Development of variable and robust brain wiring patterns in the fly visual system

Precise generation of synapse-specific neuronal connections are crucial for establishing a robust and functional brain. Neuronal wiring patterns emerge from proper spatiotemporal regulation of axon branching and synapse formation during development. Several neuropsychiatric and neurodevelopmental disorders exhibit defects in neuronal wiring owing to synapse loss and/or dys-regulated axon branching. Despite decades of research, how the two inter-dependent cellular processes: axon branching and synaptogenesis are coupled locally in the presynaptic arborizations is still unclear. In my doctoral work, I investigated the possible role of EGF receptor (EGFR) activity in coregulating axon branching and synapse formation in a spatiotemporally restricted fashion, locally in the medulla innervating Dorsal Cluster Neuron (M- DCN)/LC14 axon terminals. In this work I have explored how genetically encoded EGFR randomly recycles in the axon branch terminals, thus creating an asymmetric, non-deterministic distribution pattern. Asymmetric EGFR activity in the branches acts as a permissive signal for axon branch pruning. I observed that the M-DCN branches which stochastically becomes EGFR ‘+’ during development are synaptogenic, which means they can recruit synaptic machineries like Syd1 and Bruchpilot (Brp). My work showed that EGFR activity has a dual role in establishing proper M-DCN wiring; first in regulating primary branch consolidation possibly via actin regulation prior to synaptogenesis. Later in maintaining/protecting the levels of late Active Zone (AZ) protein Brp in the presynaptic branches by suppressing basal autophagy level during synaptogenesis. When M-DCNs lack optimal EGFR activity, the basal autophagy level increases resulting in loss of Brp marked synapses which is causal to increased exploratory branches and post-synaptic target loss. Lack of EGFR activity affects the M-DCN wiring pattern that makes adult flies more active and behave like obsessive compulsive in object fixation assay. In the second part of my doctoral work, I have asked how non-genetic factors like developmental temperature affects adult brain wiring. To test that, I increased or decreased rearing temperature which is known to inversely affect pupal developmental rate. We asked if all the noisy cellular processes of neuronal assembly: filopodial dynamics, axon branching, synapse formation and postsynaptic connections scale up or down accordingly. I observed that indeed all the cellular processes slow down at lower developmental temperature and vice versa, which changes the DCN wiring pattern accordingly. Interestingly, behavior of flies adapts to their developmental temperature, performing best at the temperature they have been raised at. This shows that optimal brain function is an adaptation of robust brain wiring patterns which are specified by noisy developmental processes. In conclusion, my doctoral work helps us better understand the developmental regulation of axon branching and synapse formation for establishing precise brain wiring pattern. We need all the cell intrinsic developmental processes to be highly regulated in space and time. It is infact a combinatorial effect of such stochastic processes and external factors that contribute to the final outcome, a functional and robust adult brain

Quantifying Glial-Glial Interactions In Drosophila Using Automated Image Analysis

Imaging is an immensely powerful tool in biomedical research. Technological advances in the last half century have led to the development of new tools for image analysis, with major strides being made in the last 20 years especially with machine and deep learning. However, researchers still often hit a bottleneck during the image analysis phase of their projects that often leads to delays and sometimes even limits the scope of their studies. In this thesis I demonstrate some of the issues that arise while quantifying images to answer a biological question by using a dataset of fly central nervous system images to elucidate interactions between different cells. I present an overview of the types of methods that can be used to perform this analysis including a discussion of their advantages and disadvantages. Finally, I present steps for creating and validating an automated image analysis pipeline that was used to analyze a large section of the fly ventral nerve cord, akin to the spinal cord. Automating image quantifying allowed us to maximize the size of the dataset analyzed, which revealed subtle patterns in cell-cell interactions that would not have been uncovered with manual quantification of a smaller dataset

Role of neuronal cell adhesion molecules in regulating filopodial dynamics and synapse formation in the drosophila visual system

Brains in all biological systems consist of large numbers of interconnected neurons. The developmental processes to wire a functional brain are controlled by many protein families that govern neuronal targeting, synaptic partner specification, and synapse formation. Using the fly visual system and the state-of-the-art live imaging of developing photoreceptors in intact Drosophila brains in ex vivo cultures, I studied the role of cell adhesion molecules (CