurochemistry of the coupling pathways between clocks is still remained to be understood more. Table 1.2. Composition profile of the contralateral neurons Medulla cell group (MC) I II III IV Reference Maximum number 5 35 6 5 Reischig and Stengl, 2002; Soehler et al. 2011 AME soma groups VNes VMNes posterior AME Nes MNes Reischig and Stengl, 2003b PDF 2(1 l-PDFME 1 m-PDFME) - - - Reischig and Stengl, 2004; Söhler et al. 2011 FMRFamide 3 - - - Söhler et al. 2011 Baratin ≤3 - - - Söhler et al. 2011 MIP 1 1 - 1 Arnold MA thesis, 2016 Orcokinin 4 3 - - Hofer and Homberg, 2006a; Söhler et al. 2011 All numbers are the maximum number of the counted cells. - : zero cells were counted. Accessory medulla (AME), median neuron (MNe), ventro-median neuron (VMNe), ventral neuron (VNe), Myoinhibitory peptide (MIP), large PDF-immunoreactive medulla neuron (l- PDFME), medium sized PDF-immunoreactive medulla neuron (m-PDFME) Introduction 16 Neuropeptide PDF as a key factor in the circadian timekeeping of the R. maderae Neuroanatomy and physiology of the PDF-ir neurons is best known in R. maderae. Several lines of evidence suggested that PDF constitutes input-, output-, and coupling pathways of both AMAE. Immunochemical staining demonstrated that there are four PDF-ir cell groups per optic lobe. Two groups are located dorsally and ventrally in the lamina (PDFLA) and another group is located near the medulla (PDFME). PDFLA group includes 50-70 somata in Figure 1.7. Reconstruction of the coupling pathways between the circadian pacemaker centers of R. maderae. A) Tract 3 runs through the anterior optic commissure (AOC) and forms loops (small black arrows) around the vertical lobe (VL) of the mushroom bodies. It enters the contralateral optic lobe. B) Tract 4 goes via the AOC and tract 7 via the posterior optic commissure (POC) connecting the two accessory medulla (AMe). The AOC and POC go via the lobula valley tract (LOVT) enter the contralateral optic lobe. Medulla cell groups I. II. III (MC I-III), superior lateral protocerebrum (SLP), superior median protocerebrum (SMP). Scale bar 200 µm. Figure modified after Reischig and Stengl 2002. Introduction 17 the posterior region of the lamina. The PDFLA cells are further subdivided into two other groups. One group is located ventrally to the lamina neuropil (vPDFLA) and the other is dorsal to the lamina (dPDFLA) (Figure 1.8 A , (Reischig and Stengl 1996, 2003b; Wei et al. 2010a). The role of PDFLA cells still remains elusive. PDF-ir neurons next to the medulla are also divided into 2 sub-groups. A group of up to 8 PDF-ir cells are located at the posterior region of the medulla (pPDFME). Apparently, the branching pattern of the pPDFME cells is limited to the ipsilateral optic lobe (Reischig and Stengl 2003b; Wei et al. 2010a; Soehler et al. 2011). The second group of PDF-ir medulla cells includes 12 cells located anteriorly to the medulla (aPDFME). They are further subdivided into four small, four medium-sized and four large cells with one cell larger than the others (Figure 1.8 A). Furthermore, the aPDFME neurons are assigned to the soma groups associated with the AME. The small aPDFMEs are assigned to the DFVNes and medium-sized and large aPDFMEs are part of the VNes group (Reischig and Stengl 2003b; Soehler et al. 2011). It seems that the small aPDFMEs are local neurons and they innervate the AME and possibly the ipsilateral optic lobe neuropils. While the anterior and the shell of the AME invaded by large aPDFMEs, the glomeruli and to a lesser extent to the interglomerular region invades by the medium-sized aPDFMEs. The aPDFME connect the AME to the medulla and the lamina through two distinct fiber systems (Figure 1.8 B). The anterior fiber fan system (AFF) via the distal most layer of the medulla connects the AME to the medulla, lamina and to the accessory laminae (ALA). The median layer fiber system (MLF) has only arborizations in the middle layer of the medulla. However, these two fiber systems connected to each other via a few fibers (Figure 1.8 B (Reischig and Stengl 2003b; Reischig et al. 2004; Wei et al. 2010a). Neuronal tracing combined with immunochemistry showed that PDF is involved in coupling function of circadian pacemakers of R. maderae (Reischig and Stengl 2002; Soehler et al. 2011). It was shown that three medium-sized and one large aPDFMEs connect both AMAE via the AOC and POC, respectively, (Figure 1.8 C). These commissural aPDFMEs were assigned to the MC I (Reischig et al. 2004) and they send processes to the output regions in central brain such as SLP and SMP. The arborization pattern of these Introduction 18 contralateral PDFME neuron is very similar to the arborization of the light- insensitive cells described by Loesel and Homberg 2001. Later, the medium- sized PDFME cells were shown to contain orcokinin and FMRFamide. Accordingly, orcokinin and FMRFamide were assumed to be part of the coupling system (Soehler et al. 2011). Moreover, aPDFME neurons are suggested to transmit contralateral light input to the clock and/or they might be a ipsi- and contralateral output to the locomotion centers in central brain ((Reischig and Stengl 2003a). Injection of PDF induces phase delay at dusk and phase advance at dawn just as light does. Therefore, PDF was also suggested to contribute to light entrainment pathways to the circadian clock (Schendzielorz and Stengl 2014). However, much remains to be uncovered about the neuronal pathways of the clock in the R. maderae. Introduction 19 Figure 1.8. Frontal 3-dimensional reconstruction of the PDF-ir neurons and their projection pattern in the optic lobe and midbrain (A-C). A) PDF-ir fiber pattern in the right optic lobe showing small-(blue), medium-sized-(green) large-(yellow), and the largest (red) anterior PDF medulla neurons (aPDFMEs). Posterior aPDFMEs are shown in orange. B) distinct PDF-ir fiber system in the medulla; the anterior fiber fan (AFF) system (dark yellow) and medial layer fiber (MLF) system (magenta). The magenta star marks fibers that connect these two fiber systems together. C) At least four aPDFMEs which all belong to the ventral neurons connect the two AMAE. The three medium-sized aPDFMEs (blue) cross to the other hemisphere via the anterior optic commissure (AOC). They also have arborization in the dorsal lateral protocerebrum (dSLP) and the posterior optic tubercles (POTU), while the large aPDFME connects both AMAE via the AOC as well as the posterior optic commissure (POC). It arborizes in all output regions of the PDF-ir fiber system such as superior lateral protocerebrum (SLP), inferior lateral protocerebrum (ILP) and POTU. Medulla (Me), lamina (La), first optic chiasm (1.OC), ventral- (vPDFLa) and dorsal PDF lamina (dPDFLa) cells, accessory medulla (aMe) Figure A-B modified after (Wei et al. 2010) and figure C from (Soehler et al. 2011). Scale bars 50 µm. Introduction 20 Photic entrainment pathways to the circadian clock: the function of GABA Much remains to be learned about the light entrainment pathways leading to the circadian clock of the R. maderae. Light is the most potent external cue that synchronizes the circadian clock. Information about the light cycle is mediated by photoreceptors which differ between organisms. In Drosophila, different photoreceptive organs such as the compound eyes, ocelli, Hofbauer Buchner eyelet (H-B eyelet), and the intracellular chromophore cryptochrome (CRY) were suggested to contribute to the entrainment pathways (Helfrich- förster 2004). In contrast, it seems that in the cockroach R. maderae, the compound eyes are the only location for light entrainment input to the clock. Bilateral covering of the compound eyes with black lacquer or cutting the optic nerves resulted in free-running locomotor rhythms, while surgical removal of the ocelli did not prevent light entrainment of the locomotor activity rhythms (Nishiitsutsuji-Uwo and Pittendrigh 1968; Roberts 1965, 1974). Later, it was shown that photoreceptor cells of the ipsi- and contralateral compound eyes are essential for transmitting photic information to the circadian clock (Page 1978). However, no direct connection between histaminergic axons of the photoreceptors and the AME was found (Loesel and Homberg 1999). Therefore, it was assumed the light information might be conveyed indirectly to the clock. The only tract known that connect the AME to other optic lobe neuropils was the distal tract (DT) (Reischig and Stengl 1996; Petri et al. 1995) and so far only GABA was detected in the DT (Petri et al. 2002). Hence, GABA was suggested to be the main neurotransmitter for light entrainment pathways. However, it was not resolved which optic neuropils the GABAergic DT connected to the AME. Immunochemical studies suggested that GABA-ir fibers in the DT connect the core of all glomeruli of the AME to different layers of the medulla and possibly, also to the lamina (Reischig and Stengl 1996; Petri et al. 1995). But it remained unknown whether there are direct synaptic connections between GABA-ir fibers of the DT with histaminergic terminals of photoreceptor neurons in the lamina and/or the medulla. Also, a substantial number of the AME neurons express GABA; however, they were never characterized into different AME soma groups (Petri et al. 2002). Introduction 21 Intracellular recordings combined with dye injections were showed that two ipsi-lateral neurons (OL1, OL2), which belong to the MNes, are light sensitive (Loesel and Homberg 2001a). The morphology of these neurons is similar to the GABA-ir neurons described by Petri et al., 2002. They connect the AME to the medulla, lamina and to the ALAE (Petri et al. 2002). The MNe neurons (56±12; (Reischig and Stengl 2003b) are a non-homogenous group that expresses different neuropeptides such as MIPs (Schulze et al. 2012), orcokinin (Hofer and Homberg 2006b), corazonin (Petri et al. 1995; Arendt et al. 2017) and allatotropin (Petri et al. 1995; Schendzielorz and Stengl 2014). Furthermore, one of the MNes, co-expressed GABA, MIP, and allatotropin. (Schendzielorz and Stengl 2014; Petri et al. 2002). This triple-labeled MNes resembled the giant GABA-ir that apparently connected all glomeruli of the AME with the medulla and the lamina (Petri et al. 2002). Another triple-labeled MNe expressed GABA, MIP, and corazonin (Schendzielorz and Stengl 2014; Arendt et al. 2017; Arendt 2016; Stengl and Arendt 2016). Because GABA injections generated a biphasic light-like PRC (Petri et al. 2002), a role of GABA in relaying ipsilateral light information was suggested. However, the neuroanatomy of these GABAergic pathways remains a large gap in our knowledge. Furthermore, several neurons were found to provide contralateral light input to the AME (Loesel and Homberg 2001b). One neuron, which belongs to the VMNes, was described as PC2 neuron and was sensitive to polarized light. The PC2 neuron connected both AMAE and both medulla via the POC and has arborizations in a distal layer of the ipsilateral medulla (Loesel and Homberg 2001a). The neuropeptide or neurotransmitter content of this neuron is still unknown. Other candidates for contralateral light input are three orcokinin-ir VMNes. One of these VMNes colocalized orcokinin and MIPs. VMNes connect the interglomerular region of the AME with median layers of the contralateral medulla via the POC (Hofer and Homberg 2006a; Schendzielorz and Stengl 2014). Also, other VMNes were showed to be GABA-ir and since VMNes were assigned to commissural MC II neurons (Hofer and Homberg 2006a; Soehler et al. 2011), it is possible that GABA is also involved in contralateral light Introduction 22 entrainment (Schendzielorz and Stengl 2014). However, electrophysiological experiments combined with dye injection and backfill experiments in combination with immunohistochemistry needs to be done to find supporting evidence for this. GABA signaling in insects GABA is the most prevalent inhibitory neurotransmitter in the brain of different species of animals (Roberts and Frankel 1950; Otsuka et al. 1966; Roberts 2000; Kravitz 1962; Florey 1991). Its inhibitory properties were first identified in the arthropods (Otsuka et al. 1966). In insects, it was shown that GABA is involved in many physiological processes such as olfactory memory formation (Stopfer et al. 1997; Strambi et al. 1998), olfactory learning (Wilson and Laurent 2005; Hamasaka et al. 2005) , vision, and processes in the circadian clock (Hamasaka et al. 2005). There is considerable evidence that GABA plays a principal role in the circadian system (Moore and Speh 1993; Albers et al. 2017). The cytosolic enzyme, glutamic acid decarboxylase (GAD) synthesizes GABA from L-glutamate (Roberts and Frankel 1950). This enzyme is specifically expressed in GABAergic neurons and thus it is used as a marker to identify GABAergic neurons. GABA is stored in small synaptic vesicles in the terminal of synapses. However, GABA also was found in the large dense core vesicles which are typical for the storage of the neuropeptides (Castel and MORRIS 2000; van den Pol 1986). Following the fusion of the GABA vesicles with the presynaptic membrane and its release into the synaptic cleft, it acts at synapses by binding to specific transmembrane receptors of both pre- and postsynaptic membranes. There are two main classes of GABA receptors: GABAA and GABAB receptors. GABAA receptors were known as ionotropic receptors, in which GABA leads to the opening of a chloride ion channel and, depending on the Cl- gradient, an influx or efflux of Cl- (Henderson et al. 1993; Mezler et al. 2001). A subunit of the GABAA receptor, resistant to dieldrin (RDL), is best studied in Drosophila (Buckingham et al. 2005), where it was shown to play an important role in sleep-wake regulation in Drosophila. Apparently, PDF-expressing neurons express GABAA receptor RDL. Introduction 23 Activation of the RDL receptor inhibits PDF release and thus promotes sleep (Sattelle et al. 1991; Harrison et al. 1996; Chung et al. 2009). GABAB receptors are metabotropic G-protein-coupled receptors (Mezler et al. 2001). Apparently, GABAB receptors were expressed on presynaptic as well as postsynaptic membrane. They work through Gi and Go proteins. Activation of the GABAB receptors indirectly results in the opening of the potassium channels on the postsynaptic membrane, or blocking of voltage-dependent calcium channels at the presynaptic membrane, therefore, inducing longer- lasting hyperpolarization (Lüscher et al. 1997; Gassmann and Bettler 2012). Activation of the presynaptic GABAB receptors regulates transmitter release by controlling the intracellular level of calcium (Kaupmann et al. 1998; Kubota et al. 2003). Moreover, the distribution and pharmacological properties of GABAB receptors were characterized in the cockroach Periplaneta americana. It was shown that activation of GABAB receptors decreases the intracellular level of cAMP (Blankenburg et al. 2015). Following the release of GABA, different transporters (vesicular GABA transporters) on the membrane of the neurons or glia reuptake GABA from the synaptic cleft and pack it into synaptic vesicles. Additionally, GABA is broken down into glutamate and glutamine by the mitochondrial enzyme GABA aminotransferase (GABA-T), which is present in GABAergic neurons and astrocytes (Bown and Shelp 1997). Although GABA is well known for its inhibitory property, there is now considerable evidence that it can serve as an excitatory neurotransmitter, too. This function of GABA appears to be mediated via GABAA receptors which are permeable to Cl- ions (Colwell 1997; Ben-Ari 2002; Watanabe and Fukuda 2015). But whether one receptor can act differently depends on intracellular concentration of the Cl-. Relative activation of two Cl- transporters called NKCC importers (Na+-K+- Cl- cotransporter) and KCC exporters (K+- Cl- cotransporter) determine whether GABA acts as an inhibitory or excitatory neurotransmitters (Myung et al. 2015; Haam et al. 2012). It was shown that GABA can change its physiological action during the course of the day. Coupling of the dorsal and ventral regions of the suprachiasmatic nuclei (SCN) is mediated by excitatory Introduction 24 action of GABA during the day and inhibitory action during the night (Albus et al. 2005; Choi et al. 2008; Colwell 1997). Regarding the clock system, there is considerable evidence that GABAergic interneurons in the SCN play a major role in the synchronization of the pacemaker neurons (Jiang et al. 1997; Strecker et al. 1997; Colwell 2000; Albers et al. 2017). It was shown that GABAA receptor-dependent inhibition is responsible for this synchronization (Liu and Reppert 2000; Shinohara et al. 2000). Apparently, GABAergic interneurons have a similar function in insects. Extracellular recordings from the AME neurons in the cockroach R. maderae showed that GABA synchronizes the firing activity of AME neurons, which leads to ensemble formation among AME neurons (Schneider and Stengl 2005, 2006). In addition, Ca2+ imaging experiments demonstrated that the AME neurons respond differently to the GABA application. Some were inhibited and some were excited by GABA (Giese et al. 2018). Additionally, in isolated AME neurons time-dependent changes in their response to GABA applications were observed (Giese 2018). Treatment of the AME neurons in primary cell cultures with GABA agonists and antagonists showed that high numbers of the AME neuros express GABAA and/or GABAB receptors in various combinations. Both, GABAB receptors and GABAA receptors could elicit excitatory and inhibitory responses (Giese et al. 2018). Moreover, backfill experiments in combination Ca2+ imaging and immunocytochemical experiments showed that all contralaterally projecting PDF-expressing neurons were inhibited by GABA indicating that GABA might play an important functional role in the mutual coupling of both AMAE (Gestrich et al. 2018). The morning- and evening-oscillators model in cockroach R. maderae Activity rhythms in rodents as well as in Drosophila express a bimodal pattern with peaks at dusk and dawn (Aschoff 1966; Helfrich-förster 2000). In the 1970s, it was suggested that two independent but coupled oscillators control bimodal activity rhythms of crepuscular rodents (Aschoff 1966; Daan and Pittendrigh 1976). An M-oscillator controls the activity in the morning and an Introduction 25 E-oscillator controls the evening activity. These two oscillators react differently to light. The M-oscillator synchronizes to dawn and is advanced by light and, therefore, controls an activity rhythm with a shorter period. Whereas the E- oscillator couples to dusk and is delayed by light resulting in an activity rhythm with a longer period. Later, electrophysiological experimentation could measure two activity peaks from the SCN, indicating two functionally organized circuts (Jagota et al. 2000). In 2004 several studies in Drosophila suggested that M- and E-oscillators are constituted by specific clock neurons (Grima et al. 2004; Stoleru et al. 2004; Stoleru et al. 2005; Stoleru et al. 2007; Rieger et al. 2006; Picot et al. 2007). It is assumed that PDF-expressing neurons contribute only to the M-oscillator, while the CRY positive LNds act as E- oscillator (Yoshii et al. 2009; Peschel and Helfrich-förster 2011). Not much is known about the cellular organization of the dual oscillators in the cockroach R. maderae. An occurrence of two activity peaks was shown at the behavioral level (Wiedenmann 1977a; Schendzielorz 2014) as well as at the cellular level (Gestrich 2018). Tracking assays displayed two activity peaks under 12:12 LD cycles, the M peak at the end of the night and the E peak at the beginning of the night (Schendzielorz 2014). Extracellular recordings of the AME neurons in vivo also exhibited a prominent E peak and a small morning peak in electrical activity (Gestrich et al. 2018). 1.5. Circadian clock in mammals and comparison with insects The circadian master clock in mammals lies in the SCN of the hypothalamus (Klein et al. 1991). The circadian clock systems of insects and mammals share astounding similarities at different organizational levels (Helfrich-förster 2004; Glossop and Hardin 2002). At the anatomical level, both clocks were connected to the visual system and contain input and output pathways enabling the clocks to synchronize with the external light-dark cycle and regulate the various physiological and behavioral processes. At the sub- structural level, the internal organization of both clocks consists of a core that receives photic and non-photic inputs and a shell that is the input and output region of the clock (Figure 1.9 A). At the neurochemical level, both clocks use different neuropeptides and neurotransmitters which partly co-localize. The Introduction 26 neurotransmitter GABA is abundant in almost all SCN neurons and many neurons associated with the AME, as demonstrated by immunostaining (Moore and Speh 1993; Petri et al. 2002). The vasoactive intestinal peptide (VIP) in mammals and the neuropeptide PDF in insects are functionally analogous, and both were expressed in the circadian pacemaker neurons (Stengl et al. 2015b; Pauls et al. 2014) (Figure 1.9 B). Both of them act via G- protein-coupled receptors, and it was suggested that they mediate the phosphorylation of clock proteins via second messengers such as cAMP or cGMP (Golombek et al. 2004; Carlezon Jr et al. 2005; Lim et al. 2007; Lee et al. 2010; Li et al. 2014; Seluzicki et al. 2014). Introduction 27 At the molecular level, there is a strong resemblance between clock genes of insects and of mammals (Rubin et al. 2006; Ingram et al. 2012). The structure and circadian expression profiles of the core feedback loop genes such as per, tim 1 and cry 2 in the cockroach R. maderae indicate similar functions in both the cockroach and the mammalian clock (Werckenthin et al. 2012). Figure 1.9. Schematic drawing of the cellular organization of the suprachiasmatic nucleus (SCN) (A) and the accessory medulla (AME) (B). A) Two sub-regions can be found in the SCN: a ventrolateral core and a dorsomedial shell. Each sub-region is densely packed with different neuropeptidergic small neurons. The major neuropeptides of the core are vasoactive intestinal polypeptide (VIP, red), gastrin releasing peptide (GRP, red) calbindin (CalB, yellow), somatostatin (SS, green). While the main neuropeptide of the shell is vasopressin (VP, black). Additionally, almost all SCN neurons express GABA (not shown in the figure). The ventral core of the SCN mainly receives light input from the eyes and the dorsal shell provides output projections. B) The AME neuropil of the cockroach R. maderae consists of a core and a shell neuropil which is densely packed with glomeruli. The shell is further divided into the interglomerular region. About 250 neurons are located in close proximity of the AME innervate different region of the AME. These neurons exhibit immunoreactivity with different antisera such as pigment-dispersing factor (PDF, black), GABA (orange), leucokinin (red), Mas- allatotropin (yellow) and FMRFamide (green). Some of these neuropeptides are shown to be colocalized (not shown). The core receives the inputs and all output signal leaves the shell of the AME via the lobula valley tract. Figure from (Helfrich-Förster 2004) (B modified after Reischig 2002). Introduction 28 1.6. Neuroanatomy of the photoreceptive organs in insects Light is the major external cue for the synchronization of the internal clock with the environment. Therefore, circadian clock and visual system are tightly interconnected. In order to understand the neuronal basis of the light input to the clock, it is important to understand the basic anatomical organization of the visual system in insects. Since the neuroanatomy of the visual system is best characterized in Drosophila melanogaster, the fruitfly’s system will be addressed. Here, the focus will be on the two main photoreceptive organs in insects as well as interconnections of the photoreceptors with the two main optic lobe neuropils, the lamina, and the medulla. Structure of the external photoreceptors, the compound eye and the ocelli The compound eye is the primary light-sensitive organ of adult insects and hemimetabolous larvae (Klowden 2013). The functional unit of the compound eye is called ommatidium. The number of ommatidia per compound eye greatly varies in different species. The cockroach P. americana and Blaberus giganteus have 2000 ommatidia (Wolken and Gupta 1961) and Gromphadorhina portentosa has 2500 ommatidia per compound eye (Mishra and Meyer‐Rochow 2008); Drosophila, though, has 750 ommatidia (Wolff and Ready 1993). In the ommatidium, the photopigment rhodopsin is located in the rhabdom which is formed by the microvilli of the eight photoreceptor cells (= retinular cells, R1-R8). Photoreceptors 1-6 (R1-R6) are at the periphery, while R7 and R8 are located in the center of each ommatidium. Several pigment cells separate neighboring ommatidia. Pigment cells confine the amount of light that rhabdomeres receive. Two types of compound eyes can be found in insects; apposition and superposition eyes. The main difference is that the individual ommatidia within the apposition eyes are isolated from each other by pigment cells (Exner 1891). Although superposition eyes were predominantly found in nocturnal insects, cockroaches possess apposition eyes even though they are nocturnal. It was hypothesized that due to the high variability of light responses found in the photoreceptor cells of the cockroach, Introduction 29 they are still able to cope with dim light environments (Butler 1971; Butler and Horridge 1973; Heimonen et al. 2006). Innervated by the photoreceptors of the compound eye, there are three interconnected neuropils, the lamina, the medulla and the lobula, which all form retinotopic maps, processing photic information while keeping their spatial relationships. Axons of all photoreceptors project towards these neuropils. Axons of the photoreceptor R1-R6 terminate in the lamina units which were called cartridges. They receive light intensity signals and mediate shape, contrast and motion detection (Morante and Desplan 2008). Photoreceptor R7 and R8 which mediate color vision terminate in different layers of the medulla depending on the species (Trujillo-Cenóz 1965; Boschek 1971). Neuronal organization of the optic lobe neuropils Neuropils of the optic lobe exhibit two types of organization; columnar and stratified. The columnar organization result from the innervation pattern of the ommatidia into the lamina, medulla, and lobula. Each lamina column (cartridge) is corresponding to one ommatidium and each medulla column is corresponding to one cartridge of the lamina. Therefore, the photic information that a single ommatidium of the compound eyes receives from its external receptive field is processed by one cartridge (=neuronal unit) in the optic lobe neuropils. This pattern is called a retinotopic map, in which the spatial image of the environment is retained in the optic neuropils. The stratified organization of the optic lobe neuropils drives from the side branches of different types of the columnar neurons. Side branches are perpendicular to the columns and consequently give a stratification look to the neuropils (Figure 1.10 B). The lamina is the most distal neuropil in the optic lobe and first neuropil which receives light information from photoreceptors of the compound eye. As mentioned above, the lamina consists of discrete units called cartridges/ retinotopic columns. There are 12 neuronal cell types that contribute to the lamina organization. These 12 neuronal types were further classified into 3 neuronal classes: columnar, multi-columnar and tangential (Table 1.3). Introduction 30 The columnar neurons innervate one cartridge (retinotopic column). Eight of twelve neuronal types are columnar. They include five lamina output neurons (monopolar cells, L1-L5) and three centrifugal feedback neurons (C2-C3 and T1) (Figure 1.10). The feedback neurons have cell bodies in the proximal and the distal side of the medulla (Figure 1.10 A). Several lines of evidence showed that C2 and C3 neurons are GABAergic in many insects (Datum et al. 1986b; Meyer et al. 1986; Buchner et al. 1988; Fei et al. 2010; Kolodziejczyk et al. 2008a). They project to multiple layers of the medulla and then send fibers back into the lamina. In Musca, it was shown that C2 cells are presynaptic to photoreceptors R1-R6, whereas C3 cells are presynaptic to the lamina monopolar L1-L2 (Strausfeld and Campos-Ortega 1977). Electron microscopy studies in flies revealed that both neuronal types receive presynaptic input from lamina monopolar cells (Takemura et al. 2008b; Kolodziejczyk et al. 2008b). The multi-columnar neurons innervate multiple columns and consist of four lamina neurons classified as either lamina intrinsic amacrine cells (Lai) or lamina wide-filed neurons (Lawf1-Lawf2). The cell bodies of these neurons were located next to the medulla, but they send axonal process back to the Figure 1.10. Camera lucida drawing of two types of lamina neurons. A) Columnar neurons and B) multi-columnar neuron are connecting the lamina to the medulla in Drosophila melanogaster. Photoreceptors (R1-R8), Centrifugal feedback neurons (C2-C3), Lamina monopolar cells (L1-L5), Lamina intrinsic (Lai), Lamina wide-filed neurons (Lawf1-Lawf2), T- shape cells (T1). Figure from (Tuthill et al. 2013). Introduction 31 lamina and project to several neighboring cartridges (Figure 1.10 B). Lastly, the group of lamina tangential neurons includes a couple of neurons with cell bodies located near the AME which arborize in the ipsilateral central brain and the AME (Helfrich‐Förster et al. 2007). Table 1.3. The neuronal types in the lamina Neuronal class Lamina neurons abbreviation Columnar neuron Feedforward (centripetal) monopolar cells Feedback centrifugal neurons Feedback T-shape cells (L1-L5) (C2-C3) (T1) Multi-columnar neuron Lamina intrinsic amacrine cells Lamina wide-filed neurons (Lai) (Lawf1-Lawf2) Tangential neuron Lamina tangential neurons (Lat) The medulla is the second visual neuropil that is connected to the lamina via first optic chiasm. It appears to be the most complex neuropil in the optic lobe. Each column in the medulla can consist of at least 40 different morphological types of neurons. The medulla is best studied in flies (Fischbach et al. 1989; Takemura et al. 2008b; Strausfeld and Nässel 1980). According to Golgi- impregnation stainings of single neurons, the medulla is subdivided into ten layers. Photoreceptor axons of R7 and R8, as well as ten lamina neurons, terminate in the medulla. The medulla neurons were classified similarly to the lamina: columnar or tangential. If the neuron’s axon is aligned parallel to the visual column of the medulla, it is considered to be a columnar neuron. Tangential neurons, on the other hand, have a wide spread lateral projection in the layers of the medulla. These neuronal types link different layers of the medulla with midbrain areas or with the contralateral optic lobe (Figure 1.11, (Fischbach et al. 1989). The types of medulla neurons were listed in Table 1.4. Introduction 32 Table 1.4. The neuronal types in the medulla Neuronal class medulla neurons abbreviation Function Columnar neuron Medulla intrinsic neurons (Mi 1-10) Connect the distal medulla to proximal medulla Transmedullary neurons (Tm 1-26) Connect the distal medulla to the lobula Transmedullary Y cells (Tm Y 1-12) Connect the distal medulla to the lobula and the lobula plate Tangential neuron Distal medulla (amacrine cells) (Dm) arborize solely in the distal medulla Proximal medulla (amacrine cells) (Pm) arborize solely in the proximal medulla Medulla tangential (Mt 1-15) arborize into the wider visual field in the medulla Introduction 33 The lobula is the third visual neuropil in the optic lobe of insects. The medulla connects to the lobula via the second optic chiasm. While in bees and cockroaches the lobula is a single neuropil, in flies, butterflies, and beetles it consists of two distinct parts: lobula and lobula plate (Figure 1.11). Together, lobula and lobula plate were called lobula complex (Strausfeld 1970; Leitinger et al. 1999). The neuroanatomy of the lobula complex was studied mainly in Drosophila. The majority of the lobula neurons are columnar neurons that are retinotopically arranged. The lobula receives input from the medulla. While the lobula plate includes both columnar and tangential neurons and receives input from the medulla as well as the lobula. Lobula and lobula plate were connected by Tm Y neurons of the medulla (Figure 1.11, (Fischbach et al. 1989). Studies in flies revealed that lobula and lobula plate have different functions. Color processing, orientation detection and small target detection are functions of Figure 1.11. A schematic drawing of the medulla neurons in Drosophila melanogaster. Horizontal view of three optic neuropils: medulla, lobula and lobula plate showing innervation pattern of six different types of medulla neuron. Only one version of each type of neuron is shown. Medulla intrinsic neurons (Mi), Transmedullary neurons (Tm), Transmedullary Y cells (TmY), Distal medulla (Dm), Proximal medulla (Pm), Medulla tangential (Mt). (adapted from Golgi drawings by (Fischbach et al. 1989). Introduction 34 the lobula, while the lobula plate serves in global motion vision (Haag and Borst 2008; Borst et al. 2010; Borst and Helmstaedter 2015). In addition to the compound eye, a lot of insects also have a simple eye, the so-called ocellus (plural: ocelli). There are two types of ocelli in insects: dorsal ocelli and lateral ocelli (or stemmata). The latter exist in larvae of insects. The dorsal ocelli were located on the dorsal surface of the insect head. The number and function of the dorsal ocelli differs in insects. In locust and dragonflies, it was shown that ocelli were involved in maintaining flight stability at dusk (TAYLOR 1981b, 1981a; STANGE and HOWARD 1979; GOODMAN 1965). The ocellus consists of a single lens (cornea) and a layer of photoreceptors. Since the refractive power of the lens is not adequate to create an image, ocelli are also sometimes classified as a non-visual organ in the insect. However, studies in some insects have demonstrated that the ocelli are capable of creating a blurry image (Warrant and Nilsson 2006; Berry et al. 2007). Furthermore, due to having a huge convergence ratio from the photoreceptors (first-order neurons) to the second-order interneurons, they are considered to have high sensitivity to light as compared to the compound eyes (Wilson 1978). Therefore, it is assumed that they are adapted to perceiving low light level and contribute to fast response at night (Weber and Renner 1976). Neuroanatomical organization and physiology of the ocelli in the brain were thoroughly studied in the cockroach P. americana (Mizunami et al. 1982; Mizunami 1995; Mizunami and Tateda 1986, 1988a, 1988b; Ohyama and Toh 1990b). The cockroach possesses a pair of ocelli which are the largest among all insects. It was shown that the ocelli in the cockroach are not responsible for light entrainment of the clock (Roberts 1965). However, extracellular recordings showed that one type of ocellar neuron that strongly responds to stationary light connects to the AME (Mizunami and Tateda 1986; Loesel and Homberg 2001a). Still the role of ocelli in the functioning of the circadian clock is unclear. 1.7. Neural network with the thoracic ganglia and premotor area in the brain Introduction 35 The brain is connected to three thoracic ganglia, pro-, meso- and metathoracic ganglia, followed by eight abdominal ganglia, displaying the characteristic knot-guide vein system of the arthropods. For the control of rhythmically alternating locomotion, so-called central pattern generators are essential. Central pattern generators could be identified in diverse species, both in vertebrates and invertebrates. A central pattern generator is a central neural network. In insects, central pattern generators in the thoracic ganglia are responsible for rhythmic behaviors such as flying, running, or breathing (Marder and Bucher 2001; Dickinson 2006). Central pattern generators are driven by excitatory inputs of the cerebral ganglion. Associated with command neurons are descending interneurons which display a tonic activity pattern that can induce rhythmic behaviors (Marder and Bucher 2001; Dickinson 2006; Hildebrandt et al. 2015). Orthopteroid insects such as the cricket, the cockroach, and the locust have a highly mobile antenna. They constantly move their antennae and capture different information from their surroundings via the mechanosensory and chemosensory receptors on the antennae. It was shown that in the cockroach, mechanosensory exploration of the environment by the antenna is performed under visual guidance. Covering the compound eyes with dye resulted in elimination of certain antennal movements (Ye et al. 2003; Comer and Baba 2011). These data suggested that to achieve the full behavioral response, such as escape or running, several sensory modalities need to be integrated (Ye et al. 2003; Comer and Baba 2011). All sensory information is evaluated by the brain to initiate adapted motor and physiological responses. Fast escape reflexes connect the sensory information of the antennas as well as the eye directly (or indirectly) to descending neurons, which activate motor neurons of central pattern generators in the thoracic ganglion. Brain areas that are innervated directly by descending neurons are called premotor areas. The information flow from the eye to premotor areas is still unknown. 1.8. The neurosecretory system of insects The neuroendocrine system of the insects includes neurosecretory cells and neurohaemal organs. Specialized neurons which are responsible for secreting Introduction 36 specific neurohormones consist of two clusters of neurosecretory cells located in the protocerebrum. One is located in the pars lateralis (PL) of the lateral superior protocerebrum and the other is located in the pars intercerebralis (PI) in the dorso-medial superior protocerebrum (Homberg et al. 1991a; Hartenstein 2006). The axons of the neurosecretory cells in PI project via the nervi corporis cardiaci I (NCC I) and the cells of the PL via the nervi corporis cardiaci II (NCC II). Both groups project to a pair of connected glands located behind the brain, the corpora cardiaca (CC) and corpora allata (CA). These glands produce and release hormones into the hemolymph and control developmental and physiological processes such as the diapause in insects. The adipokinetic hormone (AKH) and juvenile hormone (JH) are two main hormones that are produced by the CC and CA, respectively. The AKH (” insect glucagon”) is mainly responsible for the metabolism and the JH responsible for larval growth and, metamorphosis, as well as the mating behavior of insects. The biosynthesis of JH is controlled by neuropeptide and neurotransmitter content of the neurosecretory cells in the brain. This regulation is important for the development of the insect’s larva, since the developmental stage of the larva depends on the relative concentrations of JH to ecdysone in the hemolymph (Hartenstein 2006; Hildebrandt et al. 2015). Additionally, JH controls the production and release of the molting hormone, β-ecdysone (Hartenstein 2006; Hildebrandt et al. 2015). Neuropeptides and their role in the circadian and neurosecretory system The first insect neuropeptide that was sequenced was proctolin from the cockroach P. americana. Proctolin can serve as a neuromodulator or as a neurohormone. Proctolin-like-ir neurons (PL-ir neurons) in the cockroach are found in the tritocerebrum as well as in the protocerebrum. In the protocerebrum, the PL-ir neurons are localized in the median and lateral neurosecretory cells. In addition to Proctolin, other neuropeptides are found in the neurosecretory system. These include FMRFamide, corazonin, leucokinin, MIP, Allatostatin, allatotropin and amongst others (Nässel 1993). A few of the mentioned neuropeptides are described in more detail below. Introduction 37 Corazonin The neuropeptide corazonin, which was first isolated from the American cockroach, might have a neuromodulatory function in insects (Predel et al. 2007). In P. americana, in addition to this function, it seems to have a stimulating effect on the heart rate (Nässel 2002). Corazonin is synthesized in the neurosecretory cells of PI. Over the NCC I, these corazonin-ir neurons project into the CC (Predel et al. 2007). Additionally, some processes of lateral neurosecretory cells enter the CA (Veenstra and Davis 1993). Furthermore, a corazonin-ir neuron was found in the two optical lobes, that connected from the medulla to the AME (Petri et al. 1995; Nässel 2002). Myoinhibitory peptide (MIP) The MIPs have an inhibitory effect on the visceral musculature and JH synthesis. These peptides belong to the family of W (X6) Wamides and were first discovered in Locusta migratoria (Lom MIP) (Schoofs et al. 1991; Schoofs et al. 1996; Davis et al. 2003).In the cockroach, in addition to the processing of chemosensory information, they might affect the circadian system and running activity. MIP immunoreactivity was localized in the median and lateral neurosecretory cells as well as in their axons to the CC (Predel et al. 2001). In the optical lobe, MIP immunoreactivity was found in the lamina, medulla, lobula and in the AME (Schulze et al. 2012). The distribution of MIP-ir neurons in the AME are listed in Table 1.1 Leucokinin The neuropeptide leucokinin has a stimulating effect on the rectum contractions of the cockroach. Different leucokinins were isolated from the cockroach R. maderae and were subdivided into eight types (LK-I-VIII). Like corazonin and proctolin, leucokinin can also serve as a neuromodulator and neurohormone (Chen et al. 1994; Nässel 2002). In immunocytochemistry studies, leucokinin-ir neurons were identified in the neurosecretory cells of PI and PL, as well as in interneurons of the protocerebrum and optic lobe (Chen et al. 1994). The axons of the median neurosecretory cells pass over the NCC I to the CC. Whereas the axons of the lateral neurosecretory cells run over the Introduction 38 NCC II to the CC. In the two optic lobes, the AME is linked to the medulla via leucokinin-ir neurons (Nässel 1993; Lara Fricke 2016). 1.9. Aim of study Although our knowledge about the neuronal network and physiology of the circadian clock is dramatically increasing, we are still far from fully understanding the whole process. In my Ph.D. project, I further examined and characterized the neuronal pathways including light entrainment pathways, outputs, and the coupling pathways of the circadian clock in the cockroach R. maderae. In order to fill the gaps in our knowledge about GABA as an important neurotransmitter involved in different aspects of the circadian timekeeping system of the cockroach R. maderae, I was able to answer the following questions: • To which AME soma groups do GABA-ir neurons belong to? • Do the GABA-ir distal tract fibers also connect the clock to the lamina? • How does the clock interconnect with other optic neuropils via GABA-ir tracts? • Do GABA-ir neurons make contacts with histaminergic photoreceptor terminals in the lamina and in the medulla? • Is GABA involved in coupling pathways between AMAE? • To which somata do the GABA-ir processes in the lamina and medulla belong to? • What are the other types of GABA-ir somata in the optic lobe? • What does the pattern of GABA-immunoreactivity in the central brain look like? • Could PDF-ir neurons directly synapse with GABA-ir neurons in the central brain? • Using biochemical experiments, it was investigated whether the level of GABA in the optic lobe oscillates daytime-dependently and whether the level of GABA persists under different photoperiod conditions. Introduction 39 • Quantification of GABA immunoreactivity in the distal tract could be investigated to determine whether the level of GABA changes daytime dependently. Moreover, to identify possible output regions of the clock to other brain neuropils, such as premotor areas, or the neuroendocrine system, neuronal tracing from different regions such as the antennal nerve, the retrocerebral complex, and the thoracic ganglion were performed and were combined with anti-GABA and anti-PDF immunocytochemistry. Finally, using multiple-label immunocytochemistry to complete the neuropeptidergic and aminergic profile of the AME soma groups and to look for the co-occurrence of different neuropeptides and neurotransmitters, I was able to answer the following questions: • To which AME soma groups do serotonin-ir neurons belong to? • Do GABA and serotonin co-localize in circadian clock neurons? • Do FMRFamide-ir neurons of the AME neurons co-localize GABA, MIP, or serotonin? Material and Methods 40 2. Material and methods 2.1. Animals Cockroaches of the species R. maderae were reared under a 12:12 hour light/dark (LD) cycles with lights on from 08:00 to 20:00 hr at 50 % relative humidity and 25-26 °C room temperature (RT). The animals were fed with dried dog food, apples, potatoes, salad, and water ad libitum. Adult male cockroaches were used for all experiments. For immunostaining procedures (except immunostaining which were used for immunoreactive quantification), brains were dissected out at about same zeitgeber time (ZT) 02:00 to avoid ZT-dependent effect. 2.2. Immunocytochemistry Dissection and fixation Animals were cold-anesthetized and then decapitated. Heads were mounted on a wax coated petri-dish using fine pins with the head side up. Using sharp razor blade, a small square was opened into the head capsule to expose the brain (Figure 2.1). After removing fat tissue, trachea and corpora cardiaca (CC) as well as corpora allata (CA, Figure 2.4 A), by using fine forceps and ultra- fine scissor, brains were removed from the head capsule and then dissected and fixated for 2 hours at RT in 4% formaldehyde (FA) in 0.1 M sodium phosphate buffer (Rothi-Histofix; Roth, Karlsruhe, Germany). For GABA immunostaining (with anti guinea pig GABA antibody, Table 2.1), brains were fixed in 0.1% glutaraldehyde (GA; Sigma Aldrich, Munich, Germany), and 4% FA in 0.1M sodium phosphate buffer (PB; containing 78.8 mM Na2HPO4 x 2 H2O and 19 mM NaH2PO4 x H2O at pH 7.4) for 2 hours at RT. For the histamine immunochemistry, 4% N-(3-dimethylaminopropyl)-N´ethylcarbodiimide (EDAC; E-7750; Sigma-Aldrich) in PB were used for 3 hours on ice. After fixation, brains were briefly rinsed in PBS, embedded in a gelatin (6%)/ albumin (20%) mixture and post-fixated in 10% formaldehyde in PB for overnight at 4°C. Material and Methods 41 Single staining Indirect peroxidase method was employed to characterized GABA immunoreactivity distribution in the R. maderae brain. Gelatin blocks were sliced in frontal as well as horizontal plan at thickness of 30 µm with a vibrating blade microtome (VT 1000; Leica, Wetzlar, Germany). Sections were collected in a well plate containing PB. Subsequently, brain sections were rinsed four times with Tris-Buffered saline (TBS; 0.1M Tris-HCl and 0.3M NaCl at pH 7.4) containing 0.1% Triton X-100 (TrX), preincubated with 5% normal goat serum (NGS, Dianova, Hamburg, Germany) in TBS containing 0.5% (TrX) for 2 hours at RT and incubated in the rabbit anti GABA antibody solution (Table 2.1) overnight at RT. Primary antibodies were dissolved in TBS containing 2% NGS and 0.5% TrX. The subsequent day, brain sections first were rinsed four times for 10 minutes in TBS containing 0.1% TrX and then incubated with optimum dilution of horseradish peroxidase-conjugated goat anti-rabbit (GaR) antibody at concentration of 1:100 dissolved in TBS containing 1% NGS and 0.5% TrX for two hours at RT. After the washing step, peroxidase-labeled sections were stained with 0.03% 3,30-diaminobenzidine tetrahydrochloride (DAB; Sigma- Aldrich, Munich, Germany), 0.015% H2O2, and 0.6% nickel(II)sulphate- hexahydrate in Tris buffer (0.55M Tris-HCl, pH 7.6) for 5–10 minutes. After enzymatic development, sections were rinsed again and subsequently Figure 2.1. The open head capsule of the cockroach Rhyparobia maderae with the exposed brain. Material and Methods 42 mounted on chromealum/gelatin-coated microscope slides. After dehydration through a graded ethanol series, sections were covered with coverslips with Entellan (Merck, Darmstadt, Germany). Double labeling with antibodies raised in different species For immunohistochemistry using antibodies raised in two different host species, a fluorescent two-step indirect method was applied. Dissection, fixation, sectioning and preincubation steps were performed as described in 2.2.1 and 2.2.2 with the exception that for double labeling, gelatin blocks were sectioned in 40µm or 100µm thickness (see 2.2.6) or treated as whole-mount (see 2.2.5). Followed by preincubation, two antibodies generated in different animal hosts were used together and brain sections were incubated overnight at RT in antibody solution. Immunostainings with primary antibodies raised in same species For immunohistochemistry using antibodies produced in the same host species (e.g. rabbit anti-GABA and rabbit anti-histamine, rabbit anti- FMRFamide and rabbit anti-GABA, rabbit anti-FMRFamide and rabbit anti- MIP, rabbit anti-GABA and rabbit anti-5-HT or rabbit anti- FMRFamide and rabbit anti-5-HT) monovalent fab-fragment technique was performed. In this method, unlabeled- and florescent-labeled monovalent fab fragment secondary antibodies were employed to block endogenous Ig G and therefore prevent the second secondary antibody from binding to the first primary antibody. The following protocol has been modified after Arendt et al., 2016. The first primary antibody raised in rabbit, polyclonal anti-GABA antiserum (Sigma; cat. no. A2052) were applied at optimized dilution of 1:750 in 2% NGS for 24 hours at RT on a shaker. After three steps washing for 10 minutes each, goat anti rabbit (GaR)-Fab-fragment labeled with Alexa 647 (Dianova, Hamburg, Germany) at a concentration of 1:300 in 1% NGS was used for three hours to detect the first primary antibody. Subsequently, un-conjugated GaR- Fab (Dianova, Hamburg, Germany) was added at a dilution of 1:100 in 1% NGS for one additional hour to block remaining binding sites on the Fc fragment of the first primary antibody. Afterwards, sections were washed three Material and Methods 43 times for 10 minutes each. Then, the second primary antibody raised in rabbit, polyclonal anti-5-HT antiserum (Sigma Aldrich, cat. no. S5545) at a dilution of Table 2.5.list of the primary antibodies applied in this study. Antibody Immunogen Host Source Working Dilution Anti-γ-aminobutyric acid (GABA) GABA conjugated to keyhole limpet hemocyanin with glutaraldehyde Guinea pig polyclonal Protos Biotech, New York (USA) 1:1.000 Anti-γ-aminobutyric acid (GABA) GABA conjugated to bovine serum albumin with glutaraldehyde Rabbit polyclonal Sigma Aldrich, cat. no. A2052 1:750 Anti-FMRFamide FMRFamide conjugate to thyroglobulin Rabbit polyclonal Marder et al. 1987 1:3.000 Anti-Histamine Histamine conjugated to keyhole limpet hemocyanin with 1-Ethyl- 3-(3- dimethylaminopropyl)carbo dii-mid Rabbit polyclonal Merck Millipore, cat.no. AB5885 1:25.000 Anti-Pea-MIP-1 Periplaneta americana MIP-1 (GWQDLQGGWamide) conjugated to thyroglobulin Rabbit polyclonal Predel et al., 2001 1: 12.000 Anti-Drm-Pigment dispersing factor (PDF) Drosophila melanogaster PDF Peptide (NSELINSLLSLPKNMNDA a) Mouse monoclon al Development al Studies Hybridoma Bank; Cyran et al., 2005 1:500 Anti-5- Hydroxytryptamine (5-HT) serotonin creatinine sulfate complex conjugated to BSA Rabbit polyclonal Sigma Aldrich, cat. no. S5545 1:4.000 Anti-SYNORF1 (Synapsin) SYNORF1 – Drosophila synapsin I isoform Mouse monoclon al Development al Studies Hybridoma Bank 1:50 Anti-rm PER R. maderae period (NH2- MEETATHNTKISDS- COOH (1) NH2-KSSTETPPSYNQLN- COOH (2) NH2- CRRETSATNTSQGSY- CONH2(3)) Rabbit polyclonal Werckenthin 2014 1:10.000 Anti-HRP peroxidase from horseradish Rabbit polyclonal Sigma Aldrich, cat.no. P7899 1:10.000 Anti-Glutamic Acid Decarboxylase 65/67 ADD INFORMATION Rabbit polyclonal Sigma Aldrich, cat.no. G5163 1:1000 Material and Methods 44 1:3000, as well as mouse anti-PDF antibody at a dilution of 1:500 in 2 % NGS were applied simultaneously. Sections were incubated overnight. After more washing steps, sections were incubated with Cy2 conjugated GaR-FAB (Dianova, Hamburg, Germany) at a dilution of 1:300 and goat anti mouse (GaM) Cy3-conjugated at a dilution of 1:300 for 2 hours. After final washing steps for three times 10 minutes each and an additional 5 minutes rinsing in distilled water, sections were mounted on chromalum/gelatin-coated microscope slides. They were dehydrated in a graded ethanol series and embedded in Entellan (Merck, Darmstadt, Germany). Whole-mount immunohistochemistry The procedure of whole mount staining is very similar to thin section staining, except for incubation and washing steps which are much longer to allow for permeabilization of antibodies into the middle of the tissue. Dissection and fixation step were performed as described in 2.2.1. After Fixation, brains were thoroughly rinsed in Tris buffered saline (TBS; containing 300 mM NaCl, 84 mM Tris-HCl, and 16 mM Tris-Base at pH 7.4) for six times each 20 minutes. Presence of the fixative would have destroyed enzyme activity in next step. In order to improve the antibody penetration into the deep tissue, brains were incubated in 1 mg collagenase/dispase (Roche, Mannheim, Germany) dissolved in 1 ml TBS for 40 minutes at 37°C. The collagenase and dispase are mild protease which disrupt the neurilemma around the brain and therefore, enhance the permeability of the antibodies. Subsequently, brains were rinsed in TBS containing 0.1% TrX six times each 20 minutes to wash out all enzymes from the brains. Then brains were preincubated in 5% NGS in TBS containing 0.3% TrX for overnight at 4 °C. Subsequently, brains were transferred into the primary antibody solution containing optimum dilution of GABA and PDF /or Synapsin antibodies (Table 2.1), 2% NGS and 0.02% sodium azide in TBS containing 0.3% TrX and incubated for 8 days at 4 °C with gentle agitation. Sodium azide is a bacteriostatic agent which prevents bacterial and fungi growth during long incubation. Following by several washing steps with TBS containing 0.1% TrX, brains were incubated in the secondary antibody solution containing GaM Cy2 (1:300) and GaR Alexa 674 (1:500), 1% NGS and 0.02% sodium azide in TBS containing 0.3% TrX for 6 Material and Methods 45 days at 4 °C with gentle agitation. After a final washing step, brains were dehydrated using graded ethanol series 50%, 70%, 90%, 96% and 100%. Afterwards, brains were transferred to methylsalicylate 50:50 100% ethanol- mixture for 15 minutes, followed by an additional incubation in the 100% methylsalicylate. Eventually, brains, were embedded in Permount media (Permount™; Fisher Scientific, Schwerte, Germany) on self-made coverslide. Quantification of GABA immunoreactivity For quantification of GABA immunoreactivity, animals were taken out of colonies at 6-hour interval during the day (ZT 0, ZT 6, ZT 12 and ZT 18) and their brains were dissected as described above (2.2.1). In order to ensure the comparability of staining-intensities, brains from different ZTs were treated in the same way and all incubation procedures were performed in the same glass container and the same solutions. Albumin/gelatin blocks were labeled by cutting a different corner of the block so that, later, sections of different ZTs could be separated. Brains embedded in albumin/gelatin were sliced in 100µm thickness. Except incubation time, the same procedure was applied as it was explained in the 2.2.3. Brain sections were incubated in primary antibody solution for four days and in secondary antibody solution for three days. Quantitative analysis of CLSM scans The optic lobe of all brains were scanned with an CLSM (TCS SP5; Leica Wetzlar, Germany) using the same scanning parameters. All sections were scanned with a HCX PL apochromate 20X/0.7 multi immersion objective (ECPlan: Neofluar Ph2M27; Zeiss) at 1,024 X 1,024 pixels per stack in z- direction with system optimized option of step size (0.64 µm). Quantitative analysis of GABA immunoreactive intensity in the distal tract (DT) was performed using Fiji, the open source image processing software based on ImageJ (Schmid et al. 2011), and subsequently evaluated with the statistical program Prism6 (GraphPad Software, San Diego, USA). Graphs were generated by the same program and edited with the open source software Inkscape vector graphic editor (version 0.91). Material and Methods 46 Using the Bio-Format plugin of ImageJ, stacks of confocal images were imported from Leica image files and then saved in a grayscale format. Only images of the stack showing at least part of the well-recognizable distal tract (Petri et al. 2002) were considered for maximum projection. The region of interest (ROI) in the distal tract was selected by using rectangle tools. The rectangle always was drawn in 70X45 µm2 size. The signal intensity information of every pixel in the ROI was taken from the ImageJ using the Histogram and the mean value of pixel intensity of the ROI was calculated. In order to consider only the signal derived from the antibody signal, background noise intensity (or pixel intensity above a certain threshold) 0-50 or 0-100 of an 8-bit system was excluded from all mean pixel intensity values. The values obtained from different ZTs were compared. The comparison of the values at which the intensities 0-50 and 0-100 were excluded, both caused just small dispersion in different measurements of the same set of samples and thus were considered as reliable results. However, only the results of analysis excluding background intensity of 0-100 were used for statistical comparison. Data were tested for normal distribution applying a Kolmogorov–Smirnov test. To test for significant differences in normally distributed datasets, we then applied a one-way ANOVA followed by a Tukey's multiple comparisons test. Neuronal tracing (backfill) combined with immunostaining Neuronal tracers make use of axonal transport mechanisms to characterize the neuroanatomical connections and innervation pattern of the nervous system. The tracer materials are carried actively within the nerve fiber and move either from the cell body toward the axon terminals (anterograde transport) or from the fibers back to the cell bodies (retrograde transport). Two neuronal tracers were used in this study, Neurobiotin™ Tracer (Vector Laboratories Inc., Burlingame, California, USA) and Dextran conjugated with the florescent dye Tetramethylrhodamine (TMR) (Molecular Probes, 3,000 MW, lysine fixable). Neurobiotin is an amino derivative of biotin that is widely used for intracellular labeling of neurons (Köbbert et al. 2000; Oztas 2003). It is known as both an anterograde and retrograde tracer. Due to its small size, it can transport effectively, resulting in labeling of even the finest fiber branches (Köbbert et al. 2000; Oztas 2003). To visualize the unlabeled neurobiotin, Material and Methods 47 different fluorescent dyes coupled to streptavidin (Cy™-2 or an Alexa® Fluor 405 Dye (Thermo Fisher Scientific, Waltham, USA) were used. Dextran is a hydrophilic polysaccharide with good water solubility and low toxicity. It has been used as a neuronal tracer for long-term tracing in live cells. Dextran is considered predominantly an anterograde tracer, although it can be transported retrogradely (Köbbert et al. 2000). To characterize the neuroanatomical connections and innervation pattern of the brain area related to circadian clock network, neuronal tracing was performed from several regions of the R. maderae brain and subsequently combined with immunohistochemistry method using GABA and PDF antibodies. General procedure For all kinds of backfill experiments, animals were first cold-anesthetized. Using fine pins and parafilm, animals were fixed on the wax filled trough so that the head and body side up. A constant flow of CO2 was used during operation in order to keep the animal anesthetized. Single backfill from the optic stalk Backfill experiments from the optic stalk were performed as described by Reischig et al. 2002. Briefly, a small window was opened in the head capsule (Figure 2.2). Insect saline was applied to prevent hemolymph coagulation during the operation. After removing the fat tissue and trachea, the optic lobe Figure 2.2. The open head capsule with the neurobiotin (tracer) filled glass capillary slipped over the optic stalk. Material and Methods 48 was exposed. Using ultra fine scissors, the optic stalk was cut. Then a glass capillary (GC150TF-7.5, Hugo Sachs Elektronik - Harvard Apparatus GmbH, March-Hugstetten, Germany) filled with 5% (w/v) neurobiotin was slipped over the cut optic stalk (Figure 2.2). The capillary was fixed with modelling clay and the animal was kept overnight at 4 °C in a wet chamber in order to allow intracellular transport of tracer. Backfill from the antennal nerve For backfill experiments labeling neurons of the antennal nerve, animals were fixed as described above under constant Co2 flow. With a sharp razor blade small window were open so that it covered antenna nerve (Figure 2.3). Then, glass capillary filled with 5% (w/v) neurobiotin was slipped over the antennal nerve and fixed with modeling clay. The tracer was allowed to transport overnight at 4°C in wet chamber. Backfills from the neurohaemal complex Since the neurohaemal complex consisting of the CC and CA laid in the posterior region of brain, in order to reach them, animals were fixed on the wax trough with the posterior region of head facing up. Then, with a sharp razor blade, a small square in the central area of the posterior head cuticle was cut. After removing all fat tissue and residues, the X-shaped milky structure of the CC and CA was exposed (Figure 2.4 A). In other experiments, the animal was fixed in the same way as explained for the optic stalk backfill. Here, the cuticle was cut at the center of the head capsule so that after removing the fat tissue Figure 2.3. The open head capsule with the dextran (tracer) filled capillary slipped over the cut antenna nerve. Material and Methods 49 the mid-brain was exposed. (Figure 2.4 B). Using fine forceps, carefully, the neurohaemal complex was pulled in the anterior direction from the middle of the brain between the tritocerebrum so that the nerves connecting the neurohaemal complex to the brain (NCC I) stayed intact. The complex was pierced in several places using forceps. Then 5% (w/v) neurobiotin filled glass capillary slipped over the end of the severed complex (either CC or CA, Figure 2.4 B) and fixed with the modeling clay, and the tracer was allowed to run overnight at 4°C in the wet chamber. Backfill from the thoracic ganglia (TG) In another set of experiments, animals were again fixed ventral side up. In order to get the first TG exposed, the foreleg and midleg were removed. Then, using a sharp razor blade and forceps, the area between the coxa was opened. Sometimes the cut continued up adjacent to the neck part in order to see the cervical connective to subesophageal ganglion (SOG). Subsequently, all the muscles, fat tissue and trachea which covered the TG were removed. Then, a glass capillary filled with 5% (w/v) neurobiotin either was inserted to the first TG or slipped over the cut thoracic nerve (between the first and second TG Figure 2.5) and fixed with the modeling clay. The tracer was allowed to run overnight at 4°C in the wet chamber. Figure 2.4. A) The rear view of the brain of the cockroach with neurohaemal organ which consists of the corpora cardiaca (CC), corpora allata (CA) and nervi corporis cardiaci I (NCCI). B) the open head capsule with the dextran (tracer) filled capillary slipped over the CC. Material and Methods 50 Backfill from the ocelli In this type of backfill, animals were treated as described for the backfill from the optic stalk. Neurobiotin was applied in different ways. Either one drop of 5% (w/v) neurobiotin was applied on the scratched ocellus, or a glass capillary filled with 5% (w/v) neurobiotin was applied on the ocellus and fixed with the modeling clay (Figure 2.6). Double backfill from the cut optic lobe and from the thoracic ganglia (TG) Figure 2.5. Schematic representation of the nervous system of Rhyparobia maderae showing the brain, the subesophageal ganglion (SOG) and the first two thoracic ganglia (TG1-2). The tracer filled capillary was slipped over the cut connective between second and first TG (red line). Modified after Andreas Arendt. Figure 2.6. picture of the open head capsule with the neurobiotin (tracer) filled glass capillary slipped over ocellus. Material and Methods 51 For double backfills from two regions, animals were pinned the same way as described for single backfills from TG and from cut optic stalk. For these set of experiments, 5% rhodamine-Dextran solution was used for cut optic stalk backfill and 5% (w/v) neurobiotin was use for TG backfill. Animals were kept overnight at 4°C in the wet chamber to allow intracellular transport of the tracers. The following day, glass capillaries were carefully removed and the brains were dissected, and fixated and processed as explained in 2.2.1. With the exception of some experiments in which backfills from either the ocellus or from the TG were performed and processed as whole-mount (see 2.2.5.), sections of the tissue were always made (see 2.2.3.). In order to visualize neurobiotin, different fluorescent dyes coupled to streptavidin (Cy™-2 or an Alexa® Fluor 405 Dye (Thermo Fisher Scientific, Waltham, USA) were used and applied simultaneously with the secondary antibodies used for immunostaining. Microscopy imaging and evaluation Confocal laser scanning microscopy (CLSM, TCS SP5; Leica Wetzlar, Germany) was employed to evaluate the stained brain sections. Sections were scanned with a HCX PL apochromate 20X/0.7 multi immersion objective at 1,024 X 1,024 pixels per stack in z-direction with a step size of 0.5 - 1 µm. Images stacks were merged using the maximum projection option. Cy2 fluorophore were excited with an argon laser 488 nm, Alexa 647 fluorophore with a helium/neon (He-Ne) laser at 633 nm and Cy3 with the 543 nm line of a He-Ne laser. All signals were sequentially scanned to avoid false-positive signals from the fluorophores. All images were imported auto-scale via Fiji image j software and arranged with either Corel DRAW Graphics Suite X7 (Corel, Ottawa, Ontario, Canada) or the open source software inkscape vector graphic editor (version 0.91). Image processing of the indirect peroxidase staining was done with Adobe Photoshop CS5 (San Jose, CA). Reconstruction of the GABA-ir cell bodies was done from consecutive frontal sections with a Leitz microscope (SM-LUX, Leica, Wetzlar, Germany) equipped with a camera lucida attachment. A Zeiss (Jena, Germany) Hagioscope equipped with CCD Material and Methods 52 camera (ProgRes C5; Jenoptik, Jena, Germany) and objectives with 10X (A Plan Ph1; Zeiss) or 20X (ECPlan: Neofluar Ph2M27; Zeiss) were used to count the GABA immunostained somata. Somata were counted in each section using the Abercrombie correction factor (Abercrombie 1946). Antibody characterization Rabbit polyclonal GABA antiserum (Sigma Aldrich; cat. no. A2052) raised in rabbit was conjugated to the bovine serum albumin (BSA) in order to serve as an immunogen. The polyclonal 5-HT antiserum (Sigma Aldrich, cat. no. S5545) also raised in rabbits against 5-HT coupled to BSA. Specificity of both antibodies was examined by means of liquid phase pre-adsorption of diluted antisera with different concentrations (100 µM, 10 µM, 1 µM and 0.1 µM) of synthetic GABA- and 5-HT-conjugates. GABA-immunostaining was abolished after pre-adsorption of the antiserum with 10 µM of GABA-BSA conjugate. Signals of the anti-5-HT was abolished after pre-adsorption with 100 µM of 5- HT. Moreover, cross-reactivity of the anti-GABA antibody with anti-5-HT was tested through pre-adsorption (with monovalent Fab fragments of secondary antibody method) with the diluted GABA and 5-HT antibodies at various concentrations of the 5-HT or GABA-BSA conjugate, respectively. No differences in the staining intensities were observed, thus, cross-reactivity can be excluded. Another polyclonal antibody used in this study was raised in guinea pig against GABA and was coupled to keyhole limpet hemocyanin (KLH) by glutaraldehyde (Table 2.1). The specificity of this antibody on the cockroach brain’s section was also tested through pre-adsorption of GABA- glutaraldehyde-KLH conjugated. Immunostaining was abolished after pre- adsorption of the antiserum with 10µM GABA KLH-conjugated (Schendzielorz and Stengl 2014). The monoclonal anti-drosophila-PDF antibody was obtained from the mouse lymphocytes in the cell culture. This antibody was widely employed to explore the circadian system of the R. maderae (Soehler et al. 2011; Schulze et al. 2013; Wei and Stengl 2011; Arendt et al. 2015; Stengl and Arendt 2016; Arendt et al. 2017). The specificity of the PDF antibody has been determined with Material and Methods 53 liquid phase pre-adsorption test (Schulze et al. 2013). In this test, only 10 µM of the Rhm-PDF abolished the staining completely. Pre-adsorption with Rhm- PDF in double staining experiments had no effect on the rabbit anti-histamine (Arendt et al. 2017), rabbit anti-MIP (Schulze et al. 2013), Guinea pig anti- GABA and rabbit anti-GABA staining. The specificity of the rabbit anti-histamine which was histamine-conjugated KLH with EDAC has been tested with preadoption of the antiserum with the various dilution of histamine-BSA conjugated. 100µM of the conjugated- histamine were necessary to abolish the staining (Arendt et al. 2017). 2.3. Biochemical experiments Animals Cockroaches R. maderae were reared under a 12:12 hours light/ dark (LD) cycles with lights on from 08:00 to 20:00 h at 50 % relative humidity and 25- 26°C RT. The animals were fed with dried dog food, apples, potatoes, salad, and water ad libitum. Adult male cockroaches were used for all experiments. Competitive ELISA Competitive ELISA (enzyme-linked immunosorbent assay) is commonly used to measure or quantify the small molecules including lipids, hormones and small peptides. In this method, either purified antigen or a specific antibody can be labeled with tracer. If the antigen is labeled, the unlabeled antigen from the sample and tracer-labeled antigen compete for binding to the related antibody. The more antigen in the sample, the less tracer will be able to bind to related antibodies and, therefore, result in a weaker signal (Figure 2.7). Quantification of unknown samples is attained by comparing their photometric absorbance with a standard curve constructed with known standards. The most widely used tracer is the horseradish peroxidase (HRP). The HRP can be conjugated to the antibodies as well as the antigens by several different methods, including glutaraldehyde and periodate oxidation through disulfide bonds (Harlow and Lane 1988). In this study, synthetic GABA (Sigma Aldrich cat. no. A2129) was conjugated to the HRP (Sigma Aldrich cat. no P77332) by using glutaraldehyde as coupling agent. Since rabbit polyclonal GABA Material and Methods 54 antiserum (Sigma Aldrich; cat. no. A2052) was conjugated to the BSA- in order to serve as an immunogen, samples and standards were also coupled with BSA (Roth cat. no 8076.2). Although in the description of the commercial GABA antibody it is mentioned that it is able to bind with unconjugated as well as conjugated GABA, our initial ELISA experiments clearly demonstrated that it is more sensitive to the GABA-BSA conjugated. GABA measurement in the 12:12 LD cycle Sample preparation Male, adult cockroaches R. maderae were taken from the colonies at 4 hour intervals throughout 24 hours (ZT times 0, 4, 8, 12, 16 and 20). To reduce the stress of the animals, they were kept in vessels together for a short time and then were cold-anesthetized. All preparation steps were performed on ice. Cockroaches were decapitated; the head was first fixed with two dissecting Figure 2.7. Schematic representation of the competitive ELISA. A) Goat-anti rabbit IgG is bound to the well. B-C) The competition between the GABA in sample and HRP-conjugated version of GABA (tracer) for limited number of specific binding site on the rabbit anti-GABA. The concentration of tracer is held constant in all wells, while GABA concentration varies from well-to-well. Therefore, the amount of tracer that can bind to the antibody will be inversely proportional to the amount of GABA in the well, the more GABA in sample means less tracer will be able to bind to the GABA antibody. D-E) Colorless TMB in presence of hydrogen peroxide (H2O2) by horseradish peroxidase turned to blue products. Subsequently, the reaction is stopped with sulfuric acid, whereby the photosensitive TMB is stabilized and the color changes to yellow. Finally, the absorbance in the well is determined with a photometer at 450 nm. Material and Methods 55 needles and then a rectangular window cut in the head capsule using a sharp blade. To prevent dehydration, further preparation of the brain was now carried out in insect saline (128 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 2 mM calcium chloride (CaCl2),1.2 mM sodium bicarbonate (NaHCO3), dissolved in double distil H2O, pH 7.25). Under the binocular (Leica, Wetzlar, Germany), fat tissues and tracheas were carefully removed so that the brain was exposed and, with ultrafine scissors, optic lobes were carefully separated from the brain. The optic lobes of two animals (=4 optic lobes) were then transferred to individual iced Eppendorf cups containing 400 µl of the phosphate buffer (PBS; 137 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 8.1 mM sodium phosphate dibasic dehydrate (Na2HPO4), 1.47 mM Potassium phosphate monobasic (KH2PO4); pH 7.4) containing 2% perchloric acid and 10 mg of 1 mm diameter glass beads (Biospec Products, cat.no 11079110). Samples were homogenized vigorously two times for one minute using a Mini-Bead Beater (Biospec Products, Bartlesville, USA) kept in 4°C. After homogenization, the mixture was centrifuged (Heraeus Fresco 17, Thermo Scientific, Schwerte, Germany) at 900 g for 15 minutes at 4 °C to remove cells and tissue debris, and the supernatant was used for the next step. For neutralization 300 μl of the supernatant was mixed in 300 μl chloroform/trioctylamine solution (1:1) and after mixing three times for 10 seconds, centrifuged at 500 g for 5 minutes at 4 °C. 200 μl of supernatant were taken and stored at -80 ° C until further measurement. Coupling reaction In this stage, synthetic GABA and samples were coupled to the BSA via glutaraldehyde as a cross linker. In addition, synthetic GABA was coupled to the HRP via glutaraldehyde as a coupling agent. Glutaraldehyde generally couples through amino groups of GABA, BSA and HRP. In order to prepare GABA-BSA and GABA-HRP conjugation the following protocol (Carter 1996) was implemented: 1- 6 mg of GABA (Sigma Aldrich cat. no. A2129) was weighed and dissolved in 50 ml PBS pH 7.0 (240 nmol / 200 µl). Material and Methods 56 2- 2 mg BSA and 2 mg HRP were separately weighed in Eppendorf cups and each dissolved in 200 µl of the prepared GABA solution. 3- Subsequently, 16 µl of a glutaraldehyde solution (Roth, 25% solution cat. no. 4157.1) was added to each cup to start the coupling reaction. The reaction was allowed to take place for 4 hours at RT under constant, gentle agitation. 4- 24 µl of 1M Tris Base pH 7.0 was added to stop the reaction. Solutions were mixed for 3 minutes and then incubated for one more hour at RT with gentle agitation. 5- In order to remove the undesired reactions or unreacted agents after coupling step, dialyze tubes (Novagen®, D-Tube™ Dialyzer Mini, MWCO cat.no 71505-3) with pore sizes which can retain proteins with M.W. > 12-14KDa were used to purify the GABA-HRP or -BSA combinations. The whole coupling reactions were pipetted into dialyze tubes and incubated for 3 nights in PBS (and buffers were refreshed twice a day) at 4 °C with gentle agitation. 6- In the end, solutions were collected from dialyze tubes and stored at - 80 °C for next step. For samples coupling with BSA, the following step was performed: 1. 2 mg BSA weighed in an Eppendorf cup and dissolved in 200µl of supernatant. Mix for 30 seconds on vortex mixer. 2. 16 µl of 25% Glutaraldehyde solution was added to start the coupling reaction. The solutions were incubated on a shaker for 3 hours at RT with gentle agitation. 3. Added 24 µl of 1M TRIS base pH 7.0 to stop reaction and mix for 3 minutes on vortex mixer. Incubated for 1 hour at RT. 4. Pipetted the solution in dialyzing tubes. Put these tubes in 250 ml PBS for 3 days (membrane of the tubes must be surrounded by the buffer; changed PBS at least 2 times per day). 5. Weighed a new cup, transferred the whole solution of the dialyzing tube into this cup and then weighed it again in order to check the volume (weight). Material and Methods 57 GABA measurement in the constants darkness To determine if the fluctuation in GABA levels carry through a circadian basis, GABA levels were quantified at the same 4 intervals throughout the 24 hours of circadian time. Thus, animals were taken out at ZT 0, 4, 8, 12, 16, 20 and were isolated at the end of the night in separate vessels for 48 hours under constant darkness. Dissection was performed during the second day of constant darkness under red light conditions during the corresponding circadian time window as explained above. Each sample again contained 4 optic lobes (=2 animals). Sample processing were done similarly as described above. GABA measurement in the short and long day photoperiods Further experiments were conducted in which GABA levels were measured in cockroaches which acclimated to long and short photoperiod condition to establish whether duration of light phase had an influence on the GABA variation or not. Therefore, cockroaches were acclimated to two different LD cycles: 18 h light-6 h dark (LD 18:6) and 6 h light-18 h dark (LD 6:18) for at least three months. Afterwards, animals were taken from the colonies at 6 intervals throughout the 24 hours (ZT times 0, 6, 12, 18). Dissection and sample preparation were performed as explained above. Manufacturing of competitive ELISAs In the first step, microtiter plates (Nunc, Maxisorp, C-Bottom, Thermo scientific, Denmark) were coated with 125 µl (15µg/ml) goat-anti rabbit IgG (Dianova, Hamburg, Germany), dissolved overnight at 4 °C under agitation in phosphate buffer (PBS; 137 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 8.1 mM sodium phosphate dibasic dehydrate (Na2HPO4), 1.47 mM Potassium phosphate monobasic (KH2PO4); pH 7.4). Microplates were protected against evaporation with self-adhesive foil. After washing the wells one time with 250 μl washing buffer (WB; 0.05 % Tween® 20 in PBS), in order to block non-specific binding (NSB) sites, 250µl of blocking buffer (BB; 1%BSA in WB) were added in each well and incubated one hour at 37 °C. Afterward, wells were washed three times briefly with WB. Samples were diluted 1:10 in Material and Methods 58 PBS and 25 µl of each pipetted in the well, followed by 25 µl of rabbit-anti GABA antibody at a final concentration of 1: 20,000 in BB. For each sample, always 4 different wells were used for quadruple determination. GABA standards were determined in concentration range of 2.5, 5, 10, 20, 40 and 80 pmol / 25 μl dissolved in PBS, followed by 25 µl of rabbit-anti GABA antibody at a final concentration of 1: 20,000 in BB. In addition, four wells were each loaded with 25 μl of PBS and GABA antibody for the maximum signal (B0) determination and four other wells with 25 µl PBS without GABA antibody (But 25µl BB) for the determination of nonspecific binding (NSB). Microplates were covered with self-adhesive foil and incubated overnight at 4 °C with agitation. The following day, competitive reaction was carried out by quickly pipetting 25 µl of GABA-HRP conjugated at a final concentration of 1:600 in BB. After 30 minutes’ incubation time at 4 °C, without shaking, the plate was emptied and wells were washed three times with WB. For detection, 25 μl of developing solution I (0.02% H2O2, 0.3% H3PO4 dissolved in 100 mM citrate buffer, pH 5) and 50 μl of developing solution II (420 mM 3,3',5,5'-Tetramethylbenzidine (TMB), 0.7% dimethylsulfoxide (DMSO), 0.1% phosphoric acid (H3PO4), dissolved in H2O) in the wells and developed at RT for 30 minutes on the shaker. The reaction was stopped by the addition of 25 μl 2 M Sulfuric acid (H2SO4). The photometric analysis was carried out at 450 nm using microplate reader (POLARstar, BMG Labtech, Ortenberg, Germany). Analytical Specificity (Cross reactivity) To check the possible cross reaction between GABA and its analogues, competitive ELISA was performed with the two GABA metabolites glutamine and glutamate. The procedure of the coupling reaction with BSA was carried out as it was described above for GABA-BSA conjugation. A similar competitive system was constituted for these two metabolites. A competitive reaction was performed considering the concentrations 20 nmol, 2 nmol, 200 pmol, 20 pmol, 2 pmol, 200 fmol, 20 fmol and 2 fmol for both glutamine and glutamate. No significant cross reactivity or interference between GABA and glutamine or glutamate was observed. Evaluation of the Data Material and Methods 59 The amount per well was multiplied by the dilution factor and normalized by calculating a quotient of GABA concentration by corresponding protein concentration. The determined NSB value was subtracted from the other measured data points. For regression fit, LOG of applied GABA concentrations was plotted against measured values divided by B0 and multiplied by 100. GABA concentrations of samples were calculated using the following equation: y = 100 1+10(log(EC50−X)×𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻 The coefficient for determination of the regression fit was always above 0.95. Statistical analysis First, the Shapiro-Wilk normality test was used to check the distribution of data sets. Nonparametric tests were applied for data sets that were not normally distributed. Six different ZTs or CTs per day were analyzed to determine GABA concentrations by using the Kruskal-Wallis test followed by Dunn’s post hoc test. In order to get a single estimate for GABA levels during the day and during the night, the data from ZT (and CT) 4 - 8 and ZT (CT) 16-20 were pooled together. The same test was used to evaluate GABA levels in four ZTs under the long and short day photoperiod conditions. GABA levels were presented in the text as mean values and in figures as column bars with mean ± standard error. Results 60 3. Results To analyze the distribution of GABA in the brain of the Madeira cockroach R. maderae brain, indirect peroxidase immunostainings as well as fluorescence immunocytochemistry with an antibody against GABA were carried out. Additionally, double-label immunocytochemistry with antibodies against GABA and PDF as marker for circadian clock neurons was performed. Thus, it was evaluated whether GABA-immunoreactive (-ir) neurons could be pre- or postsynaptic to the PDF-expressing circadian pacemaker neurons in the cockroach brain. In search for possible direct connections between GABA-, serotonin-, or PDF-ir neurons with the terminals of the histaminergic short or long photoreceptor axons of the compound eyes, double-label immunocytochemistry with respective antisera were performed. Furthermore, with a series of backfills combined with immunocytochemistry using antisera against GABA and PDF possible direct neuronal connections were identified between the circadian clock and other neuropils that process sensory information (e.g. antennal lobe, ocellar neuropils) or premotor areas, where ascending and descending neurons arborize. 3.1. Overview of GABA immunoreactivity in the brain of the Madeira cockroach R. maderae Distribution of GABA-immunoreactivity in the midbrain GABA-ir soma groups in the midbrain In the midbrain of R. maderae 1989.56 ± 174.06 neurons (n=5 brains evaluated) exhibited GABA immunoreactivity (Table 3.1). Most GABA-ir somata were arranged in groups, termed G1-10. One conspicuous group which innervated the antennal lobes was termed lateral cluster (LC) (Figure 3.1 - Figure 3.4). All GABA-ir clusters were symmetric in both hemispheres. Since there were too many GABA-ir neurons in the central brain, reconstruction of the projection patterns of single neurons from most soma groups was not possible. However, the number of neurons in three GABA-ir cell clusters (G1, G9, LC; n=5 brains) were counted. The median dorsal (G1) and posterior median (G9) area of the protocerebrum (Table 3.1) contained Results 61 together 77.76 ± 4.70 GABA-ir neurons that could not be clearly separated. They projected to the superior median protocerebrum (SMP), to the anterior protocerebrum, and around peduncle and vertical lobe of the mushroom body (Figure 3.2 B-C, Figure 3.3 D). Several GABA-ir commissures at the midline connected the SMP of both hemispheres (Figure 3.2 B, Figure 3.3 A-B). Additionally, in the most posterior region of the brain, where there is no separation between both hemispheres, inferior neuropils were located. In its medial region, there were various GABA-ir commissures which extended laterally to each hemisphere (Figure 3.3 B). Table 3.1. Distribution of GABA-ir cell clusters (G) in the central brain (n=5); values are given as arithmetic means ± standard error of mean (SEM). Median dorsal protocerebrum neurons (MDPNes) G1 and posterior median protocerebrum neurons (PMPNes) G9, GABA-ir cell clusters (G1 and 9), lateral cluster (LC). Number (SEM) Size µm (SEM) Total 1989.56 ± 174.06 20.26 ± 0.51 Antennal lobe soma(LC) 281.68 ± 48.10 23,17 ± 0.86 MDPNes(G1), PMPNes(G9) 77.76 ± 4.70 21.89 ± 0.74 Results 62 GABA-ir neuropils in the protocerebrum In the median protocerebrum, all main neuropils such as the central complex, the mushroom bodies, superior and inferior protocerebrum showed GABA immunostaining, however, with different intensities. The GABA immunoreactivity in the central complex was particularly prominent in the lower division of the central body as well as in the bulbs (lateral triangles) of both lateral complexes (lateral accessory lobes) (Figure 3.2 C). Figure 3.1. The majority of the GABA immunoreactive(-ir) cell bodies form bilaterally symmetric clusters (G1-10) in various regions of the central brain. Frontal camera lucida drawing of GABA immunostained somata in the central brain. GABA-ir somata of the (A) anterior and (B) posterior brain. Antennal lobe (AL), calyces (CA), central body (CB), lateral cluster (LC), medial lobe (ML), peduncle (PED), vertical lobe (VL). Scale bar 100 µm. Results 63 Figure 3.2. Strongest GABA immunostaining was observed in the lower division of the central body (CBL), the bulbs, and the glomeruli of the antennal lobe. Consecutive frontal sections from anterior (A) to posterior (C) show GABA immunoreactivity in different neuropils of the central brain. A) Frontal section shows lack of GABA-ir in the vertical lobes (VLs) of the mushroom body. In the mushroom body calyx (CA) GABA immunoreactivity is confined to the outer layer of the calyces (OCA; B-D). The superior lateral protocerebrum (SLP), superior medial protocerebrum (SMP), and the antennal lobe (AL) exhibit prominent staining. The knee of the peduncle which is called peduncle divide (PEDD) contains a diffuse meshwork of immunoreactive fibers (filled arrowheads in A and B). B) A cluster of 77.76 ± 4.70 somata (G1) located in the posterior median and the dorsal median protocerebrum (G9 not shown) send arborizations to the superior median protocerebrum (SMP) and to the anterior protocerebrum, around the PED and the VL of the mushroom body. Several stained fibers in the midline connect the SMP of both hemispheres (open arrowhead). C) Strongest GABA- immunoreactivity is found in the lower division of the central body (CBL) and the bulbs (BU), next to less intense staining in the lateral accessory lobes (LALs). GABA-ir somata located in the inferior median protocerebrum (G5) as well as cell bodies more laterally, adjacent to the inferior median protocerebrum (G4, not shown) send processes into the LAL and entered the CBL. The upper division (CBU) exhibits diffuse GABA-ir staining. Several GABA-ir fibers passed through the outermost midline of the medial brain (arrows). D) Strongly stained GABA- ir neurons reside in the dorsal lateral protocerebrum (G7). A small cluster of these neurons (filled arrowhead) send projections to the calyces (CA) of the MB. These neurons have beaded ramifications throughout the OCAs. The axons of the other GABA-ir somata (open arrowhead) Results 64 Immunoreactivity in the lower part of central body originated from two bilateral cluster of tangential neurons located in the inferior median protocerebrum (G5) as well as from more laterally located cell bodies adjacent to the inferior median protocerebrum (G4) (Figure 3.2 C, Figure 3.11 D). Immunolabeled fibers of these soma groups connected the lateral accessory lobe of each hemisphere to the lower division of the central body, which was partitioned into eight subdivisions (slices) (Figure 3.2 C, Figure 3.11 D). The arborization pattern of these neurons in the lateral accessory lobes could not be differentiated any further due to the homogeneity of the staining. In contrast, the upper part of the central body contained sparse GABA-ir fine fibers (Figure 3.2 C) and the protocerebral bridge and noduli were free of GABA staining. In the mushroom bodies, GABA immunostaining was mainly observed in the outer layer of the calyces (OCA) with beaded fiber specializations. Apparently, these arborizations originated from the GABA-ir cell groups G7 and G1. The vertical lobe (VL) and medial lobes were devoid of staining. However, the peduncle divide (knee of pedunculus) was invaded by GABA-ir fibers (Figure 3.2 A-B, Figure 3.8 B). The GABA immunoreactivity of the SMP and SLP embedded the mushroom bodies and the central complex (Figure 3.2, Figure 3.3 A-B). GABA-ir cell clusters of G2 and G3 sent their primary neurites into neuropils of the superior protocerebrum, while G7-G8 innervated neuropils of the inferior protocerebrum (Figure 3.2 D, Figure 3.11 B-C). Since GABA staining was mainly uniform, different neuropils could not be distinguished clearly. Thus, GABA immunoreactivity in the SMP and SLP continued into neighboring neuropils which were located more posteriorly. innervate the SLP. The lateral protocerebrum is invaded by a fascicle of neurites that originate from a cluster of GABA-ir somata located in the posterior lateral protocerebrum (G8) (double open arrowhead). Scale bar 100 µm. Results 65 GABA-ir neuropils in the deutocerebrum and tritocerebrum In the deutocerebrum, the GABA antisera quite strongly labeled both antennal lobes (Figure 3.4). Neurobiotin backfills from the cut antennal nerve combined with anti-GABA immunocytochemistry revealed that apparently all glomeruli of the antennal lobe exhibited GABA-immunoreactivity (Figure 3.4 A-D). Neurobiotin labelled axons of the olfactory sensory neurons in the antennal Figure 3.3. Several GABA immunoreactive (-ir) fiber tracts connect both brain hemispheres in the median protocerebrum. Frontal (A, B, C) and horizontal (D) vibratome sections show GABA immunoreactivity in various areas of the mid brain. A) In the most anterior region of the central brain, several commissures (filled arrowheads) connect the superior medial protocerebrum (SMP) of both brain hemispheres. B) GABA-ir fiber bundles (filled arrowheads) in the most posterior region also connect different neuropils of the ventromedial neuropils (VMNP) including the superior posterior slope (SPS) and the inferior posterior slope (IPS). C) Uniform GABA immunostaining in the antenno-mechanosensory and motor center (AMMC) belongs to the deutocerebrum behind the antennal lobe. The AMMC is innervated (open double arrowheads) by the lateral cluster of prominent GABA-ir somata (G10) (double open arrowheads). D) GABA-ir somata residing in the median dorsal protocerebrum (open double arrowheads) send projections into the SMP, the superior lateral protocerebrum (SLP) as well as around the peduncle (PED) and the vertical lobe (VL) of the mushroom body. Scale bar 100 µm. Results 66 nerve terminated in the cap (cortex) region of each glomerulus which sparsely showed GABA- and to greater extent GAD immunoreactivity (see part 3.1.3 Figure 3.8 C). In contrast, the core area of each of the glomeruli was strongly innervated by GABA-ir interneurons and was free of neurobiotin staining. A GABA-ir soma group situated laterally to each antennal lobe (lateral cluster LC) exclusively projected to the antennal lobe and densely innervated the neuropil core of the glomeruli and the core region of the antennal lobe, the so- called hub (Figure 3.4 A-C). Apparently, the LC comprised mostly of local interneurons that restricted their arborizations to the glomeruli of the antennal lobe. But the LC contained also projection neurons that connected mostly posterior glomeruli via the medio-lateral antennal lobe tract to the lateral horn of the lateral protocerebrum and to the calyces of the mushroom body (Figure 3.4. D). Posteriorly to the antennal lobe, the antenno-mechanosensory and motor center (AMMC = dorsal lobe) displayed homogeneous GABA staining. The AMMC is an unstructured neuropil that has no clear boundary to the neighboring neuropil such as the protocerebrum and tritocerebrum. GABA immunoreactivity in the AMMC also showed uniform and continuous staining with the tritocerebrum. A big cluster of relatively small GABA positive somata (G10) lies laterally to the AMMC, innervating it (Figure 3.3 C). Due to the uniform GABA staining in the tritocerebrum, it was often difficult to distinguish GABA immunoreactivity in the glomerular lobe. However, the glomerular lobe was clearly distinguishable with GAD immunostaining. However, it remained undiscerned which soma groups innervate the glomerular lobe (see part 3.1.3. Figure 3.8 F). Results 67 Figure 3.4. In the antennal lobe (AL) GABA immunoreactivity is restricted to the core of the glomeruli (GL) as well as to the central coarse neuropil while backfilled antennal sensory neurons terminate in the cortex (cap) of the glomeruli. Frontal sections show GABA immunostaining (A, C) in the AL combined with neurobiotin backfills from the cut antennal nerve (AN, B, C). A) The core of each GL is densely filled by GABA-ir fibers. They are derived from the lateral cluster (LC) of mostly local interneurons, but also of projection neurons located anterior- dorsolaterally to the AL. B) Neurobiotin backfill from the cut antenna nerve (AN) stain axons of olfactory sensory neurons (OSNs) which form the cap of each glomerulus. The core of the glomeruli was devoid of OSN terminals. C) The overlay shows that the AL core neuropil exhibits strong GABA immunoreactivity (stars) whereas it is devoid of neurobiotin labeling. D) Frontal view of GABA staining in the most posterior