14th rauischholzhausen seminar€¦ · cockroach rhyparobia maderae (hongying wei) 11:15 larval...

14th Rauischholzhausen Seminar

Development and Plasticity of the insect nervous system

7th and 8th May 2016

Schloss Rauischolzhausen

(Schlosspark, 35085 Ebsdorfergrund-Rauischholzhausen)

http://uni-marburg.de/KfPSn

Organization:

Prof. Dr. Joachim Schachtner Philipps-University Marburg Dept. Biology – Animal Physiology Karl-von-Frisch Str. 8 35043 Marburg

Contact during the meeting Joachim Schachtner mobile: +49 175 4317502 Office Schloss: +49 6424 301-100

Program 3

PROGRAM

Saturday 7th May

Arrival from 11:30

12:30 Lunch

13:30 Welcome and introduction of participants

13:45 Neuroethology of insect social organization Jens Habenstein, Ayse Yilmaz-Heusinger, Martin Strube-Bloss (AG Rössler, Würzburg)

Olfactory-visual integration in mushroom body output neurons (Martin Strube-

Bloss)

Age-related and light-induced plasticity in the ant visual system (Ayse Yilmaz-

Heusinger)

Identification and localization of neuropeptides in the brain of Cataglyphis desert

ants using mass spectrometry imaging (Jens Habenstein)

15:00 Neural correlates and network models of olfactory processing Susanne Hindennach, Martin Nawrot (AG Nawrot, Cologne)

15:30 Coffee Break

16:15 The olfactory system of the red flour beetle Tribolium castaneum Stefan Dippel, Anika Heinze, Marlene Schmidt, Joachim Schachtner, Björn Trebels

(AG Schachtner, Marburg)

Introduction (Joachim Schachtner)

Update on the olfactory pathway of T. castaneum (Stefan Dippel)

Postmetamorphic plasticity of the mushroom bodies (Björn Trebels)

17:00 Profiling of biomolecules Sander Ließem, Marcel Bläser, Susanne Neupert (AG Predel/Neupert, Cologne)

Introduction (Susanne Neupert)

Studying of neuropeptidergic modulation of adaptive locomotor behavior in

Carasius morosus (Sander Liessem)

Analysis of the Evolution of Neuropeptides by using transcriptomes of insects,

especially Polyneoptera (Marcel Bläser)

18:15 Dinner

20:00 Functional anaylysis of cockroach antennal lobe neurons Victor Bardos, Peter Kloppenburg, Jonas Klußmann, Jan Radermacher (AG

Kloppenburg, Cologne)

Introduction (Peter Kloppenburg)

Neuron type specific transient voltage activated K+ currents (Viktor Bardos)

Specialized physiological phenotypes of central antennal lobe neurons I (Jan

Radermacher)

Specialized physiological phenotypes of central antennal lobe neurons II (Jonas

Klußmann)

Program 4

Open end – with wine and beer

Sunday 8th May

9:00 Neuropeptides in Drosophila Dennis Pauls, Radostina Lyutova, Johanna Räderscheidt, Alice Weiglein (AG Pauls,

Würzburg)

Introduction (Dennis Pauls)

Induction of associative odor memories by optogenetic activation of Kenyon cells

in Drosophila melanogaster larvae (Radostina Lyutova)

SIFa as a putative orexic peptide in Drosophila melanogaster larvae (Alice

Weiglein)

Aminergic and peptidergic control of activity and sleep in Drosophila melanogaster

(Johanna Räderscheidt)

10:15 Coffee Break

10:30 Olfactory transduction Julia Gestrich, Monika Stengl, HongYing Wei (AG Stengl, Kassel)

Introduction (Monika Stengl)

The function of Orco in pheromone transduction of the hawkmoth Manduca sexta

(Monika Stengl)

Physiological analysis of central circadian pacemaker neurons in the Madeira

Cockroach Rhyparobia maderae (HongYing Wei)

11:15 Larval development of the olfactory system of the giant robber crab Birgus

latro (Linnaeus, 1767) Tina Kirchhoff (AG Harzsch, Greifswald)

