«Dissertation zur Erlangung des Naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Thomas ...»
Aspects of neuronal plasticity in the mushroom
body calyx during adult maturation in the
honeybee Apis mellifera
Dissertation zur Erlangung des
der Bayerischen Julius-Maximilians-Universität Würzburg
Thomas Sebastian Münz
aus Rieden am Forggensee
Eingereicht am: …………………………………………………………………………………
Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. Markus Engstler (Universität Würzburg) Gutachter: Prof. Dr. Wolfgang Rössler (Universität Würzburg) Gutachter: Prof. Dr. Alison Mercer (University of Otago, New Zealand) Tag des Promotionskolloquiums: ……………………………………………………..
Doktorurkunde ausgehändigt am: …………………………………………………….
Affidavit (Eidesstattliche Erklärung) gemäß §4 Abs. 3 Ziff. 3, 5 and 8 der „Promotionsordnung der Julius-Maximilians-Universität Würzburg“ Hiermit erkläre ich an Eides statt, das die Dissertation “Aspects of neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee Apis mellifera” selbständig, unter Verwendung der angegebenen Quellen und Hilfsmittel angefertigt wurde.
Weiterhin habe ich noch keinen Promotionsversuch unternommen, oder diese Dissertation in gleicher oder ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt.
Würzburg, 25.01.2015 __________________________
This thesis is based on the following manuscripts:
• Muenz TS, Groh C, Maisonnasse A, Le Conte Y, Plettner E, Rössler W. 2015. Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences. under revision
• Pasch E*, Muenz TS*, Rössler W. 2011. CaMKII is differentially localized in synaptic regions of Kenyon cells within the mushroom bodies of the honeybee brain. Journal of Comparative Neurology 519:3700–3712.
*equally contributing first authors
• Muenz TS, Maisonnasse A, Le Conte Y, Plettner E, Rössler W. 2012. Sensory reception of the primer pheromone ethyl oleate. Naturwissenschaften 99:421-425 “Dissertation Based on Several Published Manuscripts“ Statement on individual author contributions and on legal second publication rights Publication (complete reference): Muenz TS, Groh C, Maisonnasse A, Le Conte Y, Plettner E, Rössler W. 2015. Neuronal plasticity in the mushroom-body calyx during adult maturation in the honeybee and possible pheromonal influences. under revision Pa
Publication (complete reference): Pasch E*, Muenz TS*, Rössler W. 2011. CaMKII is differentially localized in synaptic regions of Kenyon cells within the mushroom bodies of the honeybee brain. Journal of Comparative Neurology 519:3700–3712. *equally contributing first authors
Publication (complete reference): Muenz TS, Maisonnasse A, Le Conte Y, Plettner E, Rössler W. 2012. Sensory reception of the primer pheromone ethyl oleate. Naturwissenschaften 99:421-425
I confirm that I have obtained permission from both the publishers and the co-authors for legal second publication.
I also confirm my primary supervisor’s acceptance.
Table of contents Summary
Division of labor in honeybee colonies
Regulation of division of labor
The mushroom bodies as centers of multimodal integration
Structural plasticity of the mushroom bodies
Chapter I: Neuronal plasticity in the mushroom-body calyx during adult maturation in the honeybee and possible pheromonal influences
Material and Methods
Bees and cohort experiments
Pheromone treatment and behavioral observations
Laser scanning confocal microscopy, image processing, and data analysis
Statistical analysis and graphical editing
Structural plasticity in the mushroom-body calyx during adult maturation
Cellular processes underlying neuronal plasticity
Potential pheromonal modulation of synaptic maturation
Chapter II: CaMKII is differentially localized in synaptic regions of Kenyon cells within the mushroom bodies of the honeybee brain
Material and Methods
Animals and cohort experiments
Primary antibody characterization
Confocal laser scanning microscopy and image processing
General distribution of pCaMKII in the brain
Distribution of pCaMKII within microglomeruli of the mushroom body calyx
Distribution of pCaMKII at different stages during adult maturation
Characterization of the honeybee CaMKII protein
General distribution of pCaMKII in the honeybee brain
Cellular and subcellular localization of pCaMKII
Chapter III: Sensory reception of the primer pheromone ethyl oleate
Material and methods
EO reception via the olfactory pathway
Olfactory perception and learnability of EO
Detailed Material and Methods
Neuronal plasticity in the context of division of labor under natural conditions
Neuronal plasticity in the context of division of labor under manipulated conditions
Mechanisms underlying structural neuronal plasticity in the mushroom bodies
Final conclusions and outlook
Summary Division of labor represents a major advantage of social insect communities that accounts for their enormous ecological success. In colonies of the honeybee, Apis mellifera, division of labor comprises different tasks of fertile queens and drones (males) and, in general, sterile female workers. Division of labor also occurs among workers in form of an age-related polyethism. This helps them to deal with the great variety of tasks within the colony. After adult eclosion, workers spend around three weeks with various duties inside the hive such as tending the brood or cleaning and building cells. After this period workers switch to outdoor tasks and become foragers collecting nectar, pollen and water. With this behavioral transition, workers face tremendous changes in their sensory environment. In particular, visual sensory stimuli become important, but also the olfactory world changes. Foragers have to perform a completely new behavioral repertoire ranging from long distance navigation based on landmark orientation and polarized-skylight information to learning and memory tasks associated with finding profitable food sources. However, behavioral maturation is not a purely age-related internal program associated with a change, for example, in juvenile hormone titers. External factors such as primer pheromones like the brood pheromone or queen mandibular pheromone can modulate the timing of this transition. In this way colonies are able to flexibly adjust their work force distribution between indoor and outdoor tasks depending on the actual needs of the colony. Besides certain physiological changes, mainly affecting glandular tissue, the transition from indoor to outdoor tasks requires significant adaptations in sensory and higher-order integration centers of the brain.