brain regions. Most neuropils of the brain exhibit GABA immunoreactivity, including the neuropils of the optic lobes (OLs) such as the lamina (LA), medulla (ME), and the lobula (LO), the lateral protocerebrum, the superior lateral protocerebrum (SLP), the lateral horn (LH), the calyces of the mushroom body (CA), and several fiber tracts in the median protocerebrum (filled arrowheads). GABA-immunostained somata in the ME are scattered in the dorsal and ventral soma rind (rMEd and rMEv). GABA-ir fibers in the mediolateral antennal lobe tract (ml-ALT) project from the posterior region of the antennal lobe (AL) to the lateral horn (LH) and to the CA. The antenno-mechanosensory and motor center (AMMC) is strongly stained. All images are maximum projections from stacks of images. Scale bars 50 µm in A, B, C and 100 µm in D. Results 68 GABA-ir soma groups in the optic lobe All neuropils of the optic lobe showed GABA immunostaining, however, to different extends (Figure 3.5 A). Neither photoreceptor neurons nor cell bodies near the lamina exhibited GABA immunoreactivity. However, in the lamina neuropil four GABA-ir strata could be identified. Prominently stained processes were detected in the proximal lamina (Figure 3.5 B) and in the first optic chiasm. Apparently, all these GABA-ir processes derived from medulla group 1 (M1) columnar, centrifugal neurons posteriorly to the proximal face of the medulla. The profile of this type of neuron is identical to the centrifugal (C2) neuron which was described in the dipteran lamina (Strausfeld 1970; Strausfeld and Campos-Ortega 1977) and Hymenoptera (Datum et al. 1986b). These neurons mainly connect specific columns of the retinotopic map of the medulla to specific columns of the retinotopic map of the lamina, sending branches into specific layers of these optic neuropils. In addition, at least two small non-retinotopic neuropils at the ventro- and dorso-posterior edge of the lamina displayed GABA-ir varicose fibers. These two neuropils were named accessory laminae (Singular: ALA; Plural: ALAs) (Loesel and Homberg 2001b) and stained fibers extended from there over the anterior surface of the medulla through the second optic chiasm (Figure 3.5 A). However, the origin of this fibers could not be traced to any specific somata. The cell bodies of the GABA-ir neurons in the optic lobe were mainly arranged around the medulla and lobula. Based on the location and projection pattern of processes of the GABA-ir somata four medulla (M1-4) and two lobula (LO1- 2) cell groups (Figure 3.6, Figure 3.5) could be distinguished, whereby M1 and M2 were retinotopically organized. The M1group was located at the proximal side of the medulla and connected the respective columns of the retinotopic maps of the medulla and the lamina. M2 are most likely columnar transmedullary neurons. Their somata are located at the distal face of the medulla. They projected through the second optic chiasm to the lobula and connected the columns of the retinotopic maps of medulla and lobula (Figure 3.6 A). They formed side branches into one or several strata of the medulla and innervated the outermost layer of the lobula. Results 69 Figure 3.5. GABA-immunoreactive (-ir) neurons appear to contribute to many different circuits of optic lobe neuropils, with strongest immunoreactivity in two layers of the medulla (ME). Horizontal (A-D) and frontal (E) vibratome sections show GABA immunoreactivity in the optic lobe of the Madeira cockroach. A) In this overview it becomes apparent that all optic lobe neuropils, lamina (LA), ME, and lobula (LO) exhibit GABA immunostaining. B) In the LA GABA staining is most prominent in the inner layer of the LA (filled arrowheads) and in the accessory laminae (ALAs), at the proximal border of the LA. GABA-ir fibers in the first optic chiasm (OCH1) originate from the medulla centrifugal neurons (M1 in A) that connect ME and LA. C, D) Horizontal section through the ME. C) Strongly GABA- ir fibers of the distal tract (DT) bifurcate near the AME connecting the glomeruli of the AME to the ME. Additionally, thick GABA-ir processes connect the AME with the median layer fiber system of the ME, forming the medial layer fiber tract (MLFT). D) Large GABA-ir somata located anteriorly on the distal face of the LO sent their thick fibers centripetally around the LO, along with other fibers in the LOVT towards the midbrain (not shown here). E) Frontal optic lobe section shows GABA-immunoreactivity in the lamina organ (LAO), an elongated structure (filled arrowheads) which runs along the anterior margin of the lamina (LA). Scale bars 100 µm in A-E. Results Figure 3.6. GABA immunoreactive(-ir) somata of the optic lobe were grouped in the medulla 1-4 (M1-4) and lobula 1-2 (LO1-2) cell groups. Reconstruction of GABA-ir soma groups in the optic lobe in horizontal view. GABA-ir somata of the dorsal (A) and ventral (B) optic lobe. A) The ME neuropil is surrounded by numerous GABA-ir cell bodies. The ME cell group 1 (M1, red) is located on the proximal side of the ME. They centrifugally send their neurites into specific columns of the retinotopic map of the ME, continuing on through the first optic chiasm, innervating the respective columns of the retinotopic map of the lamina with arborizations mostly in the proximal lamina (LA). The M2 cells (blue) are located on the dorso- and ventro-distal face of the ME. They appear to connect corresponding columns of the ME and lobula (LO) via long processes that travel via the lobula valley tract (LOVT) along the proximal edge of the ME and through the second optic chiasm. GABA-ir lobula cell group 1 (LO1) lies on the most distal side of the LO. B) The ME cell group 3 (M3, green) has their somata dorsomedially to the ME. This cell group sends their processes to layers 7-9 of the ME. The M4 (magenta) somata are close to the ventral edge of the ME and innervate the accessory medulla (AME). A group containing big somata (LO2) ventrally between the proximal ME and the distal LO possess long neurites that pass centripetally and distally over the LO. Via the LOVT these fibers continue to the central brain regions. The distal tract (DT), median layer fiber tract (MLFT, brown) and the anterior fiber fan (AFF, light brown) are three main tracts which connect the AME to the ME and to the LA. Ventral accessory laminae (vALA). Scale bar 50 µm. The medulla cell cluster M3 are apparently amacrine cells of the distal medulla (Dm) which arborize exclusively in the distal medulla and are often restricted 70 Results 71 to one stratum (Figure 3.6 B, (Fischbach and Dittrich 1989). The GABA-ir medulla group M4 are located at the ventro-anterior border of the medulla. They belong to the AME neurons which innervate the AME (see part 3.2.1. Figure 3.6 B). They are tangential neurons with wide spread projections to ipsi- as well as contralateral neuropils of the midbrain. The lobula soma group one (LO1) cell bodies were located at the distal surface of the lobula, near the inner border of the medulla (Figure 3.6 A). The projection pattern of these neurons could not be discerned any further due to the uniform GABA staining in the lobula. However, they are possibly lobula intrinsic neurons (Li 1). The largest somata laying anteriorly to the optic lobe on the distal side of the lobula were assigned to the lobula neuron group 2 (LO2). They possessed thick neurites that run centripetally and distally around the lobula, in close contact to M4 (Figure 3.6 B, Figure 3.5 D). They are apparently lobula intrinsic neurons (Li 2) (Fischbach and Dittrich 1989; Strausfeld 1970; Strausfeld and Campos- Ortega 1977). Another structure which showed GABA immunoreactivity was the lamina organ. In some preparations an elongated structure located proximal to the lamina and between the lamina and the medulla extended across entire width from the dorsal to the ventral exhibited GABA staining (Figure 3.5 E). However, no connection between the lamina organ and any part of the optic lobe was observed. Glutamic acid decarboxylase (GAD) antibody is a reliable marker for GABAergic neurons With an antibody against GAD, the biosynthetic enzyme for GABA, GABA immunocytochemistry was checked. Double-label immunostaining with anti- GABA and anti-GAD antibody resulted in almost identical staining in the brain of the cockroach R. maderae. However, overall GAD immunostaining was more prominent especially in the fibers than anti-GABA staining (Figure 3.7). In the somata, GAD immunoreactivity was concentrated in the cytoplasm, while GABA immunoreactivity was evenly distributed in whole cell bodies (Figure 3.7). The GAD staining distribution in main neuropils of the central brain was identical to the GABA immunoreactivity (Figure 3.8). However, GAD- immunoreactivity in some regains was more prominent and clearer than GABA staining. In the medial lobe some fibers at the midline, where the two medial Results 72 lobes meet showed GAD immunoreactivity, but no GABA immunoreactivity (Figure 3.8 D). Moreover, the glomerular lobe at the border of the tritocerebrum displayed more clearly GAD-immunostaining than GABA staining (Figure 3.8 F). Figure 3.7. GABA- and glutamate acid decarboxylase (GAD)-immunocytochemistry stained the same neuropils and soma groups in the optic lobe of the Madeira cockroach. Frontal section through the optic lobe showing GABA- (A) and GAD (B)- immunoreactivity in the medulla (ME), lobula (LO), accessory medulla (AME), distal tract (DT) and medial layer fiber tract (MLFT). Images are maximum projections from stacks of optical sections. Scale bars 100 µm. Results 73 3.2. What is the role of GABA in the circadian system of R. maderae? Six of seven soma groups next to the accessory medulla exhibited GABA-immunoreactivity Figure 3.8. Glutamate acid decarboxylase (GAD) immunocytochemistry stained the same central brain neuropils and somata as shown with GABA-antisera. Confocal laser images obtained from frontal sections of different regions of the central brain of the cockroach R. maderae show GAD immunoreactivity. A, B, C, D are anterior, E and F are posterior. A-B) GABA-ir cell cluster G4 projects to the peduncle (PED) of the mushroom body (filled arrowhead). C) GAD-immunoreactivity observed in the core (star) as well as the shell (double filled-arrowhead) of the glomeruli (GL) of the antennal lobe (AL). D) prominent GAD- immunoreactivity in the lower division of the central body (CBL), the bulb (BU) and the superior median protocerebrum (SMP). Fine GAD-ir fibers project from the top of the CBL slices rise to the upper division of the central body (CBU) (open arrowhead). Additionally, some fibers at the midline between both medial lobes (ML) showed GAD staining (double open arrowheads). E) GAD-ir projection neurons of the antennal lobe connected posterior glomeruli via the medial antennal lobe tract (mALT) to the calyces (CA) of the mushroom body and to the lateral horn (LH) of the lateral protocerebrum. F) GAD immunoreactivity in the most posterior region of the brain stained the antenno-mechanosensory and motor center (AMMC) of the deutocerebrum, the inferior lateral protocerebrum (ILP), and the glomerular lobe (LG) the tritocerebrum. Images are maximum projections from stacks of 7-12 optical sections (z-distance between single sections= 0.63 µm). Scale bars in B 50 µm and 100 µm in A, C, D, E and F. Results 74 In the AME strongest GABA immunoreactivity occurred in the gloumerlar cores. The interglomelar regions and to lesser extent also the shell regions of the AME displaed sparsly but homogeneously GABA immunostaining. Total 32.76 ± 3.56 GABA-ir neurons were found in six AME soma groups (Figure 3.9, Table 3.2). Most of the GABA-ir neurons belonged to the VNe (13.56 ± 2.22). The smallest GABA-ir AME neurons belonged to the MFVNes. Other GABA-ir neurons belonged to the DFVNe, VPNe, VMNe and MNe (Table 3.2). Since GABA-ir neurons next to the AME were clusterd and closely packed next to each other, detailed assignment of the projection pattern of the most of these neurons was not possible. However, GABA-ir MNes which possesed the largest somata with strong immunostaing, send their primary neurites to the dorsal glomerulus (Figure 3.9. A-B). Moreover, the somata of GABA-ir VPNes and VMNes send long neurites to the ventro- proximal region of the AME (Figure 3.9, B-C). Figure 3.9. Six of seven identified soma groups next to the accessory medulla (AME) exhibit GABA-immunoreactivity. Frontal sections of the left optic lobe cut from anterior (A) to posterior (B, C). A total of 32.76 ± 3.56 GABA-immunoreactive (-ir) neurons appear to innervate the AME. They were assigned to median neurons (MNes), ventral neurons (VNes), medial-frontoventral neurons (MFVNes), distal-frontoventral neuron (DFVNes), ventromedial (VMNes) and ventro-posterior neurons (VPNes). A) One MNe innervates a dorsal glomerulus (white arrowhead) of the AME. B) GABA-ir fibers of the distal tract (DT) bifurcate and branch in the AME´s glomeruli (white arrowhead). To a lesser extent also the interglomerular regions and the shell of the AME are, GABA-ir. C) Some fibers of the shell project posteriorly via the lobula valley tract (LOVT) to the midbrain. The AME is encircled for clarity. Scale bars 50 µm. Results 75 Table 3.2. Distribution of GABA-ir neurons in the cell groups associated with the AME (n=8); Values are given as arithmetic means ± standard error of mean (SEM). median neurons (MNes), ventral neurons (VNes), medial-frontoventral neurons (MFVNes), distal-frontoventral neurons (DFVNes), ventromedian (VMNes), ventro-posterior neurons (VPNes). Number (SEM) Size µm (SEM) Total 32.76 ± 3.56 15.63 ± 0.70 MNes 5.11 ± 0.57 22.97 ± 1.82 VNes 13.56 ± 2.22 15.19 ± 0.42 MFVNes 3.35 ± 0.95 11.74 ± 0.66 DFVNes 3.95 ± 0.68 12.27 ± 0.68 VPNes 3.39 ± 1.09 14.21 ± 0.99 VMNes 3.46 ± 0.78 14.22 ± 1.01 Three distinct GABA-ir tracts connect the AME to the medulla and/or to the lamina To search for direct connections between GABA-ir neurons and PDF- expressing circadian pacemaker neurons in the cockroach brain, double-label immunocytochemistry with antisera against GABA and PDF was performed. In this chapter, I focus mainly on the connections between the AME and other optic lobe neuropils. Additionally, using other multiple-label staining such as GABA- combined with histamine-immunocytochemistry and acetylcholinesterase (AChE) histochemistry combined with GABA immunocytochemistry, the medulla neuropil was analyzed further. The medulla neuropil displayed a complex organization of strata and columns with widespread GABA-immunoreactivity (Rosner et al. 2017). With AChE histochemical staining combined with GABA immunostaining at least ten strata with different staining intensity and different thickness could be distinguished. Layers could be distinguished best in horizontal sections. The first medulla layer ME1 (counting from the outer chiasma) which comprises the PDF-ir AFF also contained thick GABA-ir fibers. GABA-ir fibers of the AFF connect the AME to medulla and lamina (Figure 3.10). The second GABA-ir tract is the distal tract that also projects via ME1. The GABA-ir distal tract connects the core of the glomeruli of the AME to different layers of the medulla (Figure 3.10). The distal tract can be traced to more than three-quarters of the anterior edge Results 76 of the medulla in horizontal sections (Figure 3.10 A). It connects the AME only to different layers in the medulla but does not project to the lamina. Along the anterior surface of the medulla several immunostained processes of the distal tract run perpendicularly into the medulla toward ME4 while passing ME2 and ME3 (Figure 3.15 C). Layer ME2 of the medulla which is the termination site of the long photoreceptor axons, and ME3 exhibited sparser GABA-labeling as compare to other layers (see part 3.3.1; Figure 3.15). In contrast, ME4 displayed strongest, bushy GABA staining. In ME4 also the PDF-ir medial layer fiber (MLF) system is situated (Figure 3.15). I discovered a new GABA-ir tract which I called medial layer fiber tract (MLFT). The GABA-ir MLFT is the third tract that appears to strongly connect the AME to ME4 (Figure 3.10). ME5 and ME6 had a similar pattern of GABA immunoreactivity as ME2 and ME3, but the staining was denser. The weakest GABA staining was observed in ME7. Fairly stained layers ME8 and ME10 were separated by the diffusely stained ME9 (Figure 3.5 A, Figure 3.10). In summary, three different GABA-ir tracts (Figure 4.1), the distal tract, the MLFT and the GABA-ir branch of the AFF, connect the AME to the MLF system and possibly also to other layers of the medulla. Among these three tracts, only the AFF connects the clock to the lamina and to the ALAs. Finally, GABA-ir fibers in the lobula valley tract connect the AME to midbrain targets. Results 77 Figure 3.10. Three GABA-ir tracts, the distal tract (DT), a bundle in the anterior fiber fan (AFF), and the medial layer fiber tract (MLFT), connect the AME to the medulla (ME). Confocal laser images obtained from horizontal sections of the optic lobe of the cockroach R. maderae showing GABA (magenta) and PDF (cyan) immunoreactivity (A-B). Dependent on the density of the GABA staining, three layers in the lamina (LA) and 10 layers in the ME could be identified. The strongest GABA immunoreactivity was found in medulla layers one (AFF) and four (MLFT) (filled arrowhead), where also PDF-immunoreactive (-ir) fibers arborize. The glomeruli of the AME are densely innervated by the GABA-ir DT and MLFT. All three GABA- ir tracts appear to connect the AME to layer four of the medulla, while the AFF connects it to the proximal lamina (pLA) and the accessory laminae (ALA). Images are maximum projections Results 78 Output regions of PDF-ir fibers from the optic lobe revealed remarkable overlap with GABA-ir pattern, mainly in the lateral and median protocerebrum Double-label immunocytochemistry showed that no colocalization between GABA and PDF immunoreactivity occurred in any brain area of the cockroach R. maderae (Figure 3.10, Figure 3.11). However, they were expressed in close proximity to each other in many regions of the central brain. In the posterior optic lobe, GABA- and PDF-ir fibers in the LOVT run in parallel towards midbrain regions. GABA-ir cell cluster 7 (G7) branched in the region where the PDF-ir plexus 2 (p2) is located (Figure 3.11 E-E´´). The p2 is associated with the AOC. PDF-ir fibers in the AOC, which connected both AMAE, as well as the fiber network of area 2 (a2) were in close vicinity to GABA-ir fibers in the SMP. PDF-ir fibers in a2 encircled GABA-ir neuropil in the SMP (Figure 3.11 A-A´´, D-D´´). The SMP was densely packed with GABA-ir fibers and could not be separated into different GABA-ir tracts or fiber bundles. Several GABA-ir fibers at the midline connected the SMP of both brain hemispheres (Figure 3.11 A and C). Projection of PDF-ir processes in the anterior fiber plexus (AFP) also overlapped with GABA-ir arborizations in the SLP. GABA-ir fibers of G3 somata run in parallel, near the PDF- ir fibers of the AFP (Figure 3.11 B-B´´, C-C´´). Also, in the ILP and VLP PDF- and GABA-ir fibers branched in close proximity to each other, without ever showing colocalization. from stacks of 16–22 optical sections (z-distance between single sections= 0.63 µm). Scale bars 100 μm. Results 79 Figure 3.11. Output regions of PDF immunoreactive (-ir) fibers from the optic lobe revealed remarkable overlap with GABA-ir fibers, mainly in the superior lateral (SLP) and superior median protocerebrum (SMP), without showing colocalization between GABA and PDF. Confocal laser images obtained from frontal sections through the central brain show GABA- (magenta A-E) and PDF (cyan A´-E´) immunoreactivity. A-A ´´) The SMP is innervated by a dense network of GABA-ir fibers. Several GABA immunostained fibers at the middle connect the SMPs of both brain hemispheres (arrowheads). While PDF immunoreactivity is confined to a dense fiber network in area 2 (a2), the PDF-ir fibers of a2 Results 80 GABA-ir cells express the circadian clock protein period Double-label experiments with antisera against the R. maderae (rm) clock protein PERIOD (rmPER) and GABA were performed. Anti-PER immunoreactivity was restricted to the nuclei of neurons and glia. In the optic lobe, apparently all GABA-ir soma were also rmPER-ir in the nucleus (Figure 3.12). Furthermore, numerous neurons and glia cells exhibited only PER immunoreactivity (see also part 3.2.5). encircle upper regions of GABA immunoreactivity in the SMP. Both, the vertical lobe (VL) and the medial lobe (ML) of the mushroom bodies (MB) are free of GABA- and PDF-ir fibers. B- B´´, C-C´´) GABA- and PDF immunoreactivity is prominent in the anterior lateral protocerebrum. Projections of PDF-ir in the AFP (open arrowhead) overlap with GABA-ir fibers in the SLP, especially where the GABA-ir fiber bundles originating from GABA cell clusters G2-3 branch in the lateral protocerebrum. D-D´´) Several fiber systems of the central complex express intense GABA immunoreactivity including branches from tangential GABA-ir neurons (G5) with arborizations in the lateral accessory lobes (LALs) and projections to the different columns of the lower division of the central body (CBL). None of these regions exhibits PDF immunostaining. However, PDF-ir fibers in the AOC, which connect both accessory medullae, as well as fiber networks in a2 are located in close vicinity to GABA-ir fibers in the SMP. E- E´´) GABA- and PDF-ir fibers in the LOVT run in parallel towards midbrain targets. GABA-ir cell cluster 7 (G7) branches (open arrowhead) in the region of the PDF-ir plexus 2 (p2). Images are maximum projections from stacks of 16–22 optical sections (z-distance between single sections= 0.63 µm). Scale bars 100 μm. Figure 3.12. GABA immunoreactive (-ir) cells (magenta) express the circadian clock protein period from Rhyparobia maderae (rmPER, green). Confocal laser images obtained from horizontal sections of the optic lobe. A- B) Many rmPER-ir nuclei occur in the optic lobe, belonging to neurons and glia. C) The overlay shows that in the optic lobe, all GABA-ir neurons express rmPER-immunoreactivity, however at differing staining intensity. Maximum projection (z-direction: 40 μm). Accessory medulla (AME), distal tract (DT), lamina (LA), lobula (LO), medulla (ME), optic chiasma (OCH1). Scale bars 100 μm. Results 81 Circadian clock protein PERIOD expressed in neuronal cells as well as glial With an antibody against the neuronal cell surface marker horseradish peroxidase (HRP) (Loesel et al. 2006), we tested whether both neurons and non-neuronal cells such as glia or tracheal cells are rmPER-ir in the cockroach brain. Multiple-label experiments showed that apparently all neurons as well as all glial cells (Figure 3.13- Figure 3.14) in the brain expressed the clock protein PERIOD in the nucleus with different intensity. Moreover, in the optic lobes, all nuclei of PDF-ir neurons, including the anterior aPDFMEs (Figure 3.13) and posterior (pPDFMEs) PDF-ir neurons of the medulla as well as the PDF-ir neurons in the dorsal (dPDFLA) and ventral (vPDFLA) lamina, were rmPER-ir (Werckenthin 2014). Results 82 Figure 3.13. Neurons as well as non-neuronal cells such as glia express the circadian clock protein PERIOD. Confocal laser images show period (per, green)-, horseradish peroxidase (HRP, magenta)-, PDF (cyan) immunoreactivity and DAPI (blue) labeled nuclei obtained from sections of the accessory medulla (AME). Neurons (open arrow heads), including aPDFMEs (white double arrow heads), as well as non-neuronal cells (white arrow heads) are rmPER-ir in the nucleus. Medulla (ME), lobula (LO). Scale bar 100 µm. Results 83 Figure 3.14. Central brain neurons as well as non-neuronal cells, such as glia, express the circadian clock protein PERIOD. Confocal laser images show period (per, green)-, horseradish peroxidase (HRP, magenta)-, PDF (cyan) immunoreactivity, and Results 84 3.3. GABA as a possible photic entrainment pathway to the clock GABA-ir branches overlapped with termination sites of the short and long photoreceptor axons of the compound eyes Photic entrainment pathways into the cockroach circadian clock, the AME, are not well described. To examine, whether GABA-ir processes contact histaminergic photoreceptors of the compound eye, double-label immunostainings were performed with anti-GABA and anti-histamine antisera. Multiple-label immunocytochemistry showed that no colocalization between GABA and histamine immunoreactivity occurred in the optic lobe (Figure 3.15). In the proximal lamina, the termination site of short photoreceptor axons overlapped with GABA- and PDF-ir processes. However, the ALAs were devoid of histamine staining, while they exhibited relatively strong GABA- and PDF- immunoreactivity. In the medulla, histamine immunostaining was characterized by a loose fiber network in the medulla layers ME6 and ME7. This staining originated from one histaminergic neuron that had its soma in the lateral protocerebrum of the midbrain (Loesel and Homberg 1999). It expressed also dense histamine-immunoreactivity in ME2, which is the termination site of the long photoreceptor axons of the compound eyes. Apparently, ME2 and ME7 are connected via several histamine-ir fibers (Figure 3.15 A) GABA-immunoreactivity overlapped with arborizations of the histamine-ir centrifugal neuron in layer ME2. As described above (see part 3.2.2.) several GABA immunostained processes of the distal tract could be traced. They run perpendicularly into the medulla towards layer ME4 while passing layer ME2 and ME3 (Figure 3.15 C-G). In addition, histamine-ir fibers took part in the same bundle as GABA-ir fibers in the LOVT, projecting towards the central brain (Figure 3.15 G). Neither colocalization nor overlap was observed between PDF and histamine immunoreactivity in the medulla (Figure 3.15 G). DAPI (blue) labeled nuclei in a section of the midbrain. Neurons (open arrow heads), as well as non-neuronal cells (filled white arrow heads) are rmPER-ir in the nucleus. All images are maximum projections from stacks of optical sections. Superior lateral protocerebrum (SMP), lower- and upper division of central body (CBL, CBU), calyxes (CA). Scale bar 50 µm. Results 85 Figure 3.15. In the lamina (LA) as well as medulla (ME), GABA immunoreactive-(ir) fibers overlapped with projections of short and long histaminergic photoreceptor axon terminals. Horizontal vibratome sections through the optic lobe show histamine immunoreactivity (A, B, green), GABA immunoreactivity (C, D, magenta), PDF Results 86 GABA levels cycle in the distal tract To investigate GABA level over the day, brain of adult male cockroaches was immunostained every 6h in LD 12:12 with anti-GABA (Figure 3.16) and we quantified the staining intensity in the DT. Significant cycling in staining intensity of the DT was observed at the middle of the light phase (ZT06) as compared to the beginning of the night (ZT12) (Figure 3.17). GABA levels had already increased at the beginning of the light phase (ZT00) and reached peak at ZT06. Although the GABA immunostaining in the DT at the beginning of the light phase (ZT00) was near the peak intensity, however the amount was no significant as compared to ZT12. immunoreactivity (E, F, cyan) and overlay of all stainings are shown in G and H. A, B) Histamine-ir processes of the photoreceptor neurons terminate in the LA and the second layer of the ME (filled double arrowhead). The remaining histamine immunoreactivity in ME layer six and seven originate from a neuron in the protocerebrum (not shown) that enters via the lobula valley tract (LOVT). Several histamine-ir fibers projected towards layer forth of the ME where the PDF-ir median layer fiber system (MLF) is located (not shown) and also reach the termination site of the long axon photoreceptors (open double arrowhead). The accessory laminae (ALAs) are free of histamine staining. C, D) The AME is densely innervated by a GABA-ir branch of the AFF, by the GABA-ir distal tract (DT) and by the medial layer fiber tract (MLFT). All three GABA-ir tracts appear to connect the AME and MLF of the medulla. Open arrows mark a GABA-ir fibers that branch off from the DT, arborizing perpendicular to the ME layers towards the layer two (termination of long axon photoreceptors) and the layer four. Only GABA-ir fibers of the AFF connect the AME to the lamina where they overlapped with histaminergic terminals of the short photoreceptor neurons. E, F) PDF immunoreactivity in the optic lobe showed prominent staining in the proximal lamina (pLA), ALAs, AFF, and MLF of the ME (not shown). Layer two of the ME is free of PDF staining (filled double arrowhead). G, H) The overly shows no colocalization between histamine, GABA and PDF immunoreactivity. However, in the LA, histamine-ir terminals of the short photoreceptor neurons overlapped with GABA-ir as well as with PDF-ir fibers, especially in the pLA. In the ME, only GABA-ir fibers overlapped with histamine-ir termination of the long axon photoreceptors in the ME layer two. All images are maximum projections of image stacks. Scale bars 50 µm. Results 87 Figure 3.16. GABA- immunoreactivity changes daytime-dependently in the distal tract (DT). Confocal laser images obtained from frontal sections of the optic lobe at different Zeitgeber times (A-A´ ZT00, B-B´ ZT06, C-C´ ZT12, D-D´ ZT18). Maximum projection from stacks of images containing PDF-immunoreactivity are shown in A´-D´. The AME is encircled for clarity. Accessory medulla (AME), medulla (ME). Scale bars 50 µm. Figure 3.17. Relative GABA staining intensity in the distal tract (DT) is maximal at the Zeitgeber time 06 (ZT06) as compared to ZT12. Quantification of the GABA staining intensity in the DT at ZT00, 06, 12, and 18 showed that GABA immunoreactivity increased at the beginning of the day and significantly peaked at ZT06 (p<0.05) as compared to beginning of night (ZT12). Black and white bar indicate the LD light regime. Error bars are standard errors of mean. asterisk; one-way ANOVA followed by Tukey's multiple comparisons test. Results 88 Figure 3.18. Concentration of GABA in cockroach optic lobes is higher during the day as compared to the night. Animals were kept in 12:12 light dark cycles (A), or in constant darkness (DD) (B). Only in DD a significant maximal concentration of GABA was found at CT 4-8 during the subjective day. Lights on (white bar), lights off (black bar), in DD: subjective day (gray). n= number of optic lobes. Error bars are standard error of mean, asterisk; Kruskal-Wallis test followed by Dunn´s post-hoc test P<0.01). Figure 3.19. GABA levels in optic lobes, in both short- and long-day photoperiods revealed maxima during the day. Cockroaches raised in 18:6 (A) and 6:18 (B) light/dark (LD) cycles. At long day conditions GABA concentrations in the optic lobe peaked at ZT00 and 6 compared to the middle of the night (ZT18). Whereas in short day conditions GABA levels were maximal at ZT00 and 6 compared with beginning of the night (ZT12). Lights on (white bar), lights off (black bar). n= number of the optic lobes. Error bars are standard error of mean, asterisk; Kruskal-Wallis test followed by Dunn´s post-hoc test P<0.001). Results 89 Biphasic oscillation of GABA levels only in DD2 in optic lobe neuropils In order to determine whether GABA content within the optic lobe varied temporally, the optic lobe level of GABA was determined using competitive ELISAs at 4-h intervals throughout a 24-h time period in the cockroaches that acclimated to the 12:12 light-dark cycle. Therefore, a competitive ELISAs was developed to measure GABA. Under LD, no significant differences were observed in GABA content at different ZTs (Figure 3.18 A, Kruskal-Wallis test followed by Dunn´s post-hoc test P<0.01). However, at the second day of the constant