11:40 Final discussion and group photograph

12:30 Lunch

13:30 Departure

Abstracts 5

ABSTRACTS

Saturday 7th May

Olfactory-visual integration in mushroom body output neurons

Martin Strube-Bloss

University of Würzburg, Department of Behavioral Physiology and Sociobiology (Zoology II), Theodor-Boveri-

Institute of Bioscience, Biocenter, Am Hubland, 97074 Würzburg, Germany

Brains are fascinating miracles spread all over the animal kingdom. They receive a tangle of multi

modal input and hold the capacity to simultaneously select and ignore adequate stimuli to extract

the behavioral relevant information. As a matter of course the latter reflects a multi modal

construct rather than the neural representation of a single (context less) modality. How the brain

connects the environmental indentations to guide an organism’s performance and at which neural

level the modalities interact with each other remains an open question.

Multi modal integration involves convergence of sensory pathways at a higher brain level where

in some cases single neurons displaying stimulus-evoked activity to both of two modalities that

will be integrated. The honeybee’s mushroom body (MB) represents such a higher order

integration center. Its ~170.000 Kenyon Cells (KC) are organized in layers each receiving input from

a different modality. MB output is conveyed by ~400 MB extrinsic neurons (MBON) which we

record on a reliable basis using extra cellular multi-unit recording technique. We established an

experimental design allowing characterization of olfactory, visual, as well as olfactory-visual

induced activity in the same MBONs. Preliminary results illustrate, that the layered input of the

MB is represented in a subpopulation of MBONs responding to either odors or light stimulation

whereas other MBONs were sensitive to both modalities and thus integrating olfactory-visual

information across MB input layers.

Age-related and light-induced plasticity in the ant visual system

Ayse Yilmaz, Annekathrin Lindenberg, Stefan Albert, Kornelia Grübel, Johannes Spaethe, Wolfgang Rössler, Claudia Groh

University of Würzburg, Department of Behavioral Physiology and Sociobiology (Zoology II), Theodor-Boveri-


Camponotus rufipes workers are characterized by an age-related polyethism with young ants first

performing tasks inside the nest before starting to forage outside. We tested the hypothesis that

this transition is accompanied by profound adaptations in the peripheral and central visual

systems. Our results show that C. rufipes workers express three genes encoding for ultraviolet

(UV), blue (BL), and long-wavelength (LW1) sensitive opsins in their retina. Expression levels of all

three opsin genes increased significantly within the first two weeks of adulthood and following

light exposure. Interestingly, the volumes of all three optic neuropils (lamina, medulla, and lobula)

showed corresponding volume increases. Tracer applications into the OL revealed a segregation

of visual input from the ME into the MB-calyx collar via the anterior superior optic tract (asot). The

MB collar volumes and densities of synaptic complexes (microglomeruli, MGs) increased with age.

Exposure to light for 4 days induced a decrease in MG densities followed by an increase after

extended light exposure. This shows that plasticity in retinal opsin gene expression and structural

neuroplasticity in primary and secondary visual centers comprise both “experience-independent”

and “experience-dependent” elements. We conclude that both sources of plasticity in the visual

Abstracts 6

system represent important components promoting optimal timing of the interior–forager

transition and flexibility of age-related division of labor.

Funding was provided by the DFG, SFB 1047 “Insect timing”; project B3 to J.S., B5 to C.G., and B6

to W.R.

Identification and localization of neuropeptides in the brain of Cataglyphis desert ants using mass

spectrometry imaging

Jens Habenstein1, Susanne Neupert2, Franziska Schmitt1, Reinhard Predel2, Christian Wegener3, Wolfgang Rössler1

1. University of Würzburg, Department of Behavioral Physiology and Sociobiology (Zoology II), Theodor-Boveri-


2. University of Cologne, Department for Biology, Institute of Zoology, Biocenter, Zuelpicher Strasse 47b, 50674

Cologne

3. University of Würzburg, Department of Neurobiology and Genetics, Theodor-Boveri-Institute of Bioscience,

Biocenter, Am Hubland, 97074 Würzburg, Germany

Cataglyphis desert ants undergo an age-related polyethism from interior worker with tasks like

brood care and food processing inside the dark nest to predominantly visually guided foragers.

The internal regulation of the remarkably short transition period of 1-3 days from interior worker

to forager is unknown but behavioral changes like e.g. activity levels are supposed to accompany

this transition. Changes of neuropeptide levels are associated with the behavioral transition in

honeybees and are further known to regulate a wide range of behavioral and physiological

processes including locomotor activity, feeding and metabolism. Since those processes are

potentially changing with the behavioral transition, neuropeptides might represent key signals

which are involved in this transition in Cataglyphis ants. We therefore develop mass spectrometry

imaging (MSI) to measure neuroactive peptides in the brain of C. noda. MSI enables the mass

spectrometric detection of neuropeptides on thin brain sections, providing 2-dimensional and

antibody unspecific high resolution images of the distribution of neuropeptides in an

unprecedented manner. We aim to optimize MSI on 14 μm sections for comparative and

reproducible peptide detection in different brain neuropils of C. noda. Quantitative comparison of

the distribution of individual neuropeptides in the brain of an interior worker and forager,

respectively, might indicate potential neuropeptides involved in the regulation of the behavioral

transition.

Neural correlates and network models of olfactory processing

Susanne Hindennach, Martin Nawrot

University of Cologne, Department for Biology, Institute of Zoology, Biocenter, Zuelpicher Strasse 47b, 50674

Cologne

Abstracts 7

Update on the olfactory pathway of T. castaneum

Martin Kollmann1, Stefan Dippel2, Georg Oberhofer2, Sergious Frank1, Milosz Krala1, Carolin Knoll1, Stephanie Crombach1, Ernst A. Wimmer2, Joachim Schachtner1

1. Philipps-University Marburg, Department. of Biology, Animal Physiology, 35032 Marburg, Germany

2. Georg-August-University Göttingen, Department of Developmental Biology, Johann-Friedrich-Blumenbach-

Institute of Zoology and Anthropology, 37077 Göttingen, Germany

The olfactory pathway of Tribolium castaneum, starts with the olfactory sensory neurons (OSNs)

in the chemoreceptor sensilla of the antenna, which project into the glomeruli of the antennal

lobe (AL). From the AL projection neurons send their axons in higher olfactory integration centers,

the mushroom body (MB) and the lateral horn (LH).

By creating a partial Orco-Gal4-UAS line, expressing tGFP in OSNs, which contain the Orco (the

general olfactory receptor), using the EF1-B-DsRed line (elongation factor1-alpha regulatory

region-DsRedExpress), which labels in the antenna mostly chemoreceptive neurons, RNA

sequencing,; electron scanning microscopy of the antenna, immunostainings with an antibody

against Orco, fluorescence in situ hybridization, backfills of the antenna and maxillary palps, and

dye injection into the AL, we characterized the olfactory pathway in the brain und gnathal ganglion

(GNG) of the red flour beetle Tribolium castaneum in high detail.

In the antenna as well as in the mouthparts we found many gustatory and olfactory receptors

(GRs, ORs), indicating, that there is no organotopic separation of taste and olfaction between the

two appendages. Analyzing the anatomical features of the pathway revealed no sexual

dimorphism at the level of the antenna and the AL. Backfills of the antenna labeled all AL glomeruli

but one, which is exclusively labeled by fills of maxillary palps. We indentified three antennal lobe

tracts connecting the AL with the MB and LH that were so far not known in Coleoptera. Our

analysis further revealed an unpaired, glomerular organized structure (GOC) in the GNG. The GOC

is innervated by axons originating in the maxillary and labial nerves and is labeled in the Orco-

Gal4-UAS line, indicating involvement in olfaction. The projections from the mouthparts project

further towards the brain into an area resembling the lobus glomerulatus of hemimetabolous

insects that was so far not described in holometabolous insects. Supported by the DFG priority

program SPP 1392 “Integrative analysis of olfaction” (SCHA 678/13-1, WI 1797/4-1).

Postmetamorphic plasticity of the mushroom bodies

Björn Trebels, Joachim Schachtner

Philipps-University Marburg, Department of Biology, Animal Physiology, 35032 Marburg, Germany

With its fully sequenced genome and the susceptibility for reverse genetics based upon RNA

interference (RNAi), Tribolium castaneum is best suited to study the development and plasticity

of the nervous system. While plasticity can be provided by various mechanisms, we focus on

ongoing cell proliferation in the adult brain. It is well established that neurogenesis persists in the

mushroom bodies (MB) of adult insects, including beetle T. castaneum where neuroblasts giving

birth to MB Kenyon-cells remain active for more than one month after adult eclosion. To label cell

proliferation in the adult beetle we successfully adapted the 5-ethyl-2’-deoxyuridine (EdU)

technique to living beetles. Combined with immunohistochemistry against the glia-cell marker

reversed-polarity and the use of transgenetic lines expressing neuron- and/or glia-specific

markers, we labeled the progenies of adult persisting neuroblasts, determined their identity and

counted the newborn Kenyon cells in within the first week after adult eclosion to determine the

proliferation rate.

Abstracts 8

In several studies it was proposed that newborn neurons of MBs may play a role during olfactory

processing and learning. We combined the EdU-staining with olfactory stimulation using the leaf

alcohol cis-3-hexen-1-ol and again determined the proliferation rate. Our data suggest at least two

phases. Direct after adult eclosion, proliferation is independent from stimulation with the leaf

alcohol, while after about three days, proliferation is influenced by olfactory stimulation.

To further investigate MB plasticity, we plan to use other odors, including the beetle’s aggregation

pheromone 4,8-dimeythydecanal (DMD), odor deprivation and knockdown of the general

olfactory receptor (Orco) via systemic RNAi.

Studying of neuropeptidergic modulation of adaptive locomotor behavior in Carasius morosus

Sander Ließem, Joachim Schmidt, Reinhard Predel, Ansgar Büschges


Cologne

In the past few years, an increasing number of studies have focused on the identification of

neuropeptides in the central nervous system of insects, which represent one of the largest classes

of signalling molecules. Besides the regulation of a wide range of physiological processes,

neuropeptides can act as a neuromodulator or cotransmitter modulating the neuronal activity

within neuronal circuits. For example, recent studies on insects revealed that FMRFamide-related

neuropeptides might affect the release of octopamine from dorsal-unpaired median neurons,

which is a biogenic amine that plays a major role in insect motor control. One of the most

thoroughly studied insect species with respect to locomotion is the indian stick insect Carausius

morosus. In the stick insect, detailed information exists on premotor networks in controlling

walking. Mere knowledge exists, however, on the set of neuropeptides that are involved in the

generation and modulation of motor activity, e.g. locomotion.

In the present study a biochemical approach called Imaging Mass Spectrometry (IMS) will be used

to map the spatial distribution of neuropeptides in the thoracic ganglia, which contain the

neuronal microcircuits for the generation of locomotion. However, IMS requires that the

neuropeptidome of the animal is biochemically characterized, but so far only 15 neuropeptides

were descripted. In order to investigate the distribution of neuropeptides, first a neuropeptidomic

analysis will be conducted. Subsequently, the spatial distribution of neuropeptides in the thoracic

ganglia will be examined and, hence, the peptidergic content of single neurons that belong to the

locomotor system (premotor interneurons) will be in focus of investigation. This information shall

then be used to provide insight into the neuropeptidergic modulation of motor neurons and the

premotor microcircuitry that drives locomotion.

Analysis of the Evolution of Neuropeptides by using transcriptomes of insects, especially

Polyneoptera

Marcel Bläser, Reinhard Predel


Cologne

Neuropeptides are key players in information transfer and act as important regulators of

development, growth, metabolism, and reproduction within multi-cellular animal organisms

(Metazoa). These short protein-like substances show a high degree of structural variability and are

recognized as the most diverse group of messenger molecules. We use transcriptome sequences

Abstracts 9

from the 1KITE (1K Insect Transcriptome Evolution) project to search for neuropeptide coding

sequences in more than 100 species of polyneopteran insect.

Polyneoptera is a lineage within Pterygota that rapidly evolved in the Carboniferous and radiated

into a variety of morphologically and biologically diverse orders (e.g. locusts, cockroaches,

damselflies, stick insects). Due to their fast succession of branching events, the inner phylogeny of

this group remains partially cryptic despite recent molecular and morphological attempts to solve

internal relationships. During my thesis, I am going to examine transcriptome assemblies for

neuropeptide precursor sequences; resulting alignments of selected sequences will serve as basis

for phylogenetic analyses. In this context, the evolution of FXPRLamides (pyrokinins/PBANs, CAPA)

will be studied in more detail. Genes coding for these neuropeptides are characterized by a

number of evolutionary changes occurring with the origin of Hexapoda; including new receptor

ligands, internal gene duplications and gene duplications with differentiation of cellular expression

patterns in the nervous system of hexapods. We expect that data on the evolution of the CAPA/PK

precursors of the polyneopteran insects will provide interesting and general insights into the rapid

evolution of a neuropeptide gene/precursor in general.