The mushroom bodies integrate olfactory, visual, gustatory and mechanosensory information. Furthermore, they play important roles in learning and memory processes. It is therefore not surprising that the mushroom bodies, in particular their main input region, the calyx, undergo volumetric neuronal plasticity. Similar to behavioral maturation, plastic changes of the mushroom bodies are associated with age, but are also to be affected by modulating factors such as task and experience.
In my thesis, I analyzed in detail the neuronal processes underlying volumetric plasticity in the mushroom body. Immunohistochemical labeling of synaptic proteins combined with
quantitative 3D confocal imaging revealed that the volume increase of the mushroom body calyx is largely caused by the growth of the Kenyon cell dendritic network. This outgrowth is accompanied by changes in the synaptic architecture of the mushroom body calyx, which is organized in a distinct pattern of synaptic complexes, so called microglomeruli. During the first week of natural adult maturation microglomeruli remain constant in total number. With subsequent behavioral transition from indoor duties to foraging, microglomeruli are pruned while the Kenyon cell dendritic network is still growing. As a result of these processes, the mushroom body calyx neuropil volume enlarges while the total number of microgloumeruli becomes reduced in foragers compared to indoor workers. In the visual subcompartments (calyx collar) this process is induced by visual sensory stimuli as the beginning of pruning correlates with the time window when workers start their first orientation flights. The high level of analysis of cellular and subcellular process underlying structural plasticity of the mushroom body calyx during natural maturation will serve as a framework for future investigations of behavioral plasticity in the honeybee.
The transition to foraging is not purely age-dependent, but gets modulated, for example, by the presence of foragers. Ethyl oleate, a primer pheromone that is present only in foragers, was shown to delay the onset of foraging in nurse bees. Using artificial application of additional ethyl oleate in triple cohort colonies, I tested whether it directly affects adult neuronal plasticity in the visual input region of the mushroom body calyx. As the pheromonal treatment failed to induce a clear behavioral phenotype (delayed onset of foraging) it was not possible to show a direct link between the exposure to additional ethyl oleate and neuronal plasticity in mushroom body calyx. However, the general results on synaptic maturation confirmed my data of natural maturation processes in the mushroom body calyx.
Given the result that dendritic plasticity is a major contributor to neuronal plasticity in the mushroom body calyx associated with division of labor, the question arose which proteins could be involved in mediating these effects. Calcium/calmodulin-dependent protein kinase II (CaMKII) especially in mammals, but also in insects (Drosophila, Cockroach), was shown to be involved in facilitating learning and memory processes like long-term synaptic potentiation. In addition to presynaptic effects, the protein was also revealed to directly interact with cytoskeleton elements in the postsynapse. It therefore is a likely candidate to mediate structural synaptic plasticity. As part of my thesis, the presence and distribution of
CaMKII was analyzed, and the results showed that the protein is highly concentrated in a distinct subpopulation of the mushroom body intrinsic neurons, the noncompact Kenyon cells. The dendritic network of this population arborizes in two calyx subregions: one receiving mainly olfactory input – the lip – and the collar receiving visual input. This distribution pattern did not change with age or task. The high concentration of CaMKII in dendritic spines and its overlap with f-actin indicates that CaMKII could be a key player inducing structural neuronal plasticity associated with learning and memory formation and/or behavioral transitions related to division of labor. Interestingly CaMKII immunoreactivity was absent in the basal ring, another subregion of the mushroom body calyx formed almost exclusively by the inner compact Kenyon cells and known to receive combined visual and olfactory input. This indicates differences of this mushroom body subregion regarding the molecular mechanisms controlling plastic changes in corresponding Kenyon cells.
How is timing of behavioral and neuronal plasticity regulated? The primer pheromone ethyl oleate was found in high concentrations on foragers and was shown to influence behavioral maturation by delaying the onset of foraging when artificially applied in elevated concentrations. But how is ethyl oleate transferred and how does it shift the work force distribution between indoor and outdoor tasks? Previous work showed that ethyl oleate concentrations are highest in the honeycrop of foragers and suggested that it is transferred and communicated inside the colony via trophallaxis. The results of this thesis however clearly show, that ethyl oleate was not present inside the honey crop or the regurgitate, but rather in the surrounding tissue of the honey crop. As additionally the second highest concentration of ethyl oleate was measured on the surface of the cuticle of forgers, trophallaxis was ruled out as a mode of transmission. Neurophysiological measurements at the level of the antennae (electroantennogram recordings) and the first olfactory neuropil (calcium imaging of activity in the antennal lobe) revealed that the primer pheromone ethyl oleate is received and processed as an olfactory stimulus. Appetitive olfactory conditioning using the proboscis extension response as a behavioral paradigm showed that ethyl oleate can be associated with a sugar reward. This indicates that workers are able to perceive, learn and memorize the presence of this pheromone. As ethyl oleate had to be presented by a heated stimulation device at close range, it can be concluded that this primer pheromone
acts via close range/contact chemoreception through the olfactory system. This is also supported by previous behavioral observations.