darkness (DD2) GABA levels in the optic lobe significantly increased during the subjective day compared to the beginning of the subjective night (Figure 3.18 B, Kruskal-Wallis test followed by Dunn´s post-hoc test P<0.01). These data were consistent with GABA immunostaining quantification in the DT where GABA staining levels were maximal at the middle of the light phase compared to the beginning of the night. Light-duration (photoperiod) dependent increase of GABA levels To determine whether GABA levels in the optic lobe changed in different photoperiods competitive ELISA experiments were employed to measure GABA level in the optic lobe of Madera cockroaches raised in different light regimes, 6:18 and 18:6 LD. GABA levels in the optic lobes was measured at 6-h interval throughout a 24-h time period. In long-day conditions GABA concentration in the optic lobe peaked at ZT00 and 6 compared to the middle of the night (ZT18) (Figure 3.19 A). Whereas in short-day conditions GABA level had maxima at ZT00 and 6 compared with the beginning of the night (ZT12) (Figure 3.19 B). In both long and short-term photoperiod conditions amounts of GABA peaked at ZT00 and ZT06, however, amounts of GABA during this time in long-day conditions is higher than the short-day conditions. Moreover, in the short-day condition, GABA level significantly dropped at ZT12, while, in long-day conditions GABA declined at the ZT12 were not significant compared to ZT00 and ZT06. Results 90 Double-labelled 5-HT- and PDF-ir lamina cells (PDFLAs) connect the proximal lamina, accessory laminae and accessory medulla Neuropeptidergic or neurotransmitter profile of the light entrainment pathways into the cockroach AME, are not known. Thus, multiple-label immunocytochemistry with antisera against PDF, GABA and 5-HT was performed. We searched for neurons that connect general termination sites of compound eye photoreceptors in the lamina and medulla to the AME and PDF- ir pacemakers next to the clock (PDFMEs). It was known before that PDFMEs connect the AME via the AFF to the MLF system of the medulla and to lamina neuropils. However, the arborization pattern of the PDF-ir neurons with somata in the lamina (PDFLAs), most of which colocalized FMRFamide and/or 5-HT, was not resolved (Petri et al. 1995). With multiple-label immunocytochemistry employing antisera against 5- HT and PDF (Figure 3.20 A–C´´), we confirmed the branching patterns of respective neurons described previously (Petri et al. 1995; Petri et al. 2002). Branches of 5-HT-ir neurons invaded the whole lamina and the ALAs (Figure 3.20 A). More extensive 5-HT-ir arborizations were observed in the distal lamina, while PDF-ir branches that also innervated the ALAs were restricted to the proximal lamina (Figure 3.20 A´ and A´´). A subpopulation of 5-HT-ir fibers coexpressed PDF immunoreactivity and originated from colabelled 5-HTand PDF-ir lamina cells (PDFLAs; (Petri et al. 1995), next to the ventral and dorsal ALAs (Figure 3.20 A´´; only dorsal PDFLAs were shown). The PDFLAs sent colabelled branches into the adjacent ALAs and the proximal lamina. Then, double-labelled fibers projected in parallel to single-labelled 5-HT- or PDF-ir fibers via the AFF over the anterior surface of the medulla to the AME (Figure 3.20 B–B´´). While a single side branch of the PDF-ir projections in the AFF invaded the MLF system of the medulla, no PDF/5-HT double-labelled fibers were found in the medulla (Figure 3.20 B´ and B´´). However, several layers of the medulla were 5-HT-ir. Apparently, they were innervated by 5-HT-ir neurons with somata next to the AME that projected via the AFF and possibly also via the MLFT (not shown). In the AME, double-labelled fibers were mostly abundant in the medial shell (Figure 3.20 1C–C´´). Among the AME soma groups, 1.98 ± 0.49 medial neurons (17.67 ± 5.13 µm), 2.44 ± 0.30 ventral Results 91 neurons (15.74 ± 1.44 µm), 1.74 ± 0.86 ventroposterior neurons (14.18 ± 0.77 µm) and 2.86 ± 1.46 neurons (7.94 ± 0.93 µm) of the medial group of frontoventral neurons were 5-HT-ir (n = 5 optic lobes counted). In the lobula valley tract, single-labelled 5-HT-ir fibers were observed. In summary, for the first time, it was shown that PDFLAs that coexpress PDF and 5-HT project via the AFF from lamina neuropils down to the AME, but not to the medulla. Furthermore, 5-HT-ir somata were identified in most of the soma groups of the AME. About two 5-HT-ir medial neurons and about three ventral neurons project via the AFF from the AME to the medulla and possibly also to the lamina. Results 92 Figure 3.20. Double-labelled serotonin (5-HT, magenta)- and pigment-dispersing factor-immunoreactive (PDF-ir, cyan) lamina neurons (PDFLAs) connect the proximal accessory medulla (AME) to lamina (LA) neuropils via the anterior fiber fan (AFF). Confocal images of frontal cockroach brain sections (vibratome, 40 µm) processed for multiple-label immunocytochemistry. In the right columns (A´´, B´´, C´´), double labeling appear in white colors. (A–A´´). A, A´´) The 5-HT and PDF-colabelled dorsal PDFLAs (open arrowheads) have their somata next to the accessory lamina (ALA). (A´´) Colabelled fibers (white) innervate the ALA and the proximal lamina (pLA). Only 5- HT-expressing somata innervate the proximal and distal lamina, while all single- and colabelled PDF-ir fibers (A´, A´´) restrict their arborizations to the pLA. (A–C´´) The colabelled fibers that originate from colabelled PDFLAs connect the ALA and pLA (A–A´´) and project via a distal bundle (open arrowheads in B–B´´) in the AFF (B–B´´) to the proximal shell of the AME (C–C´´). Single-labelled PDF- or 5HT-ir fibers project in more proximal bundles in the AFF. The AFF is closely associated with the most distal layer of the medulla (B-B´´). B–B´´) A single-labelled PDF-ir fiber (B´, B´´; filled arrowheads) branches off from the AFF. It traverses the medulla and ramifies in the medial layer fiber system (MLF) of the medulla, which is also innervated by single-labelled 5-HT-ir fibers (B, B´´). C–C´´) The 5-HT- and PDF-colabelled fibers of the PDFLAs terminate with varicosities in the proximal ventral shell and apparently also in some Results 93 Triple-labelled 5-HT-, FMRFamide-, and PDF-ir lamina cells (PDFLAs) connect the proximal lamina, accessory laminae, and the accessory medulla Triple-label immunohistochemistry with the new commercially available anti- serotonin antibody (Sigma Aldrich, cat. no. S5545) and with anti-FMRFamide showed that a subpopulation of PDF-ir lamina cells also exhibited 5-HT and FMRFamide immunoreactivity. The triple-labeled lamina cells were found in both the dorsal and ventral lamina cell clusters (31±9, n=5). The FMRFamide immunoreactivity of these triple-labeled lamina cells was always weaker as compared to PDF and 5-HT immunoreactivity (Figure 3.21). Moreover, PDF and FMRFamide staining in the lamina cells where mainly concentrated to the cytoplasm and 5-HT stained cytoplasm and nucleus evenly. While PDF immunoreactivity in the lamina was concentrated in the proximal lamina, 5-HT, and to lesser extent FMRFamide staining were evenly disturbed throughout the lamina (Petri et al. 1995). Although it was not possible to trace individual neurites associated with these lamina cells, triple-labeled varicosities in the ALAs and triple-labeled fibers in the lamina, especially the proximal lamina were found. In the proximal lamina, a dense sheet-like plexus of processes were found to colocalized 5-HT and FMRFamide, but not PDF (Figure 3.21, Figure 3.22). Due to the extensively and densely immunostained 5-HT and FMRFamide fiber projections in the lamina and medulla, it was difficult to distinguish and trace the double- and triple-labeled fibers. The same problem was observed for midbrain areas. ventral glomeruli of the AME (open arrowheads). However, they do not appear to project into the lobula valley tract (LOVT). Prominent 5-HT-ir branches spread throughout the medulla, embrace the AME and branch in the LOVT. Many 5-HT- and a few PDF-ir fine arborizations invade the lobula (LO; C–C´´). Scale bars: 50 µm. Results 94 Figure 3.21. Some of the ventral and dorsal lamina cells (d, vLA cells) were triple- labeled with serotonin- (5-HT), FMRFamide, and PDF immunoreactivity. Frontal vibratome section through the optic lobe shows 5-HT- (A, green), FMRFamide- (B, magenta) and PDF-immunoreactivity (C, cyan) in the lamina (LA). Somata immunoreactive (ir) to 5-HT, FMRFamide, and PDF are clustered into two groups ventrally and dorsally at the posterior edge of the LA. While most of the LA cells showed prominent PDF- and 5-HT staining, they were only weakly FMRFamide-ir. However, strong 5-HT, FMRFamide, and PDF immunoreactivity was observed in both the dorsal and ventral accessory laminae (d, vALA) (only dALA is shown). Double filled arrowheads show triple-labeled LA cells, whereas open arrowheads and filled arrowhead showing double labeled 5-HT-/PDF-, and 5-HT-/FMRFamide-ir LA cells, respectively. Scale bar 50 µm. Results 95 Figure 3.22. Proximal lamina (pLA) and accessory laminae (ALAs) colocalized FMRFamide- (magenta), 5-HT-(green), and PDF- immunoreactivity (cyan). Horizontal scheme of the optic lobe shows the scanned region (red square) of the confocal laser image presented below. Confocal laser scan images were obtained from horizontal vibratome sections showing the LA and the dorsal cluster of the LA cells (dLA cells). While 5-HT-(A) and FMRFamide (B) immunoreactivity in the LA were evenly distributed, PDF- (C) immunoreactivity was concentrated in the proximal edge of the LA. However, some PDF-ir fibers entered the distal part of the LA (small arrow). Colocalization of three substances was observed in the ALA, pLA, and some LA cells. Great overlap of 5-HT and FMRFamide fibers was also found in the distal LA. Several 5-HT-, FMRFamide-, and PDF- immunoreactive fibers cross the first optic chiasm (OCH1). All images are maximum projections from the stacks of optical sections. Coordinates: distal (di), posterior (po). Scale bar 50 µm. Results 96 5-HT- and FMRFamide immunoreactivity colocalized in two VNes and one MNes In the medulla, the AFF system which connects the lamina to the AME exhibited 5-HT-, FMRFamide-, and PDF immunoreactivity (Figure 3.23). Immunoreactive fibers of this fiber system were concentrated in bundles and spread in arrays over the distal surface of the medulla. Many immunostained fibers of the AFF system which run parallel and over each other, made it difficult to distinguish and trace the triple-labeled fibers. However, double- labeled fibers with PDF/FMRFamide or FMRFamide/5-HT were observed in the AFF system. In the posterior medulla, a strong 5-HT-ir fiber bundle in the LOVT entered the proximal medulla and projected towards the median layer of the medulla (Figure 3.23). This area was very sparsely stained with FMRFamide and devoid of PDF immunoreactivity (Figure 3.23). The origin of this 5-HT-ir fiber projection could not be determined. Several somata (3.83 ± 0.65, n=6) located at the dorsal and ventral rim of the medulla (Figure 3.23), as well as few somata dorsally to the AME, were double-labeled with 5-HT and FMRFamide (Figure 3.24). Apparently, these double-labeled medulla neurons projected to the medulla but not to the AME. In the AME, immunoreactivity of the three neuroactive substances was mainly concentrated in the shell and in interglomerular regions. Nevertheless, as previously reported (Soehler et al. 2008), one to two ventral glomeruli expressed FMRFamide (Figure 3.26 F). At the proximal edge of the AME, several FMRFamide-ir fibers colocalized with 5-HT (Figure 3.23). Here, in the AME again PDF and FMRFamide double-labeled fibers (Soehler et al. 2011) could not be traced to specific somata. However, the somata of all four medium-sized aPDFMEs next to the AME colocalized PDF with FMRFamide (Figure 3.24). Additionally, 5-HT and FMRFamide colocalized in one MNe (1 ± 0.0) and two VNes (1.50 ± 0.13, n=5) next to the AME (Figure 3.24). Results 97 Figure 3.23. Double-labeled serotonin- (5-HT, green) and FMRFamide-immunoreactive (-ir, magenta) varicosities in the accessory medulla (AME) originate from lamina (LA) cells. Frontal scheme of the optic lobe shows the scanned region (red square) of the confocal laser image presented below it. Frontal vibratome section through the optic lobe shows 5-HT- (A), FMRFamide- (B), and PDF-immunoreactivity (C, cyan) in the medulla (ME). 5-HT-, FMRFamide- and PDF-ir fibers in the anterior fiber fan (AFF) connect the AME to the ME and to the LA. 3.83 ± 0.65 somata located at the dorsal and the ventral rim of the ME (filled arrowheads) showed 5-HT- or FMRFamide immunoreactivity, but no apparent colocalization. The primary neurites of these somata project directly to the ME. Because of extensive arborizations in the ME, individually double-labeled fibers could not be traced further. A thick 5-HT-ir fiber bundle (small arrow) of the lobula valley tract (LOVT) entered the proximal ME Results 98 and projected towards median layers of the ME (star). This area sparsely stains with FMRFamide antisera and is devoid of PDF staining. Several varicosities with colocalization of 5-HT and FMRFamide were observed in the proximal side of the AME (open arrowheads). In the lobula 5-HT and FMRFamide staining greatly overlapped. All images are maximum projections from stacks of optical sections. The AME is encircled for clarity. Coordinates: distal (di), dorsal (do). Scale bar 100 µm. Figure 3.24. One median neurons (MNe) and 2 ventral neurons (VNe) next to the accessory medulla (AME) colocalized serotonin (5-HT, green) and FMRFamide (magenta) immunoreactivity. Frontal scheme of the optic lobe shows the scanned region (red rectangle) of the confocal laser image presented below. Confocal laser scanning images were obtained from frontal vibratome sections showing the anterior medulla (ME). Several ME cells located at the proximal and dorsal rim of the ME colocalized 5-HT and FMRFamide staining (stars, A-D). All medium-sized PDF-immunoreactive (-ir) ventral neurons (open double arrowheads) colocalized FMRFamide-immunoreactivity. 5- HT- and FMRFamide -immunoreactivity colocalized in one MNe (open arrowhead, A-B and D) and one VNe (filled arrowhead, E- H). All images are maximum projections from stacks of optical sections. The AME is encircled for clarity. Coordinates: distal (di), dorsal (do). Scale bar 50 µm. Results 99 GABA- and FMRFamide immunoreactivity colocalized in two VNes and one MNes Multiple-label experiments using anti-GABA, anti-FMRFamide, and anti-PDF antisera demonstrated that at least one medial neuron (MNe, 1.25±0.20) and two ventral neurons (VNes, 1.80±0.28) colocalized FMRFamide and GABA (Figure 3.25, n=5) (Figure 3.25). These somata were devoid of PDF staining. However, almost all medium-size PDF-ir cells were colocalized FMRFamide. Additionally, three (2.33±0.21) medulla cells colocalized GABA- and FMRFamide-immunoreactivity. These cells directly projected to the medulla. Due to extensive overlap between GABA- and FMRFamide-immunoreactivity, further colocalization in the optic lobe neuropils and central brain could not be discerned. One large PDF-ME neuron colocalized FMRFamide and MIP Multiple-label immunostainings utilizing different antisera against MIP, FMRFamide, and PDF showed that 3 soma groups associated with the AME colocalized FMRFamide, and MIP immunoreactivity. Out of the five evaluated optic lobes, at least three VNes (2.83 ± 0.39), four DFVNes (3.83 ± 0.30), and two MNes (1.83 ±0.30) exhibited double-labeled FMRFamide- and MIP-ir somata (Figure 3.26). The staining intensity of the double-labeled MIP-ir MNes Figure 3.25. Double-labeled accessory medulla (AME) neurons express GABA- (A, green) and FMRFamide- (B, magenta), or FMRFamide- and PDF-immunoreactivity (C, cyan). Confocal laser scan images were obtained from frontal vibratome sections of the optic lobe. One medium-sized anterior PDF cell (aPDFME) that colocalized PDF- and FMRFamide- immunoreactivity (open arrowhead) is shown. One medial neuron (MNe) (filled arrowhead), two ventral neurons (VNes) (double open arrowhead), as well as two medulla cells (double filled arrowhead) colocalized GABA- and FMRFamide-immunoreactivity. AME not shown. ME medulla; AFF anterior fiber fan system. All images are maximum projections from stacks of optical sections. Scale bars 50 µm. Results 100 was fainter as compared to the other MIP-ir somata next to the AME. Interestingly, one PDF-ir VNe was additionally FMRFamide and MIP-ir (Figure 3.26 A, D, G, J). This triple-labeled VNe showed strong MIP- and FMRFamide staining while expressing only faint PDF immunoreactivity. Considerable overlap between FMRFamide- and MIP-ir fiber projections in the medulla- especially in the AFF system-, in the lamina, as well as midbrain areas, make it difficult to distinguish and follow the double- or triple-labeled fibers in these regions. Nevertheless, one ventral glomerulus of the AME colocalized FMRFamide and MIP (Figure 3.26 C, F, I, L). Here again, several double- labeled FMRFamide and MIP-ir somata (3.17 ± 0.30) at the proximal edge of the medulla and dorsally to the AME were observed. These somata seemed to innervate directly the medulla but not AME (Figure 3.26). Additionally, all medium-sized PDF-ir cells were also FMRFamide-ir (Figure 3.26). Results 101 Figure 3.26. One medium-sized PDF-immunoreactive (-ir) neuron colocalized FMRFamide and MIP. Maximum projections of confocal laser scans of an optic lobe show MIP- (A-C, green), FMRFamide- (D-F, magenta), and PDF immunoreactivity (G-I, cyan). Colocalization of neuropeptides is shown in white (J-L). Four distal-frontoventral neurons (DFVNes) colocalized MIP and FMRFamide (filled double arrowheads). At least three ventral neurons (VNes) also showed colocalization of MIP and FMRFamide but not PDF (open arrowhead). Two median AME neurons (MNes) showed colocalization of MIP and FMRFamide (filled arrowhead). One PDF-ir VNe expressed MIP and FMRFamide immunoreactivity (A, D, G and J, yellow filled arrowhead). PDF-ir staining in this cell was weak compared to the FMRFamide and MIP staining. Open arrows showed PDF-ir neurons which Results 102 Neurochemical profile of the accessory lamine The significance of the small specialization at the border of the lamina neuropil, the ALAs, was revealed when electrophysiological experiments using light stimuli showed that two light-sensitive AME neurons project to these tiny neuropils. Therefore, it was proposed that these neuropils might function as additional light pathway to the clock (Loesel and Homberg 2001b). In order to characterize the neurochemical map of this region, several double-label immunostainings with anti-synapsin, as a neuropil marker, and antisera against different neuropeptides and neurotransmitters such as PDF, FMRFamide, GABA, 5-HT, and histamine were performed. Interestingly both dorsal- and ventral ALAs exhibited immunoreactivity to all tested antisera except anti-histamine immunoreactivity (Figure 3.27). Additionally, as previously showed (Petri et al. 1995), lamina cells only showed immunoreactivity to PDF, FMRFamide, and 5-HT. None of the lamina cells showed immunoreactivity to GABA or histamine (Figure 3.27). expressed FMRFamide but not MIP immunoreactivity (A, D, G and J). Additionally, one ventral glomerulus of the accessory medulla (AME) colocalized FMRFamide and MIP (filled arrow). Due to the extensive fiber projections in the ME, individual double-labeled AME fibers could not be traced further. Scale bar 50 µm. Results 103 Figure 3.27. Accessory laminae (ALAs) contain a striking number of different Results 104 3.4. GABA could be involved in coupling pathways between the AMAE Another candidate addition to PDF for coupling both AMAE is GABA, because some of VMNes expressed GABA. Therefore, by applying backfill experiments in combination with immunohistochemistry we investigate this assumption. At least two contralaterally projecting neurons belong to MCI (VNe) and MCII (VMNe) were GABA-ir Neurobiotin backfills from one cut optic stalk were performed and subsequently were combined with anti-GABA and anti-PDF immunocytochemistry. In five preparations, four commissural cell groups which described before were observed (Table 3.3) (Reischig and Stengl 2002; Soehler et al. 2011). Therefore, we used these samples for further evaluations. However, total numbers of the neurobiotin-labeled cells (29.41±2.42) in this study were lower compared to the previous results. Table 3.3. Medulla commissural cells labeled with neurobiotin backfills Total MC I MC II MC III MC IV Number Mean ± SEM 29.41±2.42 3.56±1.47 21.17±2.9 2.03±0.4 2.46±0.64 Size (µm) ± SEM 18.83±0.75 18.96±2.35 17.89±0.47 27.14±2.6 18.31±1 neuropeptides and neurotransmitters, however, is devoid of histamine staining, the neurotransmitter of compound eye photoreceptors. Confocal laser images obtained from the frontal (A-D) and horizontal (E) sections through the lamina (LA) showing GABA, PDF, 5-HT, FMRFamide, histamine (magenta, A-E), and synapsin (SYN) (cyan) immunoreactivity. While GABA- (A) and PDF (B) immunoreactivity in the lamina were concentrated mainly in the proximal lamina, FMRFamide (D), 5-HT (C), and histamine (E) staining were distributed evenly in the LA. Only PDF, 5-HT, and FMRFamide antisera stained the LA cells. All images are maximum projections from stacks of optical sections. Scale bar 50 µm. Results 105 Figure 3.28. One ventral neuron that projected to the contralateral optic lobe colocalized GABA and neurobiotin and, thus, belongs to MCI. Maximum projections of confocal laser scanning show neurobiotin-backfill from the contralateral optic stalk (green), GABA- (magenta), and PDF- immunoreactivity (cyan). Colocalization is shown in white (C´´´-D´´´). A-A´´´) Frontal section through the lamina shows sparse neurobiotin staining in the ventral accessory laminae (vALA) (filled arrowheads) which overlapped with GABA- and strong PDF-ir. No colocalization occurred between PDF-ir ventral lamina cells (vLA cells), neurobiotin, and GABA. B-B´´´) Frontal section at the anterior level of the midbrain shows two neurobiotin-labeled tracts. Tract 2 that was not labeled with anti-GABA or anti-PDF antibodies and tract 4 (open double arrowheads) which is associated with anterior optic commissure (AOC), showed colocalization with PDF, but not with GABA. C-D) Horizontal sections through the optic lobe. One medium-sized PDF-ir ventral neuron showed also neurobiotin staining (C´´´, open double arrowhead). Near the accessory medulla (AME) the other neurobiotin-filled neuron that could be group to MC I and to ventral neurons (VNes) (filled double arrowhead) also exhibited GABA-ir. Prominent neurobiotin- labeled branching was present in the median layer fiber system (MLF) of the medulla (ME), overlapping with GABA- and PDF-ir fibers of the MLF. Results 106 The overall number of the counted cells in each group were consistent with previous studies (Soehler et al. 2011; Arendt 2016). Since the number of the MC II neurons which were assigned to the VMNes were equal to the number of the these AME neurons (both approximately 35 neurons), it was suggested that all VMNes are commissural cells (Reischig et al. 2004; Soehler et al. 2011). The number of the MCII cells (21.17±2.9) in current study were lower than previously published data. Additionally, the neurobiotin-backfill projection pattern in the medulla and lamina were in accordance with previous studies. The MLF is strongly innervated by contralaterally projecting cells. In the AME, neurobiotin-labeled fibers were mainly observed in the interglomerular as well as shell regions (Figure 3.28). In the lamina, neurobiotin-labeled fibers concentered in the proximal lamina as well as in the ALAs (Figure 3.28 A). From 5 evaluated backfilled preparation, only two preparations contained 4 commissural cells which colocalized neurobiotin and GABA. These colabelled cells belonged to the MCI (which assigned to the VNe), MCII (which assigned to the VMNe) and one MC IV (which assigned to the MNe, not shown) (Figure 3.28). GABA and neurobiotin-labeled fibers did not colocalized in the MLF which consist of branching from MC II (Reischig and Stengl 2002). However, they greatly overlapped in this region (Figure 3.28 C-D). Additionally, the GABA-ir MLFT were in proximity of a thick backfilled fiber in the medulla (Figure 3.28 D-D´´´). GABA-ir distal tract fibers were also devoid of neurobiotin labeling. In the lamina, no colocalization occurred between neurobiotin and GABA immunoreactivity. Nevertheless, they overlapped in the proximal lamina and ALAs (Figure 3.28 A-A´´´). In the central brain, neither neurobiotin-labeled POC nor AOC colocalized with GABA (Figure 3.28 B-B´´´). Regarding the fiber projections in no case GABA/ neurobiotin colocalization was detected in the midbrain area such as the SMP, POTU. Nevertheless, GABA-ir fibers were plentiful in these areas. Regarding to the colocalization of PDF and neurobiotin-labeled commissural cells, overall (3.2± 0.3, n=5) PDF-ir VNes colocalized neurobiotin (Figure 3.28 C-C´´´). Based on the size of the cells they assigned to the one largest and three large sized PDF-ir cells. All images are maximum projections from stacks of optical sections. Scale bars A-B 100 µm, C-D 50 µm. Results 107 3.5. Output pathway of the clock and GABA In order to find the circadian clock output neurons that connect to the premotor centers first we preformed neurobiotin backfill from the first thoracic ganglion to analyze the descending and ascending neuron in the brain. Then we combine the backfill with immunohistochemistry using anti-GABA and anti- PDF antisera. Characterization of the neurobiotin backfill from the first thoracic ganglion Neurobiotin-backfills from the cut-connective between first and second thoracic ganglion in combination with immunostaining with anti-PDF antibody as a marker of the circadian network and anti-GABA antibody, the brain regions were analyzed for potential connection between the circadian system and premotor area and locomotor centers area in the cockroach R. maderae. Overall 142±30 (n=5) backfilled somata were counted in the brain. Majority of the backfilled-somata were located at the protocerebrum. They were mainly scattered along the midline posterior to the medial and lateral calyxes of both hemispheres as well as those located posteriorly to the pedunculus of the mushroom bodies. There were also several backfilled-somata located at the deutocerebrum. They were mainly located at the posterior rim of the deutocerebrum near the midline ipsilateral side to the backfilled. Additionally, few backfilled somata were observed in the tritocerebrum of the side ipsi- as well as contralateral to the backfilled (Figure 3.29). Different ascending and descending neurobiotin-stained fibers and tracts could be observed in the tritocerebrum, deutocerebrum as well as in the protocerebrum. Overall, staining intensity was higher on the side ipsilateral to the neurobiotin-backfill (Figure 3.29, Figure 3.30, A, B, D). A prominent neurobiotin-stained fiber bundles along the tritocerebrum side ipsilateral to the backfilled run up to the posterior region of the deutocerebrum and entered the region so called the antenno-mechanosensory and motor center (AMMC, Figure 3.30). The AMMC which is located at the dorsal deutocerebrum and posteriorly to the antennal lobe, also known as the dorsal lobe in some insect such as aphid, cricket and cockroach (Homberg et al. 1989). The high density of the backfilled-neurites Results 108 was observed specially in the posterior, and near the midline part of the AMMC (Figure 3.30). Figure 3.29. Partial surface reconstructions of the main neuropils of the cockroach brain and frontal reconstruction of the PDF immunoreactivity (green- blue gradient) and neurobiotin backfill from the first thoracic ganglion (TG, magenta). View of a wholemount cockroach brain in which descending/ascending neurons were stained by a neurobiotin backfill from the right TG. Prominent neurobiotin staining in the tritocerebrum (TR), posteriorly to the antennal lobe (AL) in the deutocerebrum, and in the lateral protocerebrum ipsilaterally to the TG backfill (thick white arrow). Many backfilled somata were located posteriorly to the calyxes and at the midline between both hemispheres. Thick neurobiotin-labeled fibers and several fine fibers arose from the lateral protocerebrum run towards the optic lobe and extended up to the lobula (LO). There is great overlap, but no colocalization between PDF- immunoreactive (-ir) fibers and neurobiotin-labelled fibers in the lobula valley tract (LOVT) and in the inferior lateral protocerebrum (ILP) and posterior optic tubercles (POTU). The antenno-mechanosensory and motor center (AMMC). 