Neuron type specific transient voltage activated K+ currents

Viktor Bardos, Peter Kloppenburg


Cologne

Specialized physiological phenotypes of central antennal lobe neurons I

Jan Radermacher, Peter Kloppenburg


Cologne

Specialized physiological phenotypes of central antennal lobe neurons II

Jonas Klußmann, Peter Kloppenburg


Cologne

Abstracts 10

Sunday 8th May

Induction of associative odor memories by optogenetic activation of Kenyon cells in Drosophila

melanogaster larvae

Radostina Lyuthgova1, Dennis Segebarth1, Jens Habenstein2, Christian Wegener1, Andreas Thum3, Dennis Pauls1

1. University of Würzburg, Department of Neurobiology and Genetics, Theodor-Boveri-Institute of Bioscience,


2. University of Würzburg, Department of Behavioral Physiology and Sociobiology (Zoology II), Theodor-Boveri-


3. University of Konstanz, Department of Biology, Neurobiology and Developmental Biology, 78457 Konstanz

How brains organize behavior based on internal needs on the one hand and changing

environmental information on the other is one of the key questions in neuroscience. Learning is

defined as a process leading to a lasting alteration in behavior due to experience. Even animals as

simple as the Drosophila larva are able to form and recall an association of a particular odor with

a rewarding stimulus. In the last years it turned out that - similar to the adult fly – the larval

mushroom bodies (MB) are required for diverse behavioral functions, including odor learning and

memory. The larval MB consists of about 2000 embryonic and larval born Kenyon cells. However,

in previous work we could demonstrate that only around 100 embryonic born Kenyon cells are

required for associative odor-sugar learning. Furthermore, optogenetic activation of

dopaminergic/octopaminergic neurons is sufficient to substitute the unconditional stimulus (US)

during conditioning, while optogenetic activation of specific olfactory neurons is sufficient to

substitute the conditional stimulus (CS). However, to our knowledge it is still elusive whether the

conditional activation of Kenyon cells is sufficient to form memory traces. Thus, we are interested

in whether a conditional optogenetic activation of Kenyon cells is, dependent on the set of Kenyon

cells included in the Gal4 line, sufficient to induce an appetitive or aversive memory

SIFa as a putative orexic peptide in Drosophila melanogaster larvae

Alice Weiglein, Jeffrey Wilczek, Johanna Räderscheidt, Christian Wegener, Dennis Pauls

University of Würzburg, Department of Neurobiology and Genetics, Theodor-Boveri-Institute of Bioscience,


Feeding is one of the most important physiological processes in all living organisms including the

fruit fly Drosophila melanogaster. In adult flies, metabolism and feeding behaviour is strongly

regulated by neuropeptides. We are focusing on the role of SIFamide (SIFa) as a putative orexic

peptide in larval Drosophila and investigate whether SIFa affects feeding per se or if SIFa is rather

involved in food-related chemosensory responsiveness. The first step of our work was to examine

whether the activation of SIFa-positive neurons directly leads to an altered feeding behaviour in

the larva, while we subsequently investigated if the activation of SIFa-positive neurons affects

gustatory preferences to both appetitive and aversive stimuli.

