Development and structural plasticity of the locust olfactory pathway
THESIS
submitted in partial fulfillment of the requirements for the degree
- Doctor rerum naturalium - (Dr. rer. nat.)
by
Dipl. Biol. René Eickhoff Lübeck, Germany
Hannover 2012
Supervisor: Prof. Dr. Gerd Bicker
University of Veterinary Medicine Hannover
1st Evaluation: Prof. Dr. Gerd Bicker
University of Veterinary Medicine Hannover
2nd Evaluation: Prof. Dr. Stephan Steinlechner
University of Veterinary Medicine Hannover
Date of final exam: 10.10.2012
T
ABLE OF CONTENTSAbbreviations ... I Zusammenfassung ... II Abstract ... V
Introduction ... 1
Development of the insect nervous system ... 3
Organization of the locust olfactory pathway ... 5
Thesis outline ... 7
Publications ... 8
Authors‘ contributions ... 8
Publication 1: Developmental expression of cell recognition molecules in the mushroom body and antennal lobe of the locust Locusta migratoria ... 10
Publication 2: Development of nitrergic neurons in the nervous system of the locust embryo ... 11
Publication 3: Regeneration of olfactory afferent axons in the locust brain ... 12
Publication 4: Scanning laser optical tomography resolves structural plasticity during regeneration in an insect brain ... 13
Discussion... 14
Class III (gustatory) Kenyon cells ... 15
Development of the mushroom bodies ... 15
Possible functions of axon guidance molecules and the NO/cGMP pathway ... 17
Structural plasticity of the olfactory centers ... 19
Conclusions/Outlook ... 21
References ... 22
Acknowledgements ... 29
Eidesstattliche Erklärung ... 30
I
A
BBREVIATIONS3D three dimensional
AL antennal lobe
Ca calyx
Ca2+ calcium
cAMP cyclic adenosine 3’,5’-monophosphate cGMP cyclic guanosine 3’,5’-monophosphate CLSM confocal laser scanning microscopy
DC0 catalytic subunit of the Drosophila protein kinase A DiI 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine
Fas fasciclin
GABA gamma-aminobutyric acid GMC ganglion mother cell
IBMX 3-isobutyl-1-methylxanthine
KC Kenyon cell
L1 first instar larval state L5 fifth instar larval state
Lach lachesin
LH lateral horn
LHN lateral horn neuron
MBC mushroom body calyx
MBNB mushroom body neuroblast
NADPH nicotinamide adenine dinucleotide phosphate (reduced form)
NO nitric oxide
OPN olfactory projection neuron OPT optical projection tomography ORN olfactory receptor neuron
Ped peduncle
plexA plexin A
RNAi RNA interference
Sema semaphorin
sGC soluble guanylyl cyclase
SLOTy scanning laser optical tomography
αL alpha-lobe
βL beta-lobe
Zusammenfassung
II
Z
USAMMENFASSUNGEntwicklung und strukturelle Plastizität der Heuschreckenriechbahn René Eickhoff
Die Riechbahn der Heuschrecke ist morphologisch und funktionell gut untersucht. Über die Entwicklung der Riechbahn und ihre plastischen Eigenschaften im adulten Tier ist jedoch sehr wenig bekannt.
Die Antennalloben bilden das primäre Riechzentrum der Insekten. Hier treten die olfaktorischen Rezeptorneurone der Antennen in synaptischen Kontakt mit olfaktorischen Projektionsneuronen, die die Geruchsinformation an höhere Verarbeitungszentren, die Pilzkörper weiterleiten. Pilzkörper sind aus Tausenden von Kenyonzellen aufgebaut. Sie stehen in engem Zusammenhang mit multimodaler Signalverarbeitung und der Gedächtnisbildung. Die vorliegende Arbeit befaßt sich mit der Entwicklung und dem Regenerationsvermögen der Riechzentren (Antennalloben, Pilzkörper) in der Wanderheuschrecke Locusta migratoria.
Als embryonale Marker habe ich monoklonale Antikörper verwendet, die gegen die membranassoziierten Wegweisermoleküle Semaphorin 1a (Sema 1a) und Fasciclin I (Fas I) gerichtet sind und mit ihrer Hilfe eine umfassende Beschreibung der Riechbahnentwicklung erstellt. Da auch während der Larvalentwicklung fortlaufend neue Kenyon Zellen produziert werden, habe ich die Analysen auf die postembryonale Pilzkörpergenese ausgeweitet. Ich konnte zeigen, daß auswachsende neugeborene Kenyonzellen das embryonale Expressionsmuster von Sema 1a und Fas I aufweisen. Den Entwicklungsstudien lagen anatomische Untersuchungen an Pilzkörpern adulter Tiere zu Grunde. Mit Hilfe einer Kombination von DiI-Markierungen und DC0-Immunzytochemie konnte ich bisher unbekannte Strukturen („accessory lobelets“) darstellen, die aus gustatorischen (class III) Kenyonzellen hervorgehen.
Die NO/cGMP Signalkaskade ist ein wichtiger Regulator für das Wachstum und die Wegfindung sich entwickelnder Axone. In Zusammenarbeit mit Kollegen habe ich die embryonale Entwicklung des NO/cGMP Signalweges im Heuschreckenhirn untersucht.
III Hierfür kamen eine Reihe zytochemischer Marker zum Einsatz. Putativ NO-synthetisierende Neurone wurden mit Hilfe der NADPH-Diaphorase Technik, enzymatische Aktivität der NO- Synthase via Citrullin-Immunfluoreszenz dargestellt. Um NO-sensitive Neurone zu identifizieren, wurden nach Applikation eines NO-Donors cGMP positive Neurone mittels immunzytologischer Markierung in Anwesenheit des Guanylatzyklase (sGC) Aktivators YC-1 und des Phosphodiesterase-Inhibitors Isobuthyl-Methylxanthin (IBMX) durchgeführt. Unsere Ergebnisse ergaben keine eindeutigen Hinweise darauf, daß die NO/cGMP Signal Kaskade eine Rolle während der Riechbahnentwicklung spielen könnte, da in den Riechzentren Marker für die NO-Synthase und für ihre Aktivität erst post-embryonal exprimiert wurden.