3D reconstruction was performed with AMIRA. Scale bar 150 µm. Results 109 Figure 3.30. Fibers of the GABA-immunoreactive (-ir) soma groups (magenta) overlapped to different extends with projections from the neurobiotin- backfills (green) of the ipsilateral thoracic ganglion (TG) in the superior medial protocerebrum (SMP), the inferior lateral protocerebrum (ILP), and in the antenno- mechanosensory and motor center (AMMC), while there was little overlap with PDF-ir fibers (cyan). Frontal scheme of the brain shows backfills from the ipsilateral TG (solid red line) and the regions of interest (blue squares). Maximum projections of confocal laser scans of different regions of the brain. Overlay of the stainings are shown in the right column (A´´´- D´´´). A-B) The AMMC and TG ipsilateral to the neurobiotin-filled TG showed strong immunostaining. Results 110 Double-labeling with anti-PDF antibody showed that PDF-ir fibers overlapped with neurobiotin-labeled fibers in several regions. In the tritocerebrum and the AMMC of both hemispheres, a few PDF-ir fibers brunches with a non-identified Prominent neurobiotin-labeled fiber bundles in the tritocerebrum (TR) could be traced to the AMMC and the IPL. A high density of neurobiotin-labeled fibers was observed in the region dorsally to the LH where the PDF-ir fibers are located at. GABA-ir staining in this area was moderate but homogenous. Additionally, two GABA-ir fiber bundles (filled arrowheads) extended to the AMMC and ILP (A´-B´). The cell bodies of two neurobiotin-stained neurons were located dorsolaterally and at the border of the proto- and deutocerebrum (A-B, stars). C- C´´) The lower and upper division of the central body (CBL, CBU) and medial lobe (ML) of the mushroom body are free of neurobiotin staining. The ipsilateral as well as contralateral SMP received neurobiotin-backfilled processes (open arrowheads). Here they have overlapped with PDF-ir fibers. A big neurobiotin-labeled somata located at the pars intercerebralis (star). D- D´´´) Contralateral side of the TG backfilled also showed neurobiotin staining. However, staining intensity was sparse compared to the ipsilateral side. In the TR and AMMC, a few PDF-ir fibers brunches with a non-identified soma overlapped with neurobiotin staining in both ipsi- (A´´), and contralateral side to the backfilled-TG (white filled double arrow heads). All images are maximum projections from stacks of optical sections. Dorsal lateral horn (dLH), area 1 (a1). Scale bar 100 µm. Figure 3.31. Neurobiotin-labeled (magenta) fibers from the thoracic ganglion reach the lobula in the optic lobe. Images are maximum projections from stacks of 25 optical sections obtained from wholemounts showing the posterior medulla (ME). Thick neurobiotin-labeled fibers (filled arrow head) and several fine fibers (filled double-arrow head) arise from the inferior lateral protocerebrum (ILP, open arrow head) run towards the optic lobe, and via posterior optic commissure (POC) extend to the lobula (LO). There is great overlap, but no colocalization between PDF- immunoreactive fibers and neurobiotin-labelled fibers in the POC, lobula valley tract (LOVT), ILP as well as PDF-ir plexus 3 (p3) which is associated with the anterior optic commissure (AOC). Open filled arrow head shows posterior PDF-ir ME cell. Scale bar 100 µm. Results 111 soma also overlapped with neurobiotin staining (Figure 3.30 A-D, Figure 3.32 B). In most posterior region of the protocerebrum where PDF-ir fibers in the POC connect both AMAE, neurobiotin-labeled fibers from backfilled- thoracic ganglion reached the PDF-ir fibers in the POTU and overlapped with each other (Figure 3.32). In the posterior lateral protocerebrum, a neuropil occupying region right above the deutocerebrum and dorsally to the lateral horn also received particularly strong and dense neurobiotin-stained neurites (Figure 3.30 B, Figure 3.31 A). PDF-ir fibers of the ILP greatly overlapped with this region (Figure 3.31 B). Therefore, we considered this neuropil associated with the ILP region. From the ILP neuropil few neurobiotin-stained fibers run laterally towards the ipsilateral optic lobe and via the LOVT extend dorsally to the lobula (Figure 3.31). Neurobiotin-labeled fiber arborized in the proximal area of the lobula and terminated near the distal surface of the lobula. No further neurobiotin-labeled fibers were observed in other optic lobe neuropils. These fibers were proximity of the PDF-ir fibers of the POC and LOVT. Where the PDF-ir of the LOVT bifurcate to the AOC and POC also overlapped with few neurobiotin-labeled fiber branches (Figure 3.31). The SMP of the side ipsilateral to the backfilled showed some backfilled-labeled neurites. The contralateral SMP, instead showed fewer of these processes. The PDF-ir fibers in the SMP overlapped with neurobiotin-stained neurites (Figure 3.30). In contrast, there were no neurobiotin-stained fibers was observed in the SLP and no overleaped was found with backfill and the PDF-ir fibers system of the AFP. No backfill staining were observed neither in the central complex nor in the mushroom body. However, some backfilled-processes also invade the lateral complex on the side ipsilateral to the backfilled and to lesser extent on the side contralateral (Figure 3.30). Results 112 GABA immunoreactivity in the lateral protocerebrum and the AMMC greatly overlapped with neurobiotin-labeled fibers backfilled from the thoracic ganglion Neurobiotin backfills from the first thoracic ganglion combined with immunostaining of anti-GABA and anti-PDF antisera revealed that GABA-ir staining greatly overlapped with neurobiotin-labeled regions in the brain. In the tritocerebrum neurobiotin-labeled fiber bundles from the thoracic ganglion that run towards upper brain regions, overlapped with several GABA-ir fibers (Figure 3.30 A-A´´´). In the posterior region of the deutocerebrum, where the AMMC located, GABA-immunoreactivity greatly overlapped with neurobiotin- labeled fiber projections (Figure 3.30 B-B´´´). However, no colocalization neither in the cells nor fibers in any area of the brain observed between GABA and neurobiotin immunoreactivity. Moreover, in the lateral protocerebrum ipsi side of the backfills, uniform GABA-immunoreactivity and prominent neurobiotin-labeled were greatly overlapped in the ILP (Figure 3.30 B-B´´´). The neurobiotin-labeled fibers which arose from the ILP toward the lobula in the ipsilateral optic lobe showed overlap with GABA-immunoreactivity in the Figure 3.32. Neurobiotin backfilled from the thoracic ganglion (TG) overlapped with PDF-immunoreactive (-ir) fibers in the posterior optic tubercles (POTU). Confocal laser images obtained from the frontal sections through the posterior region of the central brain show neurobiotin-labeled fibers (magenta, A) and PDF-ir fiber projections (cyan- B). A-C) Strong neurobiotin staining in the tritocerebrum (TR) ipsi side to the backfilled TG ascend toward the deuto- and protocerebrum. Fine neurobiotin-labeled fiber projections reach the POTU (white double-filled arrow heads) and overlapped with PDF-ir fibers. In the TR, a few PDF-ir fibers brunches with a non-identified soma also overlapped with neurobiotin staining (white filled arrow head). Posterior optic commissure (POC), antenno- mechanosensory and motor center (AMMC). Scale bar 100 µm. Results 113 POC, LOVT as well as in the lobula (Figure 3.30 B-B´´´). In the CBL and LAL neuropils where GABA staining are prominent, no neurobiotin staining were observed (Figure 3.30 C-C´´´). However, the SMP of both brain’s hemispheres were immunoreactive to GABA and to the neurobiotin without colocalization. On the contralateral side to the backfill, GABA- and neurobiotin immunoreactivity overlapped in the AMMC (Figure 3.30 D-D´´´). GABA and the ocelli Neurobiotin backfills from the ocellus combined with anti-GABA and anti-PDF immunohistochemistry (n=4) were performed to analyze possible connection between ocellar neurons to the circadian system. Four large second order neurons which located in the area of the pars intercerebralis (PI), above the SMP were neurobiotin backfilled. These neurons apparently have arborization in the ocellus and on the other side via thick fibers project to the ocellar tract which enter protocerebrum (Figure 3.33). Neurobiotin-labeled fiber from the second order neuros are in close vicinity of the PDF-ir a2 which is connected to the AOC (Figure 3.33). From there, it runs toward the posterior regions of the brain where PDF-ir POTU located. At the inner edge of the tritocerebrum two small cell bodies (13.3± 3.32, n=3) were stained with neurobiotin. These two cells apparently are small multimodal ocellar interneurons (Ohyama and Toh 1990b). They project up to the protocerebrum and join the ocellar tract at the posterior region where the PDF-ir POTU is located (Figure 3.35). Here due to the background staining as well as dense fasciculation of the neurobiotin- labeled fibers, it was not possible to distinguish between the small multimodal ocellar interneurons fibers and the large second order neurons fibers. From the POTU, neurobiotin-labeled fibers run parallel to the PDF-ir POC towards the optic lobe ipsilateral to the backfills. Neurobiotin-stained fibers in the POC extend to the ventro-proximal side of the lobula, proximal edge of the medulla as well as to the AME. In the lobula, neurobiotin-stained fibers overlapped with GABA immunoreactivity and PDF-ir fiber plexus 1 (Figure 3.33). Along with GABA- and PDF-ir fibers od LOVT, neurobiotin-labeled fiber entered the medulla and at the dorsal side of the AME bifurcated. One innervates small are at the proximal edge of the medulla and the other innervate the AME. Results 114 Neurobiotin-staining in the AME mostly concentrated at the interglomerular regions and lesser extent at the shell (Figure 3.33 A). The neurobiotin staining in the interglomerular region at the ventral and dorsal side encircled GABA immunoreactivity in the glomerular core of the AME (Figure 3.33 D, Figure 3.34). PDF immunoreactivity also overlapped with neurobiotin staining in the AME. However, no colocalization were occurred between GABA-, PDF-, and neurobiotin staining in the AME. Figure 3.33. Four large second-order neurons were labeled with neurobiotin backfills form the ocellus. Frontal scheme of the brain in the head capsule shows the location of the backfill (green filled capillary) and the regions of interest (blue squares) of the confocal laser images presented below. Images are maximum projections from stacks of 50 optical sections obtained from wholemounts showing neurobiotin-labeled fibers (green, A) and PDF (cyan, D) immunoreactivity in the central brain. A) Four large neurobiotin-labeled neurons (encircled area) project to the ocellar tract (white filled arrowheads). Two small backfilled neurons were observed at the inner edge of tritocerebrum. B-C) PDF immunoreactive fibers in area 2, which were connected to the anterior optic commissure (AOC), were in close vicinity to the ocellar tract. Anterior fiber plexus (AFP), area 2 (a2). Scale bars 100 µm. Results 115 Figure 3.34. Neurobiotin-labeled fibers backfilled from the ocelli greatly overlap with GABA and PDF immunoreactivity in the accessory medulla (AME), lobula valley tract (LOVT) and PDF-ir fiber plexus 1 (p1) in the lobula (LO). Frontal scheme of the brain in the head capsule shows the location of the backfill (green filled capillary) and the regions of interest (blue squares) of the confocal laser images presented below Images are maximum projections from stacks of 26 optical sections obtained from wholemounts showing neurobiotin-labeled fibers (green, A), GABA- (magenta, B) and PDF- (cyan, D) immunoreactivity in the optic lobe. A) Neurobiotin-labeled fibers backfilled from the ipsilateral ocellus in the lobula (LO) entered the proximal side of the medulla (ME) via the lobula valley tract (LOVT) and innervated the AME. In the AME neurobiotin backfills concentrated in the interglomerular and shell regions. B-D) GABA-and PDF immunoreactivity overlapped in plexus 1 (p1), LOVT. Scale bars 100 µm. Results 116 GABA and neuroendocrine system of the Madeira cockroach Information about the neuropeptide and neurotransmitter content of the neurosecretory cells in the PI and PL in the R. maderae is limited. To examine Figure 3.35. Surface reconstructions of the main neuropils of the left hemisphere of the cockroach brain and frontal reconstruction of PDF immunoreactivity (cyan) and neurobiotin backfills from the ocellus (magenta). Four large second order neurobiotin-labeled neurons project to the ocellar tract in the median protocerebrum. Neurobiotin stained fibers in the ocellar tract run posteriorly to the mushroom body where they appeared to bifurcate. They sent one projection along the posterior optic commissure (POC), overlapping with PDF-immunoreactive (-ir) fibers in the ipsilateral posterior optic tubercle (POTU). They extended few arborizations to the lateral protocerebrum, continuing on to the ventro-proximal side of the lobula (LO), the proximal edge of the medulla (ME), until branching in the accessory medulla (AME). In the LO neurobiotin stained fibers overlapped with the PDF-ir fiber plexus 1 (p1). Apparently, another projection of the neurobiotin-labeled ocellar tract fibers continued on to two small stained neurons located at the inner edge of tritocerebrum. Their axons ascended towards the ocellar tract and sent a lateral side branch posteriorly to the antennal lobe (AL). Projections of the four large second order ocellar neurons could not be clearly distinguished from the projections of the two small tritocerebrum neurons. Lamina (LA), anterior optic commissure (AOC), vertical lobe (VL), calyces (CA). The 3D reconstruction was performed with AMIRA. Scale bar 150 µm Results 117 whether neurosecretory cells express GABA, neurobiotin backfill from the retrocerebral complex was performed to identify the PI and PL cells in the brain. Subsequently, backfilled-brain were combined with GABA and PDF immunohistochemistry. Four cell clusters in the brain were neurobiotin-labeled. Two cell clusters were located at the superior lateral protocerebrum of both hemispheres and belong to the PL. Their axons run ipsilaterally via the NCC I and NCC II to the retrocerebral complex (Figure 3.36 A). Two other cell cluster were located at the median protocerebrum and gave rise to two branches of the NCC I that crossed over into the respective contralateral brain hemisphere, however still projecting down in parallel at the brain midline towards the retrocerebral complex (corpora cardiaca = CC and corpora allata = CA) (Figure 3.36 B). Both neurobiotin-labelled cells in the PI and PL were heterogenous in terms of the size. Combination of the backfill experiments with immunohistochemistry using anti-GABA and anti-PDF antisera showed that none of the neurobiotin-labeled cells neither in the PI nor PL colocalized GABA (Figure 3.36 A´-A´´)or PDF immunoreactivity (not shown). However, GABA-ir fibers were in close vicinity of the neurobiotin-labeled fibers in the NCC I and NCC II. In the anterior region of the SLP, GABA-ir fibers with unknown origin overlapped with the axon fibers NCC II of the PL cells. They run over the VL of the mushroom body. Several GABA-ir somata located at the PI region but no colocalization occurred with neurobiotin-labeled somata (Figure 3.36 B´- B´´). Results 118 Figure 3.36. GABA-immunoreactive(-ir) fibers arborize in close vicinity to the branches of neurosecretory cells in the superior protocerebrum. Frontal scheme of the brain shows the location of the backfill (solid red line) and the regions of interest (blue squares) of the confocal laser images presented below. Frontal sections through the central brain show neurobiotin-labeled fibers and somata of the pars intercerebralis (PI) and pars lateralis (PL) (green, A-B) and GABA immunostaining (magenta, A´-B´). A) Neurobiotin labeled fibers of PL neurons project via the nervus corporis cardiaci II (NCC II) anterior to the vertical lobe (VL) towards the PI. Several GABA-ir fibers (white filled arrowheads) run parallel and in close vicinity to the neurobiotin-labeled fibers in the NCC II. B) Two clusters of neurobiotin-labeled neurons in the PI were observed in the median protocerebrum. They gave rise to two branches of the NCC I that crossed over into the respective contralateral brain hemisphere, however still projecting down in parallel at the brain midline towards the retrocerebral complex (corpora cardiaca = CC and corpora allata = CA). GABA-ir fibers in the superior median protocerebrum (SMP) (white filled arrowheads) were in close vicinity to the neurobiotin-labeled fibers in the NCC I. No colocalization occurred between GABA- and neurobiotin-staining neither in the PI nor PL. Superior lateral protocerebrum (SLP). All images are maximum projections from stacks of optical sections. Scale bars 100 µm. Discussion 119 4. Discussion With neurobiotin backfills in combination with immunochemical stainings as well as biochemical experiments, the role of GABA in photic entrainment-, output pathways from the circadian clock, and in commissural pathways between both optic lobes was investigated. Apparently, GABA is involved in all these pathways. The possible function of GABA in midbrain areas and optic lobe are discussed further. 4.1. Methodological Considerations Commercial GABA immunohistochemistry fulfilled all criteria for a good staining The first aim of this work was to successfully accomplish GABA immunostaining in the cockroach R. maderae brain. Since the previously used anti-GABA antibody (provided by Dr. T.G. Kingan, US Department of Agriculture, Beltsville, Md., USA) was no longer available, an alternative was necessary. One option was the commercially available polyclonal anti-GABA antibody which is raised in the guinea pig Protos Biotech, New York (USA)]. As this staining requires a glutaraldehyde fixation, the results, especially for single enzymatic staining, yielded a poor signal-to-noise ratio as the use of glutaraldehyde often results in high autofluorescence (Pow et al. 1995). Additionally, in whole mount experiments, antibody penetration was insufficient although different dilutions of the antibody, prolonged primary antibody incubation, and use of collagenase/dispase to digest the neurilemma were tested. All experiments performed resulted in weak stainings only. In contrast, applying the antibody on thin sections with fluorescent immunocytochemistry ( (Schendzielorz 2014; Arendt 2016) similar results compared to immunocytochemistry with the Kingan antisera were obtained. Also, another commercially synthesized anti-GABA antibody (Sigma; product A2052) which was raised in rabbit was tested. This antibody was used before mainly for investigation of the GABA-ir neurons in the antennal lobe and the lateral complex in moths (Berg et al. 2009; Seki and Kanzaki 2008; Iwano and Kanzaki 2005; Reisenman et al. 2011). The specificity of the antibody in the Madeira cockroach was demonstrated by a pre-adsorption experiment on Discussion 120 brain sections, whereby GABA immunoreactivity was abolished when the primary antibody was pre-absorbed with 10 µM GABA. Additionally, in the whole mount experiments we prolonged the incubation time for this anti-GABA antibody until eight overnights to get the satisfactory results. Compared to guinea pig anti GABA antibody, rabbit anti-GABA antibody provided by Sigma revealed more details especially for the florescent staining procedure. Therefore, we choose Sigma anti GABA antibody in our current work which enabled us to analyse GABA immunoreactivity in details in the optic lobe especially in the medulla neuropil as well as the central brain. To further test whether anti-GABA antisera specifically recognize GABAergic neurons, antisera were employed against the enzyme anti-glutamate decarboxylase (GAD) which specifically occurs in GABA-synthesizing neurons (Datum et al. 1986b; Füller et al. 1989; Moore and Speh 1993; Homberg et al. 1999; Okamura et al. 1989; Kolodziejczyk et al. 2008b). Anti-GABA and anti- GAD immunostaining revealed the same pattern of labeling in the brain of R. maderae, although with different signal to noise ratio (Figure 3.7). As example, both antisera omitted the same structures such as photoreceptor neurons, or any neuron surrounding the lamina neuropil. Furthermore, in the main midbrain neuropils such as the optic lobes, central complex, antennal lobes, and mushroom bodies both antisera expressed the same staining patterns. However, GAD-antisera were in general more sensitive than GABA-antisera, e.g. at the tip of the medial lobes (Figure 3.8 D), and stained with better signal to noise ratio, such as the glomerular lobe at the border of the tritocerebrum (Figure 3.8 F). 4.2. Widespread GABA labeling in different neuropils in the central brain suggest a crucial function of GABA in various physiological and behavioral processes in the cockroach R. maderae The widespread distribution of GABA immunostaining in the central brain of the cockroach R. maderae indicates a prominent function of this mostly inhibitory neurotransmitter in regulating different neuronal and physiological processes in cockroaches as well as in other insects (Homberg et al. 1987; Berg et al. 2009; Yamazaki et al. 1998; Füller et al. 1989; Homberg et al. 1999). Discussion 121 GABA immunoreactivity was widely detected at different levels of sensory processing. For example, while it was not found in the antennal nerve, it was present at the first relay station of olfactory information: in all glomeruli of the antennal lobes, as well as the second relay in the calyces of the mushroom body and in the lateral protocerebrum. Accordingly, GABA was prominent in higher centers of information processing. Especially strongly labelled was the lower subunit of the central complex, next to the input and output regions of the mushroom bodies, as well as premotor areas such as the lateral complex. Anti-GABA staining was also observed at all processing levels of the circadian system such as inputs, local circuits, and outputs of the AME to SLP and SMP (see introduction 1.4.5; (Wei et al. 2010b). Therefore, GABA mediates synaptic activity in the whole brain and it is likely that GABA functions as an output candidate in the circadian system. GABA possibly serves via three types of inhibitory circuits in the antennal lobe (feedforward-, feedback-, and lateral inhibition) GABA immunoreactivity in the antennal lobe of the cockroach R. maderae was concentrated mostly in the core of each glomerulus, originating mostly from local interneurons and to a lesser degree from projection neurons of soma cluster LC (Figure 3.4 A). This suggests a significant function of GABA in processing olfactory information. This staining pattern resembles stainings described in the cockroach P. americana (Distler 1989), moths Heliothis virescens (Berg et al. 2009), and M. sexta (Homberg et al. 1987), in the honey bee Apis. Mellifera, and in the fruit fly D. melanogaster (Okada et al. 2009; Sinakevitch et al. 2013). To a lesser extend also in the cortex of each glomerulus sparse GABA- and more GAD immunoreactivity were observed. Neurobiotin backfills from the antennal nerve in combination with GABA immunostaining showed that the olfactory sensory neurons terminate only in the cortex of the glomeruli, in close contact to the central GABA staining. This indicates that at the interface of cortex and core of the glomeruli antennal information is processed under GABAergic control. Also consistent with other insects, GABA-ir somata of local antennal lobe neurons were located laterally to the antennal lobes. In D. melanogaster two types of local neurons were identified according to their branching patterns. The local neuron 1 (LN1) which Discussion 122 arborized only in the core and local neuron 2 (LN2) which arborized in core and cortex of the glomeruli (Okada et al. 2009). Due to the mass staining and densely compact GABA-ir cell clusters, it was not possible to differentiate these two types of local neurons in the Madeira cockroach. Another type of GABA-ir antennal lobe neuron in R. maderae were projection neurons, which constitute output pathways from the antennal lobe in all insects examined. Projection neurons leave the antennal lobe via three tracts, the so called medial-(previously known as the inner-), mediolateral- (= middle-), and lateral (= outer-) antennal lobe tracts (mALT, mlALT, lALT1) and it was described that their main target projection areas are the lateral horn, lateral protocerebrum and the calyces of the mushroom body (Ito et al. 2014; Okada et al. 2009; Berg et al. 2009; Sinakevitch et al. 2013; Kirschner et al. 2006). In moths and Drosophila (Hoskins et al. 1986; Homberg et al. 1988; Iwano and Kanzaki 2005; Okada et al. 2009; Seki et al. 2010) as well as in the Madeira cockroach GABA immunoreactivity could be observed in the projection neurons of the mlALT that connect the antennal lobe directly to the lateral horn of the lateral protocerebrum. I could not trace the origin of this tract to specific GABA-ir projection neurons, because too many neurons were stained. The functions of GABA in the antennal lobe were studied extensively in M. sexta and P. americana, while the inhibitory function of GABA in M. sexta was even described in 1987 (Boeckh and Tolbert 1993; Waldrop et al. 1987). Likewise, injections of GABA into the antennal lobe resulted in a dose-dependent reduction in excitatory responses of projection neurons to pheromones in P. americana (Boeckh and Tolbert 1993; Homberg and Müller 1999). Additionally, it was shown that both GABAA and GABAB receptors are present in the antennal lobe of D. melanogaster (Wilson and Laurent 2005). Electrophysiological experiments from projection neurons and from the olfactory sensory neurons in D. melanogaster suggested that GABA mediates presynaptic inhibition via both GABAB and ionotropic GABAA receptors. Apparently, GABA mediated gain control, adjusting the intensity of olfactory input to the projection neurons (Olsen and Wilson 2008). Synaptic connections 1 The naming and abbreviations are taken from latest systematic nomenclature for the insect brain by Ito et al. 2014. Discussion 123 between the three types of antennal lobe neurons (sensory neurons, local interneurons, and projection neurons) were thoroughly studied in P. americana, also (Distler 1989; Distler and Boeckh 1997a, 1997b). Both, input and output neurons of the glomeruli received GABAergic inhibition, apparently mediating the gain and temporal pattern of the inputs to and outputs of the antennal lobe neurons (Distler and Boeckh 1997b). Electron microscopy studies combined with neuronal labelling in the antennal lobe of P. americana demonstrated that GABAergic local multi-glomerular interneurons are synaptically interconnected, whereby one type is monosynaptic between olfactory sensory neurons and inhibitory GABAergic local neurons of the antenna. It was suggested that this type of connection provides feed-forward inhibition, or lateral inhibition between glomeruli (Distler and Boeckh 1997b). Additionally, GABAergic interneurons form polysynaptic connections to projection neurons as well as to another GABAergic interneuron. This could generate either self-inhibition to cut excitations short and allow for odor pulse tracking, or it inhibits other interneurons, possibly for contrast enhancement. In both cases, the neuronal activity in the network would be suppressed by GABA (Distler and Boeckh 1997a, 1997b). Since there is great similarity between GABA-ir distribution in the antennal lobe of R. maderae and P. americana, a similar function for GABA in the olfactory center R. maderae is likely, but needs to be investigated further. Also, since GABA can also be used as excitatory neurotransmitter (Giese et al. 2018), it needs to be examined whether it also plays this role in the antennal lobe. GABA-mediated inhibition likely occurs in the calyces of the mushroom body The calyces of the mushroom bodies are the second-order neuropil of olfactory processing, with the antennal lobes being the first order neuropil. The processes of Kenyon cells, which have their somata located right above the calyces, structure all neuropils of the mushroom bodies: calyx, peduncle, vertical, and horizontal lobes (Heisenberg 1998; Homberg 1984). In the mushroom bodies, GABA immunoreactivity was restricted to the calyces, while Kenyon cells were not GABA-ir. However, the pedunculus divide (previously known as the lobe junction or knee) also showed innervation by loose and fine Discussion 124 GABA-ir fibers (Figure 3.2 A and D). In addition, also other neuropils around the mushroom body that received antennal lobe inputs and mushroom body outputs, expressed GABA immunoreactivity, such as the ring neuropil around the vertical lobe, medial lobes and above the pedunculus of the mushroom body (= middle inferior medial protocerebrum) (Tanaka et al. 2012). Applying GAD immunostaining the origin of GABA immunoreactivity in the pedunculus divide could be traced to GABA cell cluster G4 (Figure 3.8 B). Overall, GABA immunoreactivity in the mushroom bodies closely resembled the staining pattern observed in P. americana (Yamazaki et al. 1998; Strausfeld and Li 1999; Li and Strausfeld 1999). Similar to P. americana the calyces received fiber projections from two regions, namely one from the GABA-ir projection neurons of the antennal lobe and also from the GABA-ir cell cluster G7 which is located beneath the calyces near the optic stalk (Figure 3.2 D) (Mizunami et al. 1998). In P. americana, four giant GABA-ir somata were traced to the calyces (Strausfeld and Li 1999). These neurons were located in the area beneath the lobula referred to as “optic peduncle”. The position of these cells is similar to the GABA-immunostained cell cluster G8 in R. maderae (Figure 3.2 D). However, due to the dense fasciculation of the GABA-immunostained fibers and a general uniform staining, it was not possible to follow the branching pattern from the G8 cell cluster to the calyces. Nevertheless, this is most likely the same GABA-ir cell cluster which was described to provide massive processes into the lateral protocerebrum and have finally projections to the calyces (Strausfeld and Li 1999). In contrast to the honey bee (Schäfer and Bicker 1986) and the locust (Leitch and Laurent 1996), the medial and vertical lobes of the mushroom bodies were free of GABA staining, which might indicate that the neuronal circuit in the mushroom body is different in R. maderae. Functionally, mushroom bodies are considered to be central neuropils in the control of learning and memory. They receive multimodal sensory inputs and are mainly responsible for multisensory association, for olfactory and visual learning, as well as for memory formation and retrieval in many insect species (Menzel et al. 1994; Heisenberg 1998; Ganeshina and Menzel 2001). Studies in P. americana showed that mushroom bodies are important for place Discussion 125 memory as well (Mizunami et al. 1998). Thereby, the peduncle seems to function as main output area, while the calyces are suggested to be respective input areas. The calyces mainly receive olfactory information from the antennal lobes as well as visual input from the optic lobes (Mizunami et al. 1998; Strausfeld 2009). Apparently, GABA-ir processes in the outer layer of the calyces might modulate these sensory inputs to the Kenyon cells. Nishino and Mizunami (1998) also suggested, that GABAergic input to the calyces might be important for adjusting Kenyon cells activity, keeping them in an appropriate working range via homeostatic control mechanisms such as negative feedback (Nishino and Mizunami 1998). Additionally, it was shown that both types of GABA receptors are strongly expressed in the calyces (Blankenburg et al. 2015; Sattelle et al. 2000) and it was suggested that the GABAergic giant neuron in P. americana provides inhibitory feedback to the calyces while receiving signals from output neurons of the mushroom body (Takahashi et al. 2017; Yamazaki et al. 1998). Regarding to the homology among different insect species, it was proposed that the mushroom body of the cockroach P. americana varies from other insects since GABAergic inputs to the calyces do not provide recurrent feedback to the medial or vertical lobes (Yamazaki et al. 1998; Strausfeld and Li 1999). Nevertheless, further electrophysiological studies need to be performed to determine the role of GABA in the mushroom bodies of the Madeira cockroach. Conservation of the pattern of GABA immunoreactivity in the central complex among insects GABA immunostaining in the central complex of R. maderae was restricted to the lower division of the central body (Figure 3.2 C; Figure 3.11 D). in addition, the lateral complex which is closely associated with the central body also showed strong GABA immunoreactivity. Apparently, the distribution of GABA immunoreactivity in the central complex is conserved among various insects such as A. mellifera (Schäfer and Bicker 1986), M. sexta (Homberg et al. 1987), flies (Meyer et al. 1986; Hanesch et al. 1989), P. americana (Blechschmidt et al. 1990), the beetle Tenebrio molitor (Becker and Breidbach 1993), and the locust Schistocerca gregaria (Homberg et al. 1999). Therefore, this conservation proposed that GABA possibly plays an important, Discussion 126 evolutionary conserved role in the central complex functioning (Homberg et al. 1999). Electrophysiological studies provided evidence for a role of the central complex in integration of spatial information for locomotor control, polarized light vision, and sky compass orientation (Pfeiffer and Homberg 2014). In D. melanogaster, it was shown that the central body is involved in locomotor- and flight control (Ilius et al. 2007). In addition, it was shown that the GABA receptor subunit RDL is expressed in the lower division of central body (Aronstein and Ffrench-Constant 1995; Harrison et al. 1996). These data suggest that GABA might activate chloride channel receptors in the lower division of the central body (Homberg et al. 1999). Immunocytochemical studies in P. americana also showed a widespread distribution of GABA receptor subunit 1 (PeaGB1) in the brain including the central complex (Blankenburg et al. 2015). Furthermore, electrophysiological experiments in S. gregaria showed that the GABAergic TL2 and TL3 neurons are sensitive to polarized light (Homberg and Müller 1995; Homberg et al. 1999; Vitzthum et al. 2002). Interestingly, the position of these neurons is similar to the GABA-ir cell cluster G4 and G5 described here and which showed projections to the lower division of the central body by passing the lateral complex (Figure 3.2 C, Figure 3.11 D). However, physiological and pharmacological experiments are necessary to determine the GABA function in this region of the R. maderae brain. Feedback inhibition of GABA-ir centrifugal neuron on the lamina could adjust the light sensitivity of the photoreceptors GABA immunoreactivity in the optic lobe was described in many insect species. In the cockroach R. maderae, several types of GABA-ir neurons were identified in the medulla including transmedullary, monopolar, and feedback neurons which connect different optic lobe neuropils as well as the optic lobe with the midbrain (Figure 3.5; Figure 3.6 ). GABA-ir cells in the optic lobe were assigned to different medulla cell groups (M1-4), whereby the GABA-ir medulla cell group one (M1) is of particular interest. They are most likely columnar centrifugal or feedback neurons and the profile of this group is similar to columnar feedback neurons found in diptera (Strausfeld 1971; Strausfeld 1976) and hymenoptera (Datum et al. 1986b). They project to the cartridge in the lamina and control the sensitivity of compound eye photoreceptor neurons Discussion 127 (Meyer et al. 1986; Datum et al. 1986b; Hardie 1987; Füller et al. 1989). Two types of the columnar feedback neurons, C2 and C3, are best studied in D. melanogaster (Tuthill et al. 2013). Both neurons are multicolumnar. This means that they have dendritic arborizations in the proximal and distal medulla and then send their axons to the lamina where they are presynaptic to several lamina cells including large monopolar cells (see Introduction: Figure 1.10 and Table 1.3) (Hardie 1987; Meinertzhagen and O'neil 1991; Takemura et al. 2008a). Additionally, they receive presynaptic input from lamina cells in the medulla (Takemura et al. 2008a). Both type of neurons exhibit GABA- and GAD immunoreactivity in D. melanogaster as well as many other insects such as M. sexta (Homberg et al. 1987), Calliphora, Musca Domestica, D. melanogaster ( (Datum et al. 1986a; Meyer et al. 1986; Hardie 1987) and P. americana (Füller et al. 1989). Therefore, GABA was suggested to be the neurotransmitter of these types of medulla cells (Kolodziejczyk et al. 2008b). Targeted genetic manipulations combined with behavioral assays in D. melanogaster showed that GABAergic C2 and C3 feedback neurons are important for the motion vision (Tuthill et al. 2013). Silencing C2 and C3 neurons showed that the fly’s responses to asymmetric motion stimuli changed. Therefore, it was proposed that C2 and C3 are responsible for asymmetric filtering of a luminance signal via presynaptic inhibition at the monopolar cell terminals in the medulla (Takemura et al. 2008a; Tuthill et al. 2013). Inhibitory effects of GABA on lamina cells also were analyzed in the housefly Musca domestica. Application of GABA caused depolarization and simultaneously a significant decline in the response to light in the large monopolar cells (Hardie 1987). Additionally, immunohistochemical experiments in P. americana and D. melanogaster with antisera against the GABA A subunit (anti-RDL) and GABA B receptor (anti-PeaGB1) antibody showed that all neuropils of the optic lobe express GABA receptors (Blankenburg et al. 2015; Sattelle et al. 2000). Since columnar feedback neurons C2 in Musca and other insects are GABAergic and presynaptic to lamina cells (Datum et al. 1986a; Meyer et al. 1986; Füller et al. 1989) it was suggested that GABA controls the light sensitivity and contrast of the compound eyes by lateral inhibition between cartridges in the lamina (Datum et al. 1986a; Meyer et al. 1986; Hardie 1987; Füller et al. 1989). Except for Discussion 128 blowfly Calliphora erythrocephala in which only long photoreceptor axons (7) express GABA (Datum et al. 1986a; Meyer et al. 1986), no GABA- immunostaining was observed in photoreceptor cells of the compound eyes. While in the bee GABA immunoreactivity in the lamina derived from GABA-ir somata located near the lamina (Schäfer and Bicker 1986; Meyer et al. 1986), in R. maderae as well as in C. erythrocephala, M. domestica, D. melanogaster, and P. americana processes appeared to originate from GABA-ir columnar neurons with somata located at the proximal medulla (Datum et al. 1986a; Meyer et al. 1986; Füller et al. 1989). Relatively distinct layered GABA-staining in the medulla appears to stem from tangential and transmedullary neurons which project perpendicular to the retinotopically organized columnar units. It was suggested that these GABA-ir neurons may also provide lateral inhibition between neighboring columns in the medulla. Physiological experiments will examine the functions of GABA in the optic lobe of R. maderae. 