Abstracts 11

Aminergic and peptidergic control of activity and sleep in Drosophila melanogaster

Johanna Räderscheidt, Christiane Luibl, Charlotte Förster, Markus Krischke, Martin Müller, Christian Wegener, Dennis Pauls

University of Würzburg, Department of Neurobiology and Genetics, Theodor-Boveri-Institute of Bioscience,


Vertebrate and invertebrate motor control is based upon neuronal activity in local circuits in the

spinal cord or ventral nerve cord, respectively. Descending neurons from the brain further regulate

these neuronal circuits. In insects, the central complex as the major locomotor center was shown

to control different aspects of locomotor activity such as velocity, activity maintenance and

orientation. In addition to the central complex, the mushroom bodies are suggested to participate

in locomotor control as they were shown to trigger walking behavior. Neuropeptides, key players

in the adaptation of neuronal networks to environmental changes, can modify and orchestrate

complex behaviors and were shown to influence the modulation of locomotor behavior. In detail,

adipokinetic hormone (AKH) is known to function in the regulation of hemolymph lipid and

carbohydrate levels, thus required mainly under acute locomotor activity for energy mobilization.

In this study we focus on the function of AKH for locomotor activity of flies and figure out potential

interaction of AKH with the biogenic amines octopamine and dopamine. Our approach will shed

light onto complex interactions of peptides and amines in the adaptation of fly activity to its

metabolic state and arousal, respectively.

The function of Orco in pheromone transduction of the hawkmoth Manduca sexta

Monika Stengl

University of Kassel, Department of Biology, Animal Physiology, Heinrich-Plett-Str. 40, 34132 Kassel, Germany,

Physiological analysis of central circadian pacemaker neurons in the Madeira Cockroach

Rhyparobia maderae

HongYing Wie, Monika Stengl

University of Kassel, Department of Biology, Animal Physiology, Heinrich-Plett-Str. 40, 34132 Kassel, Germany,

Larval development of the olfactory system of the giant robber crab Birgus latro (Linnaeus, 1767)

Tina Kirchhoff, Steffen Harzsch

University of Greifswald, Zoological Institute and Museum, Department of Cytology and Evolutionary Biology,

Soldmannstrasse 23, 17487 Greifswald

During the evolutionary history of Crustacea, several taxa independently invaded land to establish

a terrestrial life style. Terrestrial crustaceans include for example members of the hermit crabs

(Anomala), such as the giant robber crab Birgus latro (Linnaeus, 1767). These animals depend on

the ocean for reproduction but conquer the land during late larval development. However, the

majority of hermit crabs, like the common hermit crab Pagurus bernhardus (Linnaeus, 1758) are

fully marine. The eggs of Birgus latro are fertilized on land and carried by females under their pleon

over several months. Shortly before hatching, they are released into the ocean, where the first

larval stage (Zoëa I) hatch when in contact with salt water. Across four successive marine zoëal

Abstracts 12

stages, the marine benthic megalopa develops. This stage enters an empty gastropod shell to

finally invade the terrestrial habitat.

During larval settlement and metamorphosis as well as the transition to land, the larvae undergo

drastic changes in habitat, morphology, behavior and physiology. We are interested in the

ontogeny of the larval olfactory system of hermit crabs and its ontogenetic adaptation to

terrestrial life. Our main focus lies on the structure of the first pair of antennae and the

morphology of the brain. We analyze these structures by using X-ray microcomputed tomography,

scanning-electron microscopy as well as several histochemical and immunhistochemical stainings.

We expected to find ontogenetic changes in the morphology and arrangement of the antennular

sensilla, because terrestrial and marine animals detect different semiochemicals, which differ for

example in their chemical properties. In water, molecules have to be water-soluble to travel from

one individual to another, whereas semiochemicals on land are volatile. The brain morphology will

be mostly characterized by a substantially growth and the addition of new components. The

results obtained from studying the terrestrial species will be compared with the completely

marine common hermit crab Pagurus bernhardus. First insights into the Birgus larvae showed

drastic alterations on the first antennae at the metamorphosis to the megalopal stage. Especially

the type of setae is changed from mainly plumose setae in zoëal stages to composite and simple

setae in the megalopal stage. However, the “neuro-metamorphosis” occurred considerably later

in this stage and seems to be associated with the terrestrialization. The deutocerebral lobes for

example display conspicuous side lobes and a remarkable serotonin expression in the glomeruli

for the first time. Conclusively, our preliminary results show several transformations within the

marine and terrestrial larvae of Birgus latro and might help answer the question of evolving

terrestrial life.

Funded by the German Research Foundation (DFG: Ha 2540/13-1).

14th rauischholzhausen seminar€¦ · cockroach rhyparobia maderae (hongying wei) 11:15 larval...

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