Im zweiten Teil dieser Arbeit wurde die Riechbahn der Wanderheuschrecke als Modell für Regeneration herangezogen. Olfaktorische Rezeptorneurone wurden durch Quetschen des Antennennervs axotomiert. Die dadurch resultierende Degeneration und Regeneration im Antennallobus wurde lichtmikroskopisch via Größenmessungen und anterograder Markierungen der Rezeptoraxone dokumentiert. In den ersten drei Tagen nach dem Quetschen verloren die Antennalloben 30% ihrer Größe und erreichten innerhalb von zwei Wochen wieder nahezu Ausgangsgröße. Regenerierende Axone innervierten synaptische Glomeruli und exprimierten die embryonalen Zelloberflächenmoleküle Lachesin und Fas I.
In einem zweiten Regenerationsexperiment wurde getestet, ob Laser-gestützte Optische Tomographie (SLOTy) eine geeignete Methode darstellt, um volumetrische Veränderungen in unbehandelten (ganzen) Hirnen darzustellen. Dieses neue mikroskopische Verfahren generiert dreidimensionale Daten von Proben, die eine Größe von mehreren Millimetern überschreiten, während es simultan verschiedene Detektoren verwendet. Zusätzlich zum ursprünglichen Ansatz wurden ganze Antennen abgetrennt, um maximale Degeneration des Antennallobus zu erreichen. Neben den Antennalloben wurde das Augenmerk auch auf die Pilzkörper gerichtet. Rekonstruierte tomographische Aufnahmen lieferten glaubwürdige Daten über die strukturellen Veränderungen inmitten des Heuschreckenhirns. Es konnte zudem gezeigt werden, daß die Deafferentierung des Antennallobus trans-synaptische Veränderungen hervorruft die zu einer Größenreduktion des Pilzkörper Calyx führen.
Zusammenfassung
IV
Die vorliegende Arbeit bietet das Fundament für eine Reihe möglicher experimenteller Ansätze um die Entwicklung und das Regenerationsvermögen der Riechzentren weiter zu erforschen.
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BSTRACTDevelopment and structural plasticity of the locust olfactory pathway René Eickhoff
The locust olfactory pathway is well studied with respect to morphology and function, yet little is known about its development and its plastic capacities during adult life.
In insects the primary olfactory integration centers are the antennal lobes. Here, olfactory receptor neurons from the antennae synapse with olfactory projection neurons which relay olfactory information to second order processing centers, the mushroom bodies. These brain centers are composed of thousands of Kenyon cells. Mushroom bodies have been implicated in multimodal sensory information processing and memory formation. This thesis examines the development and the regeneration properties of the olfactory centers (antennal lobes, mushroom bodies) in the locust Locusta migratoria.
I utilized monoclonal antibodies against membrane associated developmental guidance cues such as Semaphorin 1a (Sema 1a) and Fasciclin I (Fas I) as embryonic markers and provide a comprehensive description of antennal lobe and mushroom body development. Because Kenyon cells were generated throughout larval stages I extended the analyses to postembryonic mushroom body formation demonstrating that outgrowing Kenyon cells in larvae retain the embryonic expression pattern of Sema 1a and Fas I. To obtain an anatomical reference for subsequent developmental studies I initially revisited mushroom body anatomy in adult brains. Combining DC0 immunocytochemistry with DiI labeling, I resolved previously unrecognized accessory lobelets arising from class III (gustatory) Kenyon cells.
The NO/cGMP pathway is known as critical modulator of axon outgrowth and guidance. In collaboration with colleagues I followed the embryogenesis of the NO/cGMP system in the locust brain using various cytochemical markers. Putative NO-synthesizing cells were identified using the NADPH-diaphorase technique whereas enzymatic activity of Nitric oxide synthase (NOS) was analyzed using citrulline immunolabeling. To visualize NO-sensitive neurons I used cGMP immunocytochemistry after bath application of an NO-donor in
Abstract
VI
presence of the soluble guanylyl cyclase (cGC) activator YC-1 and the phosphodiesterase- inhibitor isobuthyl-methylxanthine (IBMX). For the olfactory pathway these markers did not provide conclusive evidence for a role of the NO/cGMP system in axon extension or growth cone navigation, because NOS expression and activity occurred only at the end of embryogenesis.
In the second section of this work the locust olfactory pathway was introduced as model system for regeneration. Olfactory receptor neurons were axotomized by crushing the antennal nerve. Using conventional microscopy, we followed the resulting degeneration and regeneration in the antennal lobe, by measuring its size and by anterograde dye labeling of regenerating axons. Within three days post crush antennal lobe size was reduced by 30%
and from then onward regained size almost back to normal within two weeks. Regenerating olfactory receptor axons re-innervated antennal lobe glomeruli and expressed the developmental cell surface molecules Lachesin and Fas I.
In a second regeneration experiment we tested whether Scanning Laser Optical Tomography (SLOTy) might serve as a suitable method to monitor structural changes in whole mount brain preparations. This novel microscopic technique generates three-dimensional information of samples above millimeter size by using two different imaging modalities at the same time. In addition to the initial regeneration approach we ablated entire antennae to follow maximum degeneration and concentrated not only on the antennal lobe but also on the mushroom body calyx. Reconstructed tomographic images reliably resolved volumetric changes deep in the locust brain, thus allowing for rapid screening of structural plasticity in high throughput applications. Remarkably, SLOTy revealed that deprivation of olfactory input has a trans-synaptic effect leading to size reduction of the mushroom body calyx.