4.3. The role of GABA in the circadian timekeeping system of Madeira cockroach The possible roles of GABA-ir neurons in the circadian clock of R. maderae As previously shown by Petri and Stengl (2001) GABA distribution is widespread in the circadian network of R. maderae. However, GABA-ir cells in the soma groups next to the AME were not known. In this project, we showed that GABA-ir somata belong to all soma groups except the ANes. Therefore, GABA was not only expressed in local neurons (DFVNes), but also in projection neurons (=output) of the AME. The total number of the GABA-ir neurons next to the AME was higher than what it was published before. Dense GABA immunoreactivity in the glomerular core of the AME implied that GABA acts as main inhibitory neurotransmitter via synaptic interactions between the AME neurons. GABA application to excised AMAE caused dose- dependent inhibition of electrical activity. Since GABA antagonist, picrotoxin desynchronized AME neurons, while GABA application caused neuronal Discussion 129 assemble formation, this suggested that GABA is important for the synchronization of the AME neurons (Schneider and Stengl 2005). In addition to assemble formation GABA is able to phase shift the cockroach’s clock, it was found that GABAergic synaptic interactions resulted in forming phase- locked assembles in the AME neurons (Schneider and Stengl 2005). Also, in mammals GABA mediated synchronization of the circadian pacemaker neurons via GABAA receptor activation (Strecker et al. 1997; Liu and Reppert 2000; Shinohara et al. 2000; Michel and Colwell 2001). In the cockroach P. americana, GABAB receptors were observed in all three neuropils of the optic lobe (Blankenburg et al. 2015). These findings already emphasized the importance role of GABA in circadian time-keeping in R. madarea. The importance of GABA for circadian time-keeping is further supported by the overlapping of GABA-ir fibers with PDF-ir fibers in the central brain. Arborization of PDF-immunostained fibers in two putative output regions of the circadian clock, the SMP and SLP (Homberg et al. 1991b; Reischig and Stengl 2003a; Renn et al. 1999), greatly overlapped with GABA immunostaining in the flies and cockroach. Although no colocalization occurred between both immunoreactivities, GABA-ir arborizations seem to partially overlap with PDF- ir arborizations, hence allowing for direct synaptic contact between each other. Besides, GABA- and PDF-ir fibers connect the AME via the lobula valley tract to the midbrain (Figure 3.11 E-E´´). Whether these are outputs of the clock to midbrain targets or input pathways from the midbrain into the AME, as known for PDF (Petri et al. 1995; Petri et al. 2002), will have to be determined. Strong connection of the AME to the multipeptidergic layer 4 of the medulla via GABA-ir medial layer fiber tract While only the AFF connected the AME to lamina neuropils, several tracts connected it to the medulla. The newly described GABA-ir MLFT connected the AME with the MLF system of the medulla, where PDFMEs and many other neuropeptidergic neurons arborize (Figure 4.1). Also, the GABA-ir distal tract which was shown previously to connect the glomeruli of the AME to the medulla (Petri et al. 1995; Petri et al. 2002), interconnected the AME and the MLF system of the medulla. We show here that GABA-ir fibers in the distal Discussion 130 tract terminate in the medulla and do not continue on to the lamina (Figure 4.1). Thus, the strong GABA-ir MLF system of the medulla, where as many neuropeptide immunoreactivity were detected as in circadian clock neurons, is the layer of the medulla that is most strongly interconnected with the AME (review: (Stengl et al. 2015a). Numerous peptidergic ipsi- and contralaterally projecting neurons of the circadian clock contribute to these connections. Furthermore, the MLF system of the medulla also receives ipsi- and contralateral midbrain input (review: (Stengl et al. 2015a). Among them are polarization- sensitive ventromedial AME neurons. They also arborize in the posterior optic tubercles (POTU) that are input regions to the protocerebral bridge of the central complex. Figure 4.1. Frontal view of a 3D reconstruction of GABA-immunoreactive (-ir) tracts (arrowheads), connecting the accessory medulla (AME) to lamina (open arrowheads) and medulla neuropils. A) The 3D reconstructions of lamina (LA) and medulla (ME) with yellow GABA-ir fiber tracts. Open arrowheads: a bundle of GABA-ir fibers of the anterior fiber fan projects from the AME into the most distal layer of the medulla. It sends side branches towards the medial layer fiber system (MLF) of the medulla and continues on to the proximal lamina. Double arrowhead: GABA-ir fibers of the distal tract also project into the most distal layer of the medulla, connecting the AME to the medulla, only. Fine branches of the distal tract turn towards the MLF (asterisk) of the medulla. Filled arrowheads: GABA-ir fibers of the MLFT originate from somata next to the AME and connect the MLFS (asterisk) directly with the AME, omitting the distal-most layer of the medulla. B) GABA-ir fiber tracts implemented into a confocal image (maximum projection of whole left optic lobe, stained with anti-GABA antisera). Dense GABA-ir innervation lightens up the MLFS (asterisk) of the medulla. Scale bars 100 µm. Discussion 131 The central complex is an important center for sky compass orientation that also controls locomotor programs (Loesel and Homberg 2001b; Homberg 2015; Varga et al. 2017). Thus, we hypothesize that many photic and non- photic inputs, also from locomotor control areas, reach the AME from the MLF system of the medulla and may be involved in phase resetting of the clock. In addition, circadian clock outputs may control information flow via the MLF system of the medulla, also via GABA-ir pathways, that could also guarantee mechanisms of gain control. The strong GABA-mediated connections between the cockroach clock and the MLF system are reminiscent of the GABAergic geniculohypothalamic tract that connects the suprachiasmatic nucleus to the intergeniculate leaflet (Albers et al. 2017). Both, the mammals intergeniculate leaflet of the lateral geniculate nucleus and the cockroach MLF system of the medulla are second-order optic neuropils that are closely connected to the circadian clock. Both receive photic input from the eye and both receive prominent input from 5-HT-ir, GABA-ir and neuropeptidergic projections. Furthermore, serotonergic projections from the nucleus raphe mediate non- photic inputs to the intergeniculate leaflet (Moore and Klein 1974; Shioiri et al. 1991; Morin 1999; Takemura et al. 2008a; Bridge 2011). Similar to the 5-HT effects on the cockroach clock in vivo and 5-HT effects in the circadian system of the cricket (Saifullah and Tomioka 2002), 5-HT also modulated rhythmic firing in the suprachiasmatic nucleus clock (Prosser et al. 1993; Jiang et al. 2000). Therefore, we hypothesize that photic and non-photic phase information is exchanged between the cockroach clock and the MLF system of the medulla. Could GABA-ir cells be circadian oscillators? Additionally, here we showed that almost all GABA-ir somata in the optic lobe including GABA-ir somata next to the AME (Figure 3.12) as well as in central brain regions were stained with the rmPER antisera. Oscillations in expression of the per and tim genes is required for endogenous circadian oscillation in D. melanogaster (Hall 1998). Studies in the cockroach R. maderae also showed that expression of per and tim1 genes oscillate in a circadian manner (Werckenthin et al. 2012). Since levels of GABA oscillated daytime- Discussion 132 dependently in the optic lobe as well as in the distal tract (see part 4.4.2), we propose that GABA-ir neuron next to the AME are circadian oscillators. In flies it was shown that the circadian clock protein PERIOD is expressed in neurons and glial cells. Moreover, PERIOD displayed robust circadian oscillations in both cell types (Jackson 2011). Since these findings were in accordance to data from mammals, it was suggested that glial cells have similar function in circadian timing system of both mammals and insects (Jackson 2011). It was demonstrated that the glial-specific factor so called Ebony is required for normal circadian behavior in Drosophila (Suh and Jackson 2007). Glial-specific genetic manipulations result in arrhythmic locomotor activity in Drosophila. It appears that astrocytes could modulate clock neurons and circadian rhythms in a calcium-dependent manner (Ng et al. 2011). Here, we showed that PERIOD similar to Drosophila and mammals is expressed in neuron and glial. However, in the cockroach R. maderae the clock protein PERIOD could not be shown to cycle in a circadian manner. Therefore, the level of rmPER appears not to be synchronized in the majority of cells in the central nervous system (Werckenthin et al. 2012). There is no information about the function of the glial or glial-neuron communication in the circadian system of the R. maderae. 4.4. GABA as a possible photic entrainment pathway to the clock Technical considerations Fab-fragment technique provides an opportunity to check the presence and distribution of several antisera that is raised in the same animal species in the same experiment. Here in this project, several multiple-label experiments with anti-GABA and anti-histamine or anti-serotonin antisera were performed. In all multiple-label experiments with the Fab method, anti-PDF antisera were also used as circadian marker. Additionally, we checked the colocalization of the anti-FMRFamide antibody with other antisera which were raised in the same species such as anti-5-HT, anti-MIP, and anti-GABA antisera. Even though several colocalizations in the cell body of the neurons next to the lamina, medulla and also neurons next to the AME were found, double- or triple immunostained fiber processes were rarely observed. Some reasons were Discussion 133 already mentioned for this phenomenon in different publications (Sossin et al. 1990; Reischig and Stengl 2003b; Soehler et al. 2011). One is the process called “peptide sorting”. Soehler et al. (2011) suggest that circadian clocks neurons operate via axonal peptide-sorting to regulate the phase of the physiological processes at the different time of the day. Generally, neuropeptides are transcribed in the cell nucleus. After processing in the endoplasmic reticulum of the soma they are packed in the Golgi apparatus into vesicles. Then, they are transferred down the axons to the terminals and stored there before release (Eipper 2005). It was also shown that the neuropeptide can be store and release at the varicosities (also named non-synaptic sites) (Nässel and Winther 2010). Additionally, there is now enough evidence that some neurons contain and release more than one neurotransmitters. They can be packed into different vesicles or the same vesicle (Vaaga et al. 2014). Therefore, according to the peptide sorting process, although several neuropeptides and neurotransmitters can be present in the same neuron, particular transmitters can be moved to specific terminals (Purves et al. 2001). This results in uneven distribution of neurochemicals throughout the neurons and subsequent identification of the different peptides and colocalization between them in the fiber projections become more difficult. Furthermore, multiple-stainings with the Fab-fragment method could cause crosstalk on two levels, on the level of the primary antibody, and on level of the secondary antibody that is conjugated with a fluorescence dye. Basically, Fab-fragment antibodies are used for double-immunostainings in which both antisera were obtained from the same host species. Because of the monovalent aspect of the Fab-fragments, they are not able to bind to any extra IgG antibodies. Therefore, they avoid binding to the second or third primary antibody. However, if too many of the conjugated-fab fragments bind to the first primary antibody, they might serve as divalent antibodies and pick up some of the second primary antibodies which subsequently lead to overlapping detections. Especially, when too high concentrations of antisera were used. Thus, I performed experiments with different dilutions of the antisera and also changed the order of the primary antisera to double-check my results. I noticed that when the anti-FMRFamide antibody was used as the first primary antibody Discussion 134 I obtained better data. Also, prolonged incubation times for low concentrations of Fab-fragments (conjugated as well as unconjugated) allowed for better results as compared to shorter incubation times with higher concentrations of Fab-fragments. Another reason for unspecific crosstalk is related to the detection level. Because three substances were labeled in the same sample, in order to prevent the crosstalk between the fluorescent dyes, the dyes required to be selected cautiously. Although the preparations were analyzed sequentially, the number of the analyzed substances were confined by the spectrum of the available lasers of the applied CLSM. Additionally, despite the fact that the CLSM provides high depth resolution, nevertheless, the size of the scanned individual points is restricted by the optic resolution of the utilized objective or the amount of excited fluorescent dyes (Pawley 2006). This issue is even more problematic in the area of fine fiber arborizations in which at some point detection of colocalization was no longer possible. Finally, because of dense and voluminous (or concentrated) overlapping immunoreactive fiber projections of some of the neuropeptides and neurotransmitters such as FMRFamide, serotonin, and GABA and because of similarity between the immunoreactive projection patterns, it was difficult to separate the fibers from each other. Most of the times, they appeared to overlap rather than co-localize. This was often observed in the optic lobe neuropils as well as in some midbrain areas such as the lateral protocerebrum. The GABA-ir neurons in the optic lobe could transmit photic signals to the accessory medulla Similar to other insects such as flies (Rieger et al. 2003; Yoshii et al. 2016), also in the Madeira cockroach R. maderae the histaminergic photoreceptors of the compound eyes do not directly project to the circadian clock (Loesel and Homberg 1999). Therefore, interneurons intercalate between photoreceptor terminals and the circadian clock. Only the GABA-ir distal tract was shown before to connect the circadian clock to other optic lobe neuropils (Reischig and Stengl 1996; Petri et al. 2002). Also, GABA injections into the vicinity of the AME resulted in a biphasic light-like phase response curve (Petri et al. Discussion 135 2002). Therefore, the GABA-ir distal tract was suggested to be involved in circuits conveying light information to the circadian clock. However, it was not clear where the distal tracts originated from and whether light information is encoded in the GABAergic distal tract. To investigate whether GABA might serve as a photic entrainment of the circadian clock via the ipsilateral compound eye, double-label immunostaining with anti-GABA and anti- histamine antisera were performed. With double labeling with anti-GABA and anti-histamine antisera, we showed that GABA-ir fibers in the distal tract might contact the terminals of the long histaminergic photoreceptor neurons in the medulla layer ME 2. Strong GABA immunoreactivity from neurons that project via the AFF to the proximal lamina, the termination site of the short histaminergic photoreceptors axons, could be another path to relay light information GABA-dependently to the AME. The AFF projections connect the AME to the medulla and to the proximal lamina. Soma groups in the lamina as well as next to the AME project via the AFF, such as GABA-ir AME neurons. In accordance with our results, intracellular recording from one of the AME neurons, which resembled a large-field GABA- ir neuron (Petri et al. 2002), showed that this neuron strongly responds to the light (Loesel and Homberg 2001b). Thus our results support the hypothesis of Petri et al. (2001) that suggests that the large-field GABA-ir neurons are candidates for light entrainment pathways to the circadian clock. Moreover, ELISA experiments showed that GABA content in the optic lobe peaked during the subjective day when animals are resting and sensitivity of the eyes is minimal (Figure 3.18 B ). The intensity of GABA-immunoreactivity in the DT also showed a maximum during the light phase (ZT06) (Figure 3.17). This finding is consistent with the pervious data regarding the inhibitory effect of GABA and its role as a sleep-promoting factor in mammals as well as insect (Roth 2007; Parisky et al. 2008; Chung et al. 2009; Gmeiner et al. 2013). In other insects such as M. domestica, it was proposed that GABA might control the light sensitivity of the compound eyes by presynaptic inhibition on cartridges of the lamina (Hardie 1987). So apparently GABA influences the photosensitivity by mainly presynaptic inhibition at the level of the lamina. Discussion 136 Application of GABA caused depolarization and simultaneously a significant decline in the response to light in the large monopolar cells (Hardie 1987). The large monopolar cells are directly postsynaptic to the photoreceptors and receive GABAergic input from columnar feedback neurons of the medulla. Therefore, the possible function for GABA in the lamina is modulation of transmission of light information via pathways between lamina and the clock. With respect to the function of the GABAergic neurons in the medulla we could only speculate that the GABAergic DT modulates processed visual information in the different layers of the medulla. However, at this point we cannot say where connections are made between the DT fibers and the any layers of the medulla. Further EM studies can clarify this topic. Since we hypothesize that light duration regulates neuropeptide-synthesis in the circadian clock neurons which release these neuropeptides daytime- dependently (Soehler et al. 2011; Stengl et al. 2015a; Stengl and Arendt 2016), together with previous findings it seems that the neurotransmitter GABA mainly provides light dependent inhibition to the AME. Besides, recent studies showed that the PDF level is also high during the day (Arendt 2016) and as PDF might have dual effects (inhibitory or excitatory) on the AME (Gestrich et al. 2018), we suggest that GABA and PDF both synchronize firing of clock neurons and thereby are able to adjust the neuropeptide release via gain control mechanisms. In support for an indirect effect of GABA on the circadian system is the finding that in the grasshopper Oedipoda caerulescens, it was shown that GABA levels showed significant daily variations in the optic lobe with maxima at the end of the light phase as well as a peak at the end of the dark phase (Vieira et al. 2005). This result suggests that GABA increase during the day/night transition might contribute to the integration of light information and subsequently to the control of daily melatonin synthesis (Vieira et al. 2005). Possible roles of melatonin in conveying photoperiodic information was suggested in invertebrates (Vivien-Roels and Pévet 1993). It was shown that melatonin is synthetized in a rhythmic manner in the optic lobe of the cockroach (Page 1987). These finding is consistent with our assumption that GABA might be involved in the regulation of rhythmic neuropeptide release. Discussion 137 GABA might receive light information from an extraretinal photoreceptive organ, the lamina organ In 2001 Fleissner and colleges described two putative extraretinal organs namely lamina and lobula organs in the cockroach R. maderea (Fleissner et al. 2001). Electron microscopy studies showed that both organs are composed of rhabdom-like microvilli. This indicated that these organs might have a photoreceptive function. Additionally, they exhibited CRY immunoreactivity. CRY is a blue light photoreceptor in many insects (Fleissner et al. 2001; Helfrich-förster et al. 2001) which could receive light through the light permeable head capsule of insects. Further studies on both organs revealed that they were also strongly immunoreactive to M. sexta UV-opsin antisera. This further supported the assumption of the photoreceptive role for the lamina and the lobula organs (Hofer 2004b). However, electrophysiological recordings combined with the application of light are necessary to test this assumption. Until now only Leucokinin showed immunostaining in the lamina organ. Apparently, few Leucokinin-ir fibers from the lamina organ project to the distal medulla (Lara Fricke 2016). In the beetle was shown that fiber fascicles connect lamina and lobula organs to the AME (Fleissner et al. 1993; Frisch et al. 1996). However, in the cockroach R. maderae this has not been shown yet. Here I showed for the first time that the lamina organ is also GABA-ir (Figure 3.5 E). But, no connection was found between GABA-immunostaining in the lamina organ neither to the lamina nor medulla neuropils. Nevertheless, we assume that GABA-ir fibers in the proximal lamina, the ALA and most likely in the first optic chiasm could receive light information from the lamina organ and relay it to the AME indirectly (Figure 4.2). Discussion 138 Possible role of GABA and 5-HT in gating circadian clock inputs and outputs Parallel physiological studies with this project demonstrated that GABA- and 5- HT-immunoreactive neurons constitute parallel excitatory or inhibitory pathways connecting the circadian clock either to the lamina or medulla where photic information from the compound eye is processed (Giese 2018; Gestrich 2018). With Ca2+ imaging on primary cell cultures of the AME and with loose- patch-clamp recordings in vivo, we showed that both neurotransmitters either excite or inhibit AME clock neurons (Giese et al. 2018). Because we found no colocalization of GABA and 5-HT in any optic lobe neuron, GABA- and 5-HT neurons form separate clock input circuits. Among others, both pathways Figure 4.2. Hypothetical ipsilateral photic input pathways to the cockroach circadian clock (accessory medulla = AME) with focus on PDF (cyan)-, GABA (yellow)- and 5-HT (magenta)-expressing neurons. Anti-PDF and anti-5-HT colabelled fibers of PDFLAs innervate the accessory laminae (ALAs) and the proximal lamina (pLA). We suggest that they receive inputs from extraretinal photoreceptors of the lamina organ (LAO) in the ALA and from short green-sensitive photoreceptor axons of the compound eye in the pLA. Via the anterior fiber fan, they send branches to the ventral shell and to ventral glomeruli of the AME, possibly controlling sleep–wake cycles. In contrast, the GABA-ir distal tract (DT), which does not coexpress neuropeptides or 5-HT, connects all glomeruli of the AME to the median layer fiber system (MLF) of the medulla. We suggest that this pathway gates photic and non-photic clock inputs and outputs via GABA-dependent ensemble formation/ dissipation. Furthermore, the MLFT interconnects the MLF of the medulla directly with the AME, possibly supplying homeostatic plasticity to control the gain of clock in- and output pathways. Coordinates: an, anterior; di, distal; ve, ventral. Discussion 139 converged also on AME neurons that coexpressed mostly inhibitory GABA- and excitatory 5-HT receptors. Our morphological and physiological findings suggested that GABA and 5-HT play important roles in photic- and non-photic input pathways that phase shift the clock. In addition, all tracts described could also carry GABA- and 5-HT-ir clock output pathways from the clock back to these input areas in the lamina and MLF system of the medulla, serving as gates for these inputs. Finally, GABA and 5-HT are also suited to serve as gates for other clock outputs to midbrain targets (Figure 4.2). Multiple-labeled medial neurons (MNes) are candidates for the ipsilateral/contralateral photic entrainment pathways to the circadian clock Using multiple-label experiments we analyzed the colocalization of FMRFamide with GABA, 5-HT, and MIP. The overall distribution of the tested antisera including MIP, 5-HT, PDF, and FMRFamide was consistent with previous works (Petri et al. 1995; Petri et al. 2002; Soehler et al. 2008; Soehler et al. 2011; Schulze et al. 2012). Additionally, as previously reported (Soehler et al. 2008) FMRFamide immunoreactivity was found in five soma groups next to the AME including DFVNes, MNes, VPNes, VNes, and ANes. In all triple- labeling experiments, almost all medium-sized PDF-ir soma colocalized with FMRFamide which is in accordance with result from previous work (Soehler et al. 2011). However, in just three preparations colocalization of PDF and FMRFamide was observed in small PDF-ir soma (2.8 ± 1.7). Also consistent with Petri et al. (1995), FMRFamide staining intensity in the lamina cells revealed only faint immunoreactivity in the cytoplasm while 5-HT immunoreactivity was stronger and even in whole cell bodies. Nevertheless, FMRFamide-ir lamina cells were only observed when anti-FMRFamide antisera were used at higher concentrations. In this work, we showed that FMRFamide coexpressed with 5-HT, GABA, and MIP in some of the neurons next to the AME. Interestingly, in all three experiments, FMRFamide showed colocalization in the MNe neurons (2 MNes with MIP, 1 MNe with GABA and 1 MNe with 5-HT). Since it was suggested that MNe neurons are involved in the ipsilateral light entrainment pathway to the AME (Loesel and Homberg Discussion 140 2001b), our result further indicated that MNe neurons might use these three transmitters to relay light information to the clock. It was speculated that photic entrainment pathway to the clock in the R. maderae happen via two different pathways at dawn and dusk, according to specific neuropeptidergic neurons that either advance or delay the circadian clock (Soehler et al. 2011; Stengl et al. 2015a). The MNes next to the AME are of particular interest. About 56 MNes lie next to the AME (Reischig and Stengl 2003b). Apparently, this group of neurons are a diverse group with respect to neuropeptide content as well as type of neurons. GABA, allatotropin, corazonin, FMRFamide, MIP, orcokinin were identified in MNes (Reischig and Stengl 2003b; Stengl and Arendt 2016; Soehler et al. 2008; Schulze et al. 2012; Hofer and Homberg 2006b). One of MNe neurons is assumed to be a good candidate for M-oscillator in the R. maderae. Although the composition of the M- and E-oscillator at the cellular level is still unknown, however, there are evidences for M-oscillator cells. The triple-labeled corazonin-, GABA-, and MIP-ir MNe was suggested to function as M-cell in the integration and transmission of light from contralateral to the ipsilateral AME (Stengl and Arendt 2016), since injection of each of these substances advanced the circadian clock during the late subjective night/early subjective day (Petri et al. 2002; Schendzielorz and Stengl 2014; Stengl and Arendt 2016). Additionally, it seems that one of GABA-ir MNes is light sensitive (Loesel and Homberg 2001b; Petri et al. 2002). This neuron connects the core of AME glomeruli to different