This thesis provides the groundwork for various experimental approaches regarding both development and regeneration of the olfactory centers.
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NTRODUCTIONSeveral fundamental mechanisms of early nervous system development have originally been described in insects such as fruit flies (Drosophila melanogaster) and locusts (Schistocerca gregaria, Locusta migratoria). However, the majority of insights came from investigations of the ventral nerve cord and the peripheral nervous system. Hence, our understanding of insect brain development remains fragmentary. To elucidate the ontogeny of the more complex brain, neuroscientists can now take advantage of what is known from the ventral nerve cord and apply similar approaches to insect brain preparations, such as mapping neuroblast patterns, tracing specific cell lineages, or monitoring the spatiotemporal sequence of neuropil formation (Boyan et al., 1995; Urbach and Technau, 2003; Boyan and Reichert, 2011; Kunz et al., 2012).
The olfactory pathway is particularly suitable for analyzing insect brain development because it is clearly organized in characteristic neuropils. Olfactory information from the antennal receptors is transmitted to higher order integration centers, e.g. the mushroom bodies, via only two consecutive synaptic stages (Fig. 2). Thus, all integral parts of the system are uniquely identifiable during morphogenesis. So far, the formation of the olfactory pathway has predominantly been analyzed in the holometabolous species Drosophila melanogaster and Apis mellifera (reviewed in Farris and Sinakevitch, 2003; Jefferis and Hummel, 2006). Whereas in the locust the olfactory system has thoroughly been analyzed with respect to morphology and function (Laurent and Naraghi, 1994; Anton and Hansson, 1996; Leitch and Laurent, 1996; Ignell et al., 2001), only little is known about its development.
Central nervous systems can change throughout lifetime. It is well accepted that insect brains retain plastic capacities after development has been completed (Groh and Meinertzhagen, 2010). For instance, structural changes in the nervous system occur after injury, sometimes associated with regeneration. As a remarkable exception to vertebrates, insects are able to regenerate neurites both in the peripheral and the central nervous system (Jacobs and Lakes-Harlan, 1999; Pätschke et al., 2004; Ayaz et al., 2008; Stern and Bicker, 2008). Thus, mechanistic regeneration studies can largely benefit from insect models.
Introduction
2
So far, the locust olfactory system has not been investigated with respect to its regeneration capacities. However, this system could provide a useful model to study neural plasticity for the same reasons that predestine it for developmental studies. Moreover, the locust olfactory neuropils show an exceptional degree of intraspecific plasticity during the phenomenon of phase polyphenism, that accompanies the conversion from solitary lifestyle to the swarm phase (Ott and Rogers, 2010).
The visualization of phenotypic changes deep in the locust brain is hampered by its relatively large size. Due to limitations in focal depth, confocal laser scanning microscopy (CLSM) requires to physically section the sample inducing distortions to the tissue that compromise the validity of quantitative data. Novel microscopic techniques allowing 3D imaging of whole mount samples above millimeter size are now gradually emerging (Sharpe, 2004; Lorbeer et al., 2011). These methods could likely help minimizing artefacts and reducing the effort needed to image structural changes in the brain. Their suitability for insect brain preparations remains to be shown.
This thesis examines the development and the plastic properties of the olfactory neuropils in the hemimetabolous insect Locusta migratoria. The first section (publications 1,2) addresses development. It is focussed on the developmental expression patterns of common key regulators of axon guidance, such as the cell surface glycoproteins Fasciclin I and Semaphorin 1a as well as molecules of the NO/cGMP signaling cascade. To obtain an anatomical reference for subsequent developmental studies, I initially revisited mushroom body anatomy in adult brains. In the second section (publications 3,4) the focus is on phenotypic plasticity. In close collaboration with colleagues, I axotomized antennal afferents and followed the resulting degeneration and regeneration in the olfactory centers. To quantify volumetric changes in whole mount brain preparations, the novel microscopic technique of Scanning Laser Optical Tomography (SLOTy) was utilized.
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Development of the insect nervous system
The formation of the nervous system is a sequence of consecutive developmental processes including neuronal differentiation, cell migration, axon outgrowth, synapse formation, cell death and, lastly, adaptations to the external environment (Sanes et al. 2006).
Neurogenesis commences shortly after gastrulation with the segregation of neural/glial progenitor cells from neurogenic zones of the embryonic ectoderm. Lateral inhibition by Notch and Delta signaling leads to the differentiation of only one neuroblast out of a proneural cluster. In contrast to vertebrates, whose neural precursors maintain contact to the epithelium, insect neuroblasts delaminate from the neurectoderm (Arendt and Nübler- Jung, 1999). Insect neuroblasts divide asymmetrically in a stem cell mode and generate a stereotyped sequence of ganglion mother cells (GMC). Eventually, the ganglion mother cells divide symmetrically to differentiate into neurons or glia (Doe and Goodman, 1985 a,b).
Once a neuron is born, its outgrowing neurite has to cover considerable distances to connect to an appropriate target (Araújo and Tear, 2003). An extension at the tip of the axon, the growth cone, navigates through the tissue by “sensing” different molecules in its environment. During axon outgrowth, correct path finding is accomplished by cell adhesion molecules and secreted or membrane associated guidance molecules which either serve as chemoattractants or chemorepellants (Araújo and Tear, 2003). Among the first axonal guidance cues that were found in neurodevelopmental studies, two members of the immunoglobulin super family, Fasciclins (Fas) and Semaphorins (Sema), have originally been identified in the locust (Bastiani et al., 1987; Snow et al., 1988; Kolodkin et al., 1992). Their developmental functions have been characterized focusing on the ventral nerve cord and the peripheral nervous system of locusts and fruit flies (Bastiani et al., 1987; Snow et al., 1988; Harrelson and Goodman, 1988; Zinn et al., 1988; Grenningloh et al., 1991). Virtually nothing is known about Fasciclin and Semaphorin expression in the olfactory pathway of the locust brain.
The signal transduction pathways by which external stimuli are transduced to the cytoskeleton are gradually becoming identified. It is well established that growth cone motility (collapse, protrusion, directed steering) is mediated by intracellular Ca2+ levels which
Introduction
4
in turn affect downstream enzymes and/or signaling cascades that trigger the reorganization of the cytoskeleton (Henley and Poo, 2004; Gomez and Zheng, 2006). In this context, the second messengers cAMP (cyclic adenosine 3’,5’-monophosphate) and cGMP (cyclic guanosine 3’,5’-monophosphate) are implicated in regulating how the growth cone reacts to an external stimulus. For instance, growth cone response to Sema 3a in Xenopus can be converted from repulsion to attraction by increasing the cGMP level (Song et al., 1998). In Drosophila, both cAMP and cGMP are likely to regulate growth cone response to Sema 1a (Ayoop et al., 2004; Terman and Kolodkin, 2004). An increasing number of studies implicate the NO/cGMP signaling cascade in growth cone behavior of insect neurons, e.g. the locust antennal pioneers (Seidel and Bicker, 2000; Bicker, 2005; 2007). Using NADPH-diaphorase staining and NO-induced cGMP immunoreactivity, Seidel and Bicker (2002) showed that parts of the NO/cGMP signaling cascade are expressed in the embryonic locust brain. In the last decade, considerable methodological progress has been made in visualizing components of this pathway (Martinelli et al., 2002; Ott and Elphick, 2003; Ott et al., 2004), thus allowing to investigate NO producing cells during formation of the locust brain in more detail.
Locusts (Locusta migratoria, Schistocerca gregaria) are useful insect models for neurodevelopmental studies. They posses a highly accessible nervous system composed of relatively large neurons that can individually be identified. Moreover, they are easily cultured allowing to maintain relatively large populations with minimal care (Boyan and Ball, 1993). Embryonic locusts are exceptionally robust, thus allowing being cultured in vitro.
Thanks to detailed literature, the development of the locust embryo can be staged in live animals on the basis of morphological features (Bentley at al., 1979; Ball and Truman, 1998).
Developmental stages are given as percentage of time between egg laying (0%) and hatching of the larva (100%). From 20-30% of embryonic development, the ectoderm of the ventral midline differentiates into a neuroepithelium (Boyan and Ball, 1993). Postembryonic development in the hemimetabolous locust is represented by subsequent moults into 5 larval stages (instars L1-L5). Figure 1 shows the life cycle of the locust as well as photographs of the locust embryo.
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Figure 1: Development of the locust. A: Life cycle of the locust (source: http://www.daff.gov.au/animal-plant- health/locusts/about/about_locusts/lifecycle; 06.08.2012). The female lays about 40 eggs into the soil. When embryogenesis is completed, the first instar (nymph, L1) hatches. It resembles a tiny and wingless version of the adult. With successive moults the wing buds gradually gain size. The 5th instar (L5) undergoes the imaginal moult developing into the full-winged imago. The sexual organs maturate within ca. two weeks thereafter. B:
Locust embryo at 35% of embryogenesis. Dorsal view. Anterior is up. C: Locust embryo at 95% of embryogenesis in its eggshell which has been cleared with sodium hypochloride. Ventral view, anterior is up.
Organization of the locust olfactory pathway
The organization of the locust olfactory pathway is shown in Figure 2. Olfactory information from 50,000 olfactory receptors on the antenna is transferred to the antennal lobe, the analogue to the vertebrate olfactory bulb (Ernst et al., 1977). Here, the olfactory receptor neurons synapse with local interneurons and olfactory projection neurons. The synaptic organization in the locust antennal lobe is unique among the insect species analyzed thus far. Whereas in most insects olfactory receptor neurons of a given odor converge onto one of 50-200 glomeruli, in the locust, most olfactory receptor neurons synapse in multiple microglomeruli (Hansson and Stensmyr, 2011). The olfactory projection neurons relay olfactory information from the antennal lobe to second-order integration centers, the mushroom body and the lateral horn. In the dendritic compartment of the mushroom body,
Introduction
6
the calyx, 830 olfactory projection neurons diverge to 50,000 densely packed mushroom body intrinsic neurons, the Kenyon cells (Laurent and Naraghi, 1994; Anton and Hansson, 1996; Leitch and Laurent, 1996). The antennal lobe generates oscillatory output shaped by GABAergic local interneurons (Stopfer et al., 1997). The output of the excitatory projection neurons is decoded in the mushroom body. Here, each projection neuron contacts on average 600 Kenyon cells while one Kenyon cell receives input from several projection neurons. The Kenyon cells filter the olfactory input by coincidence detection while mushroom body output responses are kept sparse due to normalizing inhibitory feedback loop from the lateral horn (Perez-Orive et al. 2002; Papadopoulou et al., 2011).
Figure 2: Simplified schematic drawing of the locust olfactory pathway. In the antennal lobe (AL), the primary olfactory processing center, olfactory receptor neurons from the antenna (ORN, purple) synapse with interneurons (not shown) and olfactory projection neurons (OPN, green) in multiple microglomeruli. The olfactory projection neurons project toward second-order integration centers, the mushroom body calyx (MBC) and the lateral horn (LH). In the calyx, olfactory projection neurons diverge to ca. 50,000 mushroom body intrinsic neurons, the Kenyon cells (KC, red). Kenyon cell axons bifurcate to form a α-lobe and a β-lobe (αL, βL, red). Lateral horn neurons (LHN, blue) provide an inhibitory feedback loop to the Kenyon cells. αL, α-lobe;
βL, β-lobe; AL, antennal lobe; MBC, mushroom body calyx; KC, Kenyon cell; LH, lateral horn; LHN, lateral horn neurons; OPN, olfactory projection neuron; ORN, olfactory receptor neuron.
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Thesis outline
This thesis was prepared as a cumulative dissertation comprising four original publications two of which I have first-authored. For clarity, the articles are not presented in chronological order of publication.
Publications
8
P
UBLICATIONSAuthors‘ contributions
1) René Eickhoff, Gerd Bicker. 2012. Developmental expression of cell recognition molecules in the mushroom body and antennal lobe of the locust Locusta migratoria. J Comp Neurol 520: 2021-40. DOI: 10.1002/cne.23026.
designed experiments: RE, GB; performed experiments: RE; analyzed data: RE; wrote the article: RE (with input from GB); corrected and improved manuscript: GB
2) Michael Stern, Nicole Böger, René Eickhoff, Christina Lorbeer, Ulrike Kerssen, Maren Ziegler, Giorgio P. Martinelli, Gay R. Holstein, Gerd Bicker. 2010. Development of nitrergic neurons in the nervous system of the locust embryo. J Comp Neurol 518:1157-75. DOI: 10.1002/cne.22303
designed experiments: MS, RE (Citrulline and c-GMP immonoreactivity in the brain), GB; performed experiments: MS, NB, RE (Citrulline and c-GMP immonocytochemistry in the brain), CL, UK, MZ; analyzed data: MS, NB, RE (Citrulline and c-GMP immonoreactivity in the brain); wrote the article: MS; corrected and improved manuscript: all authors
3) Michael Stern, Hannah Scheiblich, René Eickhoff, Nadine Didwischus, Gerd Bicker.
2012. Regeneration of olfactory afferent axons in the locust brain. J Comp Neurol 520: 679-93. DOI: 10.1002/cne.22770.
designed experiments: MS, RE, GB; performed experiments: MS, HS, RE (involved in all experiments; Sema 1a and Lachesin immonocytochemistry), ND; analyzed data:
MS, HS, RE; wrote the article: MS; corrected and improved manuscript: all authors
9 4) René Eickhoff, Raoul-Amadeus Lorbeer, Hannah Scheiblich, Alexander Heisterkamp, Heiko Meyer, Michael Stern, Gerd Bicker. 2012. Scanning laser optical tomography resolves structural plasticity during regeneration in an insect brain. PLoS ONE 7:
e41236. doi:10.1371/journal.pone.0041236
designed experiments: RE, RAL, AH, HM, MS, GB; performed experiments: RE, RAL, HS, MS; analyzed data: RE, MS; wrote the article: RE (with input from RAL, GB);
corrected and improved manuscript: all authors
Publications
10
Publication 1
René Eickhoff, Gerd Bicker. 2012. Developmental expression of cell recognition molecules in the mushroom body and antennal lobe of the locust Locusta migratoria. J Comp Neurol 520: 2021-40. DOI: 10.1002/cne.23026.
http://onlinelibrary.wiley.com/doi/10.1002/cne.23026/pdf
Abstract
We examined the development of olfactory neuropils in the hemimetabolous insect Locusta migratoria with an emphasis on the mushroom bodies, protocerebral integration centers implicated in memory formation. Using a marker of the cyclic adenosine monophosphate (cAMP) signaling cascade and lipophilic dye labeling, we obtained new insights into mushroom body organization by resolving previously unrecognized accessory lobelets arising from Class III Kenyon cells. We utilized antibodies against axonal guidance cues, such as the cell surface glycoproteins Semaphorin 1a (Sema 1a) and Fasciclin I (Fas I), as embryonic markers to compile a comprehensive atlas of mushroom body development. During embryogenesis, all neuropils of the olfactory pathway transiently expressed Sema 1a. The immunoreactivity was particularly strong in developing mushroom bodies. During late embryonic stages, Sema 1a expression in the mushroom bodies became restricted to a subset of Kenyon cells in the core region of the peduncle. Sema 1a was differentially sorted to the Kenyon cell axons and absent in the dendrites. In contrast to Drosophila, locust mushroom bodies and antennal lobes expressed Fas I, but not Fas II. While Fas I immunoreactivity was widely distributed in the midbrain during embryogenesis, labeling persisted into adulthood only in the mushroom bodies and antennal lobes. Kenyon cells proliferated throughout the larval stages. Their neurites retained the embryonic expression pattern of Sema 1a and Fas I, suggesting a role for these molecules in developmental mushroom body plasticity. Our study serves as an initial step toward functional analyses of Sema 1a and Fas I expression during locust mushroom body formation.
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Publication 2
Michael Stern, Nicole Böger, René Eickhoff, Christina Lorbeer, Ulrike Kerssen, Maren Ziegler, Giorgio P. Martinelli, Gay R. Holstein, Gerd Bicker. 2010. Development of nitrergic neurons in the nervous system of the locust embryo. J Comp Neurol 518:1157-75.
DOI: 10.1002/cne.22303
http://onlinelibrary.wiley.com/doi/10.1002/cne.22303/pdf
Abstract
We followed the development of the nitric oxide-cyclic guanosine monophosphate (NO- cGMP) system during locust embryogenesis in whole mount nervous systems and brain sections by using various cytochemical techniques. We visualized NO-sensitive neurons by cGMP immunofluorescence after incubation with an NO donor in the presence of the soluble guanylyl cyclase (sGC) activator YC-1 and the phosphodiesterase-inhibitor isobutyl-methyl- xanthine (IBMX). Central nervous system (CNS) cells respond to NO as early as 38%
embryogenesis. By using the NADPH-diaphorase technique, we identified somata and neurites of possible NO-synthesizing cells in the CNS. The first NADPH-diaphorase-positive cell bodies appear around 40% embryogenesis in the brain and at 47% in the ventral nerve cord. The number of positive cells reaches the full complement of adult cells at 80%. In the brain, some structures, e.g., the mushroom bodies acquire NADPH-diaphorase staining only postembryonically. Immunolocalization of L-citrulline confirmed the presence of NOS in NADPH-diaphorase-stained neurons and, in addition, indicated enzymatic activity in vivo. In whole mount ventral nerve cords, citrulline immunolabeling was present in varying subsets of NADPH-diaphorase-positive cells, but staining was very variable and often weak.
However, in a regeneration paradigm in which one of the two connectives between ganglia had been crushed, strong, reliable staining was observed as early as 60% embryogenesis.
Thus, citrulline immunolabeling appears to reflect specific activity of NOS. However, in younger embryos, NOS may not always be constitutively active or may be so at a very low level, below the citrulline antibody detection threshold. For the CNS, histochemical markers for NOS do not provide conclusive evidence for a developmental role of this enzyme.
Publications
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Publication 3
Michael Stern, Hannah Scheiblich, René Eickhoff, Nadine Didwischus, Gerd Bicker. 2012.
Regeneration of olfactory afferent axons in the locust brain. J Comp Neurol 520: 679-93.
DOI: 10.1002/cne.22770.
http://onlinelibrary.wiley.com/doi/10.1002/cne.22770/pdf
Abstract
The insect olfactory system consists of thousands of sensory neurons on each antenna, which project into the primary olfactory center, the glomerular antennnal lobe. There, they form synapses with local interneurons and projection neurons, which relay olfactory information to the second-order olfactory center, the mushroom body. Olfactory afferents of adult locusts (Locusta migratoria) were axotomized by crushing the base of the antenna.
We studied the resulting degeneration and regeneration in the antennal lobe by size measurements, anterograde dye labeling through the antennal nerve, and immunofluorescence staining of cell surface markers. Within 3 days postcrush, the antennal lobe size was reduced by 30% and from then onward regained size back to normal by 2 weeks postinjury. Concomitantly, anterograde labeling revealed regenerating afferents reaching the antennal lobe by day 4 postcrush, and reinnervating the olfactory neuropil almost back to normal within 2 weeks. Regenerated fibers were directed precisely into the antennal lobe, where they reinnervated glomeruli. As a remarkable exception, a few regenerating fibers projected erroneously into the mushroom body on a pathway that is normally chosen by second-order projection neurons. Regenerating afferents expressed the cell surface proteins lachesin and fasciclin I. The antennal lobe neuropil expressed the cell surface marker semaphorin 1a. In conclusion, axonal regeneration in the locust olfactory system appears to be possible, precise, and fast, opening the possibility of future functional and mechanistic studies.
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Publication 4
René Eickhoff, Raoul-Amadeus Lorbeer, Hannah Scheiblich, Alexander Heisterkamp, Heiko Meyer, Michael Stern, Gerd Bicker. 2012. Scanning laser optical tomography resolves structural plasticity during regeneration in an insect brain. PLoS ONE 7: e41236.
doi:10.1371/journal.pone.0041236
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0041236
Abstract
BACKGROUND:
Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system.
METHODOLOGY:
We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx.
CONCLUSIONS:
This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.
Discussion
14
D
ISCUSSIONThis thesis provides a comprehensive description of antennal lobe and mushroom body development as well as novel anatomical details on mushroom body organization in the locust. Moreover, the locust olfactory pathway was introduced as model system for brain plasticity. To quantify volumetric changes in the olfactory centers, the novel microscopic technique of Scanning Laser Optical Tomography (SLOTy) was used in a functional analysis for the first time. Key-findings of this work are:
• During embryogenesis developing olfactory processing centers express the cell surface molecules Fasciclin I and Semaphorin 1a.
• Both olfactory and gustatory Kenyon cells are generated throughout the larval stages.
• Newborn Kenyon cells in larvae retain the embryonic expression pattern of cell surface molecules.
• Cytochemical markers for the NO/cGMP pathway do not provide evidence for a role of this signaling cascade during embryonic development of the olfactory centers.
• Antennal olfactory afferents regenerate upon axotomy. Regeneration is fast and precise.
• Regenerating olfactory afferents re-express the developmental cell surface molecules Fasciclin I and Lachesin.
• Deafferentation of olfactory input affects second order integration centers and leads to size reduction of the mushroom body calyx.
• Scanning Laser Optical Tomography (SLOTy) is a suitable method for rapid screening of volumetric plasticity deep in the relatively large locust brain.
• Class III (gustatory) Kenyon cells form accessory lobelets separate from the α/β- lobe system.
In the following, these findings are discussed in light of future perspectives and open questions.
15
Class III (gustatory) Kenyon cells
Class III Kenyon cells are sporadically distributed across both holometabolous and hemimetabolous insect species (Farris, 2005). The existence of gustatory Kenyon cells in Orthoptera is well known, but only a limited amount of data is available on the axonal projections and the development of class III Kenyon cells in the locust (Weiss, 1981). In this study, DiI-labeling of the accessory calyx revealed the projection profile of class III Kenyon cells in the adult locust brain. These insights further helped to distinguish between olfactory and gustatory Kenyon cells during mushroom body formation. In insects possessing class III Kenyon cells, these cells are generally thought to be the first Kenyon cells to occur during development and to be generated exclusively during embryogenesis (Farris and Sinakevich, 2003; Farris and Strausfeld, 2003; Farris, 2005). Here, I provide evidence that the development of locust class III Kenyon cells differs from what is known from other species, including the related cricket Acheta domestica (Malaterre et al., 2002; Farris, 2005). In the locust the development of class III Kenyon cells commences only after the first occurrence of olfactory Kenyon cells. Moreover, the generation of locust class III Kenyon cells is apparently not restricted to embryogenesis. By contrast, gustatory Kenyon cells are gradually added to the mushroom body throughout the larval stages similar to olfactory Kenyon cells. Recently, Farris et al. (2011) reported that in the tobacco hornworm Manduca sexta class III Kenyon cells are generated during larval stages by neuroblasts residing outside the mushroom bodies, indicating that class III Kenyon cells are not progeny of mushroom body neuroblasts (MBNB). In light of this study it would be interesting to localize the neuroblasts that give rise to class III Kenyon cells in the locust to examine whether or not gustatory Kenyon cells share the same developmental origin as olfactory Kenyon cells.