layers of the medulla as well as to the proximal lamina and ALAs. Therefore, it was suggested that the core of the AME receives ipsilateral photic input. Additionally, at least 2 MNes are assigned to the commissural cell group MC IV which connects the two AMAE. The peptide content of commissural MNes remains elusive. So far, only the neuropeptide MIP was found in this group (Thordis Arnold 2016). Here we showed that FMRFamide-ir MNes colocalized with MIP, GABA and 5-HT (2 MNes with MIP, 1 MNe with GABA and 1 MNe with 5-HT). Since, one ventral glomerulus of the AME colocalized FMRFamide and MIP, it is possible that these MNes belong to those two the triple-labeled MNes found by Schendzielorz and Stengl (2014) which contains FMRFamide too. It is more Discussion 141 likely that the colabelled FMRFamide- and GABA-ir MNe also be the identical to colabelled FMRFamide- and MIP-ir MNe, given to that both triple-label MNes express GABA as well. Nevertheless, multiple-label immunocytochemical experiments required to show this colocalization. As described above, MNes are apparently responsible for ipsilateral light input to the AME and apparently, they contain different neuropeptides and neurotransmitters (see end of this part). Moreover, next to the double-labeled lamina cells, one of the two FMRFamide- ir MNes next to AME also expressed 5-HT. Injections of 5-HT and FMRFamide generated monophasic phase-response curves that differed in phase and shape from light-dependent phase shifts. The respective 5-HT- and FMRFamide-ir neurons might, therefore, relay non-photic clock inputs (Stengl et al. 2015a). Thus, it is possible that there are parallel phase-resetting input pathways to the AME. While it was shown that some of the AME neurons express more than one neuropeptide and neurotransmitter, we still do not have the complete picture. Further studies such as mass spectroscopy of individual AME neurons could provide missing information. Nevertheless, our results support the previous data that showed that the clock neurons express different neurochemicals that are differentially located and released via mechanisms of peptide sorting, possibly to phase-control physiological processes at specific times of the day (Soehler et al. 2011). Triple-labeled large-sized PDF-immunoreactive(-ir) neuron colocalizing FMRFamide and MIP as an important labeled line Out of five evaluated preparations, only in one optic lobe one triple-labeled large-sized PDF PDFME colocalized PDF with MIP- and FMRFamide immunoreactivity. The PDF-staining intensity of this triple-labeled cell was really weak and using high laser intensity we were able to see the cell. Therefore, we assume that maybe this triple-labeled cell often overlooked due to the weak staining intensity of the PDF. Discussion 142 Immunocytochemistry using different antisera showed that the medium-sized PDF-ir neurons colocalized with other neuropeptides such as orcokinin ( (Hofer and Homberg 2006a), FMRFamide (Soehler et al. 2011) and MIP (Schendzielorz and Stengl 2014). Additionally, neuronal tracing from cut optic stalks in combination with immunohistochemistry revealed that at least two medium-sized and one large PDF-ir neurons belong to the MC I contralaterally projecting neurons which coupled two AMAE (Reischig et al. 2004; Soehler et al. 2011). The medium-sized PDF-ir neurons also express orcokinin and FMRFamide (Hofer and Homberg 2006a; Soehler et al. 2011), and they connect both AMAE via the AOC and they have arborizations in output regions in the SLP and SMP. Therefore, it is assumed that in addition to PDF, FMRFamide and orcokinin serve a function in the coupling of both pacemakers (Schendzielorz and Stengl 2014). However, later in another study PDF-ir commissural MC I cells were assigned to the large-sized PDF-ir cells. One of this cells colocalized with MIP-immunoreactivity and another contralateral projecting large-sized PDF-ir cell colocalized MIP- and orcokinin- immunoreactivity (Thordis Arnold 2016). In current studied we also assigned the triple-labeled PDF-ir medulla cell to the large-sized, as the size of the soma (21.5 µm) were consistent to the size of this group of cells reported previously (Reischig and Stengl 2003a, 2003b). Therefore, it is likely that this cell is identical to either the commissural quadruple- or triple-labeled cells which contain PDF, MIP and orcokinin (Thordis Arnold 2016). However, additional backfill experiments in combination with multiple-label staining is required to identified this cell as contralaterally projecting neuron. Behavioral experiments with injection of MIP (Schulze et al. 2013), orcokinin (Hofer and Homberg 2006b, 2006a), and PDF (Schendzielorz and Stengl 2014) proposed a function of these neuropeptides in the photic entrainment pathways to the circadian clock. The above mentioned neuropeptides are also possible candidates for transmission of the contralateral light information as one backfilled commissural MC II (which assigned to the VMNes) colocalized MIP- and/or orcokinin immunoreactivity and one backfilled commissural MC IV (which assigned to the MNes) colocalized MIP. If the triple-labeled large medium- sized PDFME (PDF, FMRFamide, and MIP) found in the current study is identical with the multiple-labeled commissural cell of previous studies Discussion 143 (backfilled, MIP-, ORC-, and PDF-ir; MC I, (Thordis Arnold 2016), we further support the hypothesis in which circadian timed behavior is daytime- dependently gated by neuropeptide release (Stengl et al. 2015a). Based on this hypothesis, release of various neuropeptides or neurotransmitters at specific times of the day might recruit various cell groups and form ensembles of synchronized cells and thus promote gating of outputs. Extracellular recordings showed that application of PDF resulted in a new ensemble PDF- dependently (Schneider and Stengl 2005). Therefore, multipeptidergic large medium-sized PDFME of MC I most likely is responsible for photic entrainment as well as synchronization of both pacemakers via coordination of neuropeptides release. The likely mechanism of this neuron is that it releases neuropeptides day-time dependently with different phase. Then, the specific neuropeptide could influence postsynaptic cell ensembles via activation or inhibition and subsequently this specific “labeled line” results in the activation or inhibition of certain behavior (Stengl et al. 2015a; Thordis Arnold 2016). Nevertheless, further experiments are required to prove this hypothesis. Multipeptidergic accessory laminae might provide light input to the clock Distinct and small regions at the proximal edge of the lamina close to the ventral and dorsal lamina cell clusters were found in several studies (Petri et al. 1995). These tiny specializations were termed accessory laminae (ALAs) (Loesel and Homberg 2001b). The function of this neuropil is not known yet. However electrophysiological experiment in combination with the immunostainings showed that these structures receive arborizations from the light-sensitive local interneurons (ipsi) as well as contralateral neurons (Loesel and Homberg 2001b; Soehler et al. 2011). Additionally, they directly receive neurites from the lamina cells (Petri et al. 1995). Therefore, it was suggested that these structures might contribute to the photic entrainment pathway to the clock. In order to map the neuropeptidergic profile of the ALAs, double-labeled immunohistochemistry was performed. To visualized the neuropil structure, double staining with antisera against synapsin and different antisera against histamine, PDF, FMRFamide, serotonin, and GABA were performed. It appears that this small neuropil is abundant of various neurosubstances. Discussion 144 Interestingly, the ALAs are devoid of histamine staining, the neurotransmitter of the photoreceptors. Ca2+ imaging experiment on primary cell cultures of lamina cells (ventral and dorsal) that might also contain cells that innervate the ALAs showed that the majority of them responded to PDF, GABA, 5-HT, histamine and acetylcholine (Nali Hussein 2018). Despite the fact that neither ALAs nor lamina cells expressed histamine but they responded to it, indicated that these LA cells received light input from histaminergic photoreceptors of the compound eye. It was suggested that light resulted in PDF release in the lamina via histaminergic photoreceptors, subsequently recruiting several neuropeptide-dependent circuits. Via positive feedforward mechanisms the light-response will be amplified and passed on to the AME (Stengl and Arendt 2016). This circuit was suggested to control sleep and therefore, PDF is assumed to function as a sleep signal for cockroach. With the beginning of the night and in the absence of light result in termination of PDF signal and subsequently initiation of the activity cycle (Stengl and Arendt 2016; Gestrich et al. 2018). In addition, although none of the lamina cells were GABA-ir, however, they responded to the GABA application with either Ca+-influx or Ca+-efflux. This response of the LA cells to GABA was increased when PDF was applied before (Gestrich et al. 2018). Therefore, it was hypothesized that PDF-expressing neurons could regulate or gate photic entrainment in the lamina GABA-dependently (Arendt 2016; Giese et al. 2018). Additionally, GABA probably has a homeostatic function, so that the lamina cells are always receptive (gain control). In the beetle Pachymorpha sexguttata it was shown that there is a connection between the accessory neuropil associated to the lamina and the lamina organs (Fleissner et al. 1993; Frisch et al. 1996). In summary, these data together with current results indicate the importance of the ALAs and possible role in the light input pathway to the clock. Electrophysiology in combination with dye injections need to be performed in the future to determine the function and neuroanatomical connections of the ALAs and lamina organs with other regions of the optic lobe. Discussion 145 4.5. Possible role of GABA in coupling pathways between the AMAE To characterize the neurons involved in coupling of both AMAE and to determine whether the neurotransmitter GABA is involved in the coupling of both AMAE, neurobiotin backfills from one optic stalk were performed. Additionally, we analyzed the number of the commissural cells to make a reevaluation of the previously published labeled cells. A new polyclonal anti- GABA antibody (Sigma) was employed for the first time in the cockroach. The commissural cell number in each group corresponded to previous work (Table 3.3 ) (Soehler et al. 2011; Arendt 2016). The GABA immunoreactivity showed some variability from sample to sample when it was combined with backfill experiments (see also parts 3.5.2., 3.5.4., 3.5.5). GABA immunoreactivity exhibited more uniform and homogenous staining in many regions such as SMP, SLP, POTU, and neuropils posterior to the central brain as compared to when it was applied alone. One possibility might stem from the fact that since most of the backfills were done by severing the tissue, it might result in firing and subsequently discharging the GABAergic neurons. Therefore, less GABA content is left in the network to be recognized with GABA antisera. Another reason could be, since incubation time with tracer took overnight in the ringer solution, this might interfere with GABA epitopes in the tissue. In the brain region contralaterally to the backfilled side, neurobiotin-labeled fibers without GABA immunoreactivity were found in the SMP, SLP, ILP, and POTU. Nevertheless, they greatly overlapped. Based on the electrophysiological and neuroanatomical experiments it was suggested that contralaterally projecting PDFMEs of MC I convey phase information while contralaterally projecting VMNes (MC II) possibly transmit contralateral light information. It seems that MC I cells provide clock- dependent phase information via the AFF projecting to the lamina and ALAs whereas MC II cells project to the MLF system of the medulla and supply light information. Therefore, it was proposed that bilaterally symmetric AMAE are connected via two functionally different pathways (Reischig and Stengl 2002; Reischig et al. 2004). FMRFamide, orcokinin, and MIP are also candidates for Discussion 146 coupling pathways of both AMAE as they colocalized in contralaterally projecting PDFMEs (Soehler et al. 2011; Arendt 2016; Thordis Arnold 2016). In summary, Since GABA-immunoreactive cells were also found in the VNes and VMNes, and additionally colocalized in one MC I (VNe) and one MC II (VMNe) and one MC IV (MNe), this might indicate a function of GABA in coupling pathways. This further indicates the complexity of interactions in the same functional circuits. 4.6. Output pathway and GABA ILP, SMP, and POTU are potential premotor areas that relay circadian information to locomotor centers in the thoracic ganglia Central pattern generators in the thoracic ganglia of the ventral nerve cord control motor programs of wings and legs. Descending neurons transmit multi- sensory information from different brain centers to orchestrate central pattern generator activity. The central pattern generators in turn give feedback about activation states through ascending neurons (Marder et al. 2005). In the brain, ascending and descending neurons arborize in different neuropil regions, termed premotor areas. For the cockroach P. americana it is proposed that there are several parallel direct or indirect processing pathways from sensory centers in the brain to locomotor control centers in the thoracic ganglia (Okada et al. 2003). To generate appropriately timed rhythmic motor patterns, stimulus-controlled or endogenous clock-controlled time information input is also necessary. However, not much is known about information flow from the circadian system to locomotor control centers in the thoracic ganglia. In search for potential premotor areas where circadian information is relayed to the descending/ascending neurons, neurobiotin-backfill from the TG combined with immunocytochemical stainings using anti-GABA and anti-PDF antisera could identify potential synaptic connections between these two systems. Since my backfill experiments do not distinguish between descending and ascending neurons, neuropil areas with either one were identified. Distribution of somata and dendrites of descending and ascending neurons in the R. maderae was consistent with those described in P. americana (Okada et al. 2003). However, the number of the neurobiotin-labeled descending or Discussion 147 ascending neurons in R. madarea was less than in P. americana, most likely due to the fact that I performed fewer preparations and did not identify all of them. The large variation of neurobiotin-stained somata between preparations most likely was due to difference in up-take and transport of the tracer along the axons and not because of the actual variation in the number of the descending or ascending neurons. Additionally, due to the high background staining in some of preparations, it was often difficult to distinguish specific neurobiotin-stained somata from unspecific ones. As in P. americana, in R. maderae the fiber projection of the descending and ascending neurons were mainly concentrated in the tritocerebrum, the AMMC, ILP, lateral, and medial protocerebrum and lateral complex (Figure 3.30). While no neurobiotin-labeled processes were found in the antennal lobe, mushroom body and central complex. Additionally, we found that the ipsilateral lobula also receive fiber projections from the descending/ascending neurons. Backfill experiments from the thoracic ganglion in combination with immunohistochemistry using GABA and PDF antisera showed that several regions could serve as potential connection between the circadian system and descending/ascending neurons. Neurobiotin-labeled fibers are in close vicinity to the PDF-ir fibers in the LOVT, the lobula, POC, p3, ILP and SMP (Wei et al. 2010b). In all of these neuropils also GABA immunoreactivity are present. Of particular interest is the SMP since in Drosophila this neuropil was identified as output of the circadian clock to locomotor control areas ( (Homberg et al. 1991a; Reischig and Stengl 2003a; Renn et al. 1999). In the Madeira cockroach the SMP is a bilaterally symmetric neuropil. Both PDF-ir SMP and SLP are connected to the optic lobe via the AOC (Reischig and Stengl 2002). Based on the overlap between PDF-ir and neurobiotin-labeled fibers, it can be assumed that the SMP is an important premotor area with connections between the circadian system and descending/ascending neurons (downstream neurons) of locomotor control centers. In orthopteroid brains such as grasshoppers brains it was shown that the dendrites of descending neurons that contribute to flight stabilization were located in the posterior brain such as the inferior- and ventrolateral Discussion 148 protocerebrum (ILP and VLP) (Homberg et al. 1991b; Williams 1975). In the Madeira cockroach PDF-ir fiber projections in the ILP greatly overlapped with neurobiotin-labeled fibers from the thoracic ganglion. Therefore, PDF-ir ILP could be another potential premotor area receiving circadian information via PDF-pathways and relaying it to locomotor control centers in thoracic ganglia. Another putative area connecting the circadian system to the locomotor control center is the POTU. PDF-ir fibers from the largest PDFME arborize in the POTU overlapping with neurobiotin-labeled fibers ipsi- and contralateral side of backfills. There is not much known about the function of the POTU in the Madeira cockroach. However, polarization-sensitive VMNes also arborize in the POTU (Loesel and Homberg 2001b; Soehler et al. 2011). Also in the locust it is involved in sky compass navigation (Beetz et al. 2015). In the locust the AME is connected directly to the protocerebral bridge with additional arborizations in the POTU (Homberg et al. 2011). Thus, the POTU in the Madeira cockroach might relay visual information to the central complex as well as being a premotor area transmitting circadian information to downstream locomotor control centers. In all potential premotor areas that were mentioned so far, GABA immunoreactivity was prominent. GABA immunoreactivity in the SMP, ILP, and POTU overlapped with neurobiotin-labeled fibers from the TG. The role of GABA in these regions is not understood yet. Additionally, GABA immunoreactivity in the lateral complex, AMMC and tritocerebrum greatly overlapped with neurobiotin-labeled fibers that branched on both, ipsi- and contralateral sides of the backfills. There is not much known about the functions of GABA in the AMMC in insects. In crickets, the AMMC is involved in the control of antenna movements during the flight and walking (Horseman et al. 1997; Cardona et al. 2009). The AMMC receives dendritic arborizations from descending neurons, and thus seems to convey mechanosensory information to the flight center in the thoracic ganglia. (Horseman et al. 1997; Cardona et al. 2009). The lateral complex mainly contributes to polarized light-guided flight. Apparently this neuropil receives ascending afferent fibers from the thoracic ganglia as well as dendritic Discussion 149 arborizations from descending neurons (Homberg 1994a; Homberg et al. 2004; Homberg 2004). Whether GABA plays a role in the AMMC and lateral complex in the Madeira cockroach needs to be studied further. GABA might increase photosensitivity of the ocellar photoreceptors to provide entrainment at low light levels In 1977 Page and colleagues showed that the ocelli in the R. maderae have no function in the light entrainment pathway since surgical removal of the ocelli did not affect entrainment by a light cycle. However, intracellular recordings combined with dye injection showed that one light-sensitive ocellar neuron arborized in the AME (Loesel and Homberg 2001b). Later, behavioral assays under dim light/dark conditions demonstrated, however, that cockroaches without ocelli were not able to entrain to low light conditions (Hofer 2004a, 2004b). This finding indicates that ocelli are required for light entrainment of the circadian clock under low light conditions. With backfill experiments we were able to label 2 types of ocellar neurons. The morphology of these neurons was similar to large second-order neurons (Mizunami and Tateda 1986; Loesel and Homberg 2001b), and small multimodal ocellar interneurons (Ohyama and Toh 1990a, 1990b). These two neuronal types join the ocellar tract and from there project to the ipsilateral optic lobe and enter the AME. Although projections of the large second order ocellar neurons could not be clearly distinguished from the projections of the two small multimodal neurons, apparently both neuron types project towards the optic lobe (Ohyama and Toh 1990b; Loesel and Homberg 2001b; Ohyama and Toh 1990a). In the optic lobe neurobiotin-labeled fibers from the ocellus greatly overlapped with PDF- and GABA-ir fibers in the LOVT. These fibers entered the medulla and innervated the AME. Electrophysiological experiments in the dragonfly suggested that there might be synaptic feedback interactions between the ocellar photoreceptors and second- or third order cells (Klingman and Chappell 1978; Stone and Chappell 1981). At the beginning of the light stimulus presentation, the ocellar photoreceptors released a transmitter and elicited an inhibitory response in the second-order neurons. At the same time, the second-order neurons released Discussion 150 feedback neurotransmitters onto photoreceptor terminals. This process is in turn inhibited by the release of the neurotransmitter by the photoreceptors upon illumination. GABA is suggested to be a possible candidate for the feedback neurotransmitter since GABA application resulted in an increased amplitude of the response and facilitated the release of neurotransmitter from photoreceptor terminals. It was proposed that this feedback mechanism results in a higher sensitivity of the photoreceptor cells and therefore enables responses to dim light (Klingman and Chappell 1978; Stone and Chappell 1981). Additionally, release of GABA from the ocellar neuron dendrites decreased the time that the cell requires to return to the baseline level after hyperpolarization (Klingman and Chappell 1978; Stone and Chappell 1981). This might decrease the response time to the next light stimulus. Later, in locusts it was shown that ocellar second order neurons (s-neurons) are GABAergic and responsible for feedback to the ocellar photoreceptors (Ammermüller and Weiler 1985). Ocelli in Drosophila contribute to photic entrainment. Mutant flies without functional compound eyes, ocelli and Hofbauer-Buchner eyelet are not able to entrain to the LD cycle (Rieger et al. 2003). However, no direct connection was found between ocelli and the circadian clock. Apparently, ocellar neurons contact projections of the circadian neurons in the dorsal protocerebrum where circadian information is relayed to downstream neurons (Helfrich‐Förster 2003). Regarding roles of GABA in ocellar functions, in P. americana it was shown that the large ocellar neurons express GABA receptors. Application of GABA and its agonists elicited an excitatory response in these neurons (Lee et al. 2007). Additionally, activation induced by cercal stimulation in the large ocellar neurons was attenuated by applying GABA A receptor antagonists. This finding suggested that sensory information transmission might be modulated by GABAergic input to the large ocellar neurons (Lee et al. 2007). In the Madeira cockroach, it seems that light information from the ocelli is relayed directly to the AME and contributed to entrainment at low light levels. The neurobiotin-labeled fibers from the ocellar neuron in the AME overlapped Discussion 151 with GABA immunoreactivity, therefore, GABA might also have a modulatory function to input from the ocellar neurons. Nevertheless, this function of GABA is not known yet. To further understand how the ocellar system contributes to the circadian system of R. maderae electrophysiological experiments with different light intensities in combination with dye injections could characterize functions of the ocellar neurons which innervate the clock. Additionally, more backfill experiment combine with immunohistochemistry are required to characterize all three level ocellar neurons and projection patterns in R. maderae. GABA might be involved in the temporal control of the neurosecretory system via the circadian system GABA is the predominant neurotransmitter of the circadian system as well as the main inhibitory neurotransmitter in the central brain of insects and mammals alike. It was hypothesized that light duration regulates neuropeptide- synthesis in the circadian clock neurons which in turn release different neuropeptides daytime-dependently. The released neuropeptides then synchronize firing of GABAergic clock neurons thereby activating clock outputs to orchestrate hormone release (Schneider and Stengl 2005; Stengl et al. 2015a). In search for connections between the circadian clock and neurosecretory cells, neuronal backfills from the neurohaemal organs were combined with GABA- and PDF immunohistochemistry. Our experiments showed that none of the neurosecretory cells neither in the PL nor PI showed GABA- immunoreactivity. However, neurobiotin-labeled fibers in the nerves from the CC, NCC I and NCC II were located closely to the GABA-ir fibers. The absence of GABA immunoreactivity in the neurosecretory cells were also reported in the M. sexta (Homberg et al. 1987; Homberg et al. 1991a). Neuroendocrine systems in insects are important for long-term functions such as development and metabolism. For insects seasonal timing is as important as daily timing because it enables them to predict environmental changes in different seasons, because some seasons are more suitable for reproduction Discussion 152 and development than others. Therefore, photoperiodic information from internal clocks must reach the neuroendocrine system (Meuti and Denlinger 2013). There is little known about how output pathways of the circadian clock connect to the neurosecretory cells in the brain of the R. maderae. A connection between the circadian system and the endocrine system was demonstrated in the cricket Acheta domesticus (Cymborowski 1981). In particular, the ablation of the PI diminished the diurnal locomotor rhythm and the implantation of PIs could rescue it with the same circadian period as the donor (Cymborowski 1981). Cells in the PI of cricket also expressed the clock proteins PER and CRY (Shao et al. 2006). Additionally, PER- and PDF-ir neurons with connections to the CC were detected in the German cockroach Blattella germanica (Wen and Lee 2008), indicating a connection between neurosecretory centers and the circadian system. Employing immunocytochemistry, many neuropeptides which were shown to be involved in regulations of circadian clock processes (Petri et al. 1995) were also found in neurons of the PI and PL terminating in the retrocerebral complex. Detailed descriptions were shown for FMRFamide-related peptides (FaRPs) (Nässel 1993), allatostatin ( (Stay et al. 1992), and corazonin (Veenstra and Davis 1993; Predel et al. 1994). However, it was not known which neurotransmitters the neurosecretory cells in the PL and PI express. In the Madeira cockroach, the SMP and SLP could provide connections between circadian system and neurosecretory system. Multiple-label immunohistochemistry using anti-PDF and anti- corazonin antisera showed that projection of the neurosecretory corazonin-ir neuron overlapped with PDF- ir fibers in the SLP and SMP (Arendt et al. 2017; Arendt 2016). Respectively, PDF-ir fibers in the SLP also overlapped with GABA-ir fibers. The function of GABA in the neurosecretory circuits in the brain of Madeira cockroach is not known yet. The functions of GABA in neurosecretory cells that are located in the abdominal ganglion of Periplaneta were studied. It was shown that in Periplaneta the dorsal unpaired median (DUM) neurons which also are neurosecretory cells have GABA receptors (Dubreil et al. 1994). Application of GABA hyperpolarized the DUM cells. Additionally, immunohistochemical Discussion 153 experiments showed that GABA-ir fine processes were near the DUM neurosecretory cells which could indicate possible synaptic connections. Therefore, it was suggested that the DUM cells receive inhibitory inputs from GABAergic interneurons and modulate neurosecretion in DUM cells (Dubreil et al. 1994). A similar function could be suggested for neurosecretory cells in the PI and PL, however, not much is known about the presence of GABA receptors this brain area. In summary, backfill experiment combined with immunohistochemistry showed that apparently GABA directly or indirectly could modulate the outputs of the circadian clock to the downstream neurons. In the ocellar system apparently GABA influence input and output of the both the circadian system and photosensitivity of the ocellar photoreceptors. GABA has a similar modulatory effect on the information flow between the circadian system and the neurosecretory system as well as the central pattern generators in thoracic ganglia via premotor areas. Nevertheless, further experiments are required to prove these assumptions. 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I wish to thank my committee members, Prof. Dr. Maniak and Prof. Dr. Mayer, who were more than generous with their expertise and precious time. I would also like to thank Prof. Dr. Uwe Homberg and her technical assistant Mrs. Jutta Seyfarth for their kind support and comment. This dissertation would not have been possible without the help and support of a number of people. I feel myself very fortunate for having had the opportunity to work along with so many talented and friendly colleagues. I would like to thank my friends and former co-workers, Dr. Achim Werckenthin, Dr. Julia Schendzielorz, Dr. Thomas Schendzielorz, Dr. Andreas Arendt and Dr. Petra Gawalek for imparting their knowledge to me at the beginning of my project. Their continued support allowed me to finish my thesis. Special thanks to my colleague and best friend, Thordis Arnold, for her friendship and all the great moments that we have had together in and out of the laboratory. I sincerely appreciate Katrin Schröder for her critical suggestions and comments during practical work in the laboratory. A special thanks to Dr. Jenny Plath, whose comments and questions were very helpful in the completion of my discussion manuscript. I thank Karin Große-Mohr, Romy Freund, and Christa Uthof for their expert technical assistance. I owe my deepest gratitude to Christina Wollenhaupt, who helped me throughout the administrative processes. 179 I also want to express my gratitude to former animal keeper Christin Sender and present animal keeper André Arand for providing me with the animal housing facilities necessary to carry out my dissertation work. I sincerely appreciate the support and friendship of all the former and present colleagues and students: Sebastain Korek, Fransiska Gronow, Ragna-Maja v. Berlepsch, Alena Kött, Susanne Koziarek, Dr. Nico Funk, Dr. HongYing Wei, Dr. Maria Giese, Rany Karls, David Eschstruth, Robin Schumann, Dr. Andreas Nolte. I cannot find words to express my gratitude to Carmen Muresan and Ashish Dewan for the positive influence they’ve had on my life. Their words of encouragement inspired me during a difficult time when I needed them the most. Thank you both for your concern and support! I’ll be forever grateful. As a final word, I would like to thank each and every individual I might have forgotten, but whose support and encouragement nonetheless helped me to achieve my goal and complete my dissertation work successfully.