Development of the mushroom bodies
By using the cell surface glycoproteins Fas I and Sema 1a as embryonic markers, I resolved the development of the olfactory pathway with an emphasis on the mushroom bodies. Even though much effort has been devoted toward mapping neuroblasts in the locust brain
Discussion
16
(Zacharias et al., 1993; Boyan and Williams, 2000; Urbach and Technau, 2003) virtually no information is available on mushroom body neuroblasts (MBNB). In the locust embryo (Schistocerca gregaria) an estimated number of 130 neuroblasts contribute to the formation of the brain excluding the mushroom bodies (Zacharias et al., 1993; Boyan and Williams, 2000; Urbach and Technau, 2003). Future investigations will now have to unravel the identity, number, and spatiotemporal development of mushroom body precursors in the locust. However, the large number of Kenyon cells (50,000 per brain hemisphere) assumes also a large number of MBNB (Farris et al, 1999; Farris and Sinakevitch, 2003; Urbach and Technau, 2003). An interesting question is by which division-mode MBNB in the locust generate the many Kenyon cells. It has been shown that in insects with relatively low Kenyon cell numbers, e.g. Drosophila (~ 2000 KCs), large solitary MBNB divide asymmetrically in a stem cell mode (Ito and Hotta, 1992; Ito et al., 1997; Urbach and Technau, 2003). By contrast, insects with complex mushroom bodies, e.g. Apis mellifera (~ 170,000 KCs), exhibit clusters of relatively small MBNB that are thought to be generated from initial sets of neuroblasts as a result of symmetric divisions (Malun, 1998; Farris et al., 1999). Recent studies provide evidence for a novel mode of neurogenesis in the Drosophila brain (albeit not in MBNB) involving amplification of neuroblast proliferation via intermediate progenitors that repeatedly divide to produce multiple clones (Bello et al., 2008; Boone and Doe, 2008).
These divisions are morphologically symmetric but molecularly asymmetric, because dividing intermediate progenitors segregate the cell fate determinants Miranda and Prospero to only one of the two siblings which in turn develops into a ganglion mother cell (Bello et al, 2008).
More recently, secondary amplifying, Prospero-negative progenitor cells have also been found in the locust brain where they give rise to the central complex (Boyan et al., 2010). It is not unlikely that the generation of the considerably large number of Kenyon cells in the locust is accomplished by comparable mechanisms of neurogenesis, yet the actual division- mode of locust MBNB remains to be elucidated in future examinations. Though currently there are no cytochemical markers available, that selectively label MBNB, Fas I immunoreactivity (this study) as well as the combinatory expression of the transcription factors Eyeless, Dachshund and Retinal Homeobox might help tracing MBNB (Kunz et al.,
17 2012). Secondary amplifying progenitors can be distinguished via spindle orientation (Boyan et al., 2010).
Possible functions of axon guidance molecules and the NO/cGMP pathway
Locusts and fruit flies show remarkable similarities in the way developmental guidance molecules contribute to the formation of the ventral nerve cord (Thomas et al. 1984;
Goodman et al. 1984; Zinn et al., 1988). The expression pattern of cell surface molecules in the locust olfactory pathway now allows for a direct comparison with the expression of orthologues in the Drosophila brain. Since this study follows a descriptive approach, functional implications of Sema 1a and Fas I expression remain speculative. However, transient expression of Sema 1a as well as striking similarities between embryonic and larval expression patterns of Sema 1a and Fas I in outgrowing Kenyon cells support a developmental significance of these molecules, albeit Fas I might also be important for maintenance of the mature system (see discussion in: Eickhoff and Bicker, 2012).
Compiling evidences implicate Semaphorins as bifunctional regulators of axon guidance mediating both axon attraction and repulsion (reviewed in: Zhou et al., 2008). The transmembrane Sema 1a has been shown to mediate cell adhesion in the peripheral nervous system of the locust (Isbister et al., 1999; Wong et al., 1999) whereas in Drosophila it is characterized as repulsive ligand to its receptor plexin A (plexA; Zhou et al., 2008). Studies of the Drosophila visual system provide evidence that Sema 1a might also serve as a receptor regulating axon guidance via plexA-to-Sema 1a reverse signaling (Cafferty et al., 2006, Yu et al., 2010). Tracking for plexA expression might be an initial step to shed light on the molecular basis of Sema 1a signaling in the locust olfactory pathway. Preliminary experiments using an antiserum against Drosophila plexA (Sweeney et al., 2007; kind gift from Liqun Luo), however, revealed no immunoreactivity in the locust brain (not shown).
To test for the function of Sema 1a and Fas I in the developing locust brain various experimental approaches might help to perturb the function of these molecules, such as antibody blocking experiments in embryo culture (Kolodkin et al., 1992, Isbister et al., 1999).
Additionally, larval brain culture might serve as an appropriate model system to analyze
Discussion
18
postembryonic neurodevelopment. Recent studies have shown that insect brains can be maintained under culture conditions for several days (Ayaz et al., 2008).
RNA interference (RNAi; Fire et al., 1998) is a powerful tool to specifically suppress gene activity in vivo. Due to limited sequence information, genetic manipulation has for a long time hardly been available for hemimetabolous species. Only recently RNAi-mediated loss of function experiments have successfully been used to manipulate development in the locust (Dong and Friedrich, 2005) and the cricket Gryllus bimaculatus (Nakamura et al., 2008). This technique appears to be a promising tool to analyse the function of Sema 1a and Fas I during development of the locust olfactory pathway.
Semaphorin-mediated axon guidance is strongly linked to cGMP and cAMP signaling cascades (Zhou et al., 2008). To better understand the mechanisms of Sema 1a signaling in the locust brain it might thus be helpful to pharmacologically alter the cyclic nucleotide levels or to manipulate the activity of the corresponding target enzymes (protein kinases) in vivo.
The NO/cGMP pathway has been shown to regulate several mechanisms of neural development, such as neurite outgrowth (Seidel and Bicker, 2000), neural migration (Haase and Bicker, 2003) and axonal regeneration (Stern and Bicker, 2008). In this study cytochemical markers for nitric oxide synthase (NOS) did not provide conclusive evidence for a role of this signaling cascade in axon extension or growth cone navigation because NOS expression and activity occurred only at the end of embryogenesis (discussed in: Stern et al., 2010). This is reflected in postembryonic mushroom body development. As shown in figure 3, outgrowing, newborn Kenyon cells do not produce cGMP in response to bath application of a NO-donor. Instead Kenyon cells surrounding the inner core show NO-dependent cGMP immunoreactivity (unpublished). Thus, both embryonic and larval mushroom body development apparently seem to be mediated by similar mechanisms (figure 7 in Stern et al., 2010; figure 3).
19
Figure 3: Newborn Kenyon cells in L1 larvae do not respond to Nitric oxide (NO) by producing cGMP. Outgrowing neurites of newborn Kenyon cells (revealed by prominent Phalloidin staining; arrow) in the core region of the peduncle (Ped) show no cGMP immunoreactivity after application of an NO-donor.
Instead, Kenyon cells surrounding the inner core show NO- dependent cGMP immunoreactivity (arrowheads). Ca, calyx;
Ped, peduncle.
Structural plasticity of the olfactory centers
We have shown that locust olfactory afferents can regenerate after injury. Regeneration is fast and, with respect to recovery of antennal lobe volume, very effective. It could be shown that, following neurite outgrowth, antennal lobe glomeruli are re-established. Whether regenerating neurites form synapses with the correct targets and indeed provide restoration of function has to be elucidated in future analyses (discussed in: Stern et al., 2012).
However, the locust olfactory pathway constitutes a promising test system for drugs that might enhance or inhibit neuronal regeneration. Because current microscopic methods are limited in focal depth, we utilized Scanning Laser Optical Tomography to quantify structural plasticity in the relatively large locust brain. This technique allowed rapid screening of structural changes in whole mount preparations making it suitable for high throughput applications.
Some of the antibodies used as developmental markers, were also employed in the regeneration study. Interestingly, regenerating olfactory afferents express the developmental cell surface molecules Lachesin and Fas I, suggesting that regeneration recapitulates development.
A striking finding of this study is that degeneration of the antennal lobe is transduced to second-order neuropils leading to shrinkage of the mushroom body calyx. This is to our notion the first study that demonstrates trans-synaptic effects of degeneration in the insect
Discussion
20
brain. So far the cellular basis underlying the size reduction of the locust calyx is unknown.
However, the synaptic organisation within the mushroom body calyx is well studied. In the calyx olfactory receptor neurons terminate in (pre-) synaptic boutons. These boutons are surrounded by multiple Kenyon cell dendritic spines, forming minute microglomeruli (figure 4). Comparative analyses indicate that calyx microglomeruli represent a common feature of neopteran insects (Groh and Rössler, 2011). These structures can be visualized by a combinatory application of Synapsin immunocytochemistry and Phalloidin staining of f-actin (Frambach et al., 2004; figure 4). It has been shown that alterations in microglomeruli number and/or size account for calyx volume plasticity in the honey bee and the fruit fly (Groh et al., 2004; 2006; 2012; Kremer et al., 2010). Using high resolution confocal microscopy, Synapsin immunostaining in combination with Phalloidin staining allows distinguishing microglomeruli also in the locust calyx (figure 4). Thus, we can now elucidate whether volume changes in the locust calyx are caused indeed by alterations of microglomeruli number or size.
Figure 4: Microglomeruli in the mushroom body calyx. A: Confocal image showing the microglomerular organization of the mushroom body calyx. Presynaptic projection neuron boutons are labeled with an antibody against Synapsin (Synorph 1, red). Boutons are enwrapped by Kenyon cell dendritic spines which are rich in f- actin (Phalloidin, green). B: Close up of a single mushroom body microglomerulus constituting a synapsin- positive bouton (red) encircled by f-actin-rich dendritic spines (green). C: Schematic drawing of a mushroom body microglomerulus, as shown in B (modified after Groh et al., 2006).
21
Conclusions/Outlook
This thesis describes the anatomy and development of the locust olfactory pathway. It provides the groundwork for comparative analyses with different insect species and thus contributes to better understand the basic principles of insect brain development. The expression patterns of cell surface guidance molecules, on which parts of the developmental studies are based on, provide the basis for future functional analysis, e.g. on Semaphorin signaling during mushroom body formation. Moreover, the locust olfactory pathway was introduced as model system for regeneration. Its accessibility and robustness predestines this preparation as a test system for target drugs that might potentially enhance neuronal regeneration capacities. The novel microscopic technique Scanning Laser Optical Tomography allows for rapid screening of structural plasticity in the relatively large locust brain, suggesting its application also to vertebrate preparations. Finally, this study contributes to understanding the biology of a pest insect that via its great population densities is responsible for severe economical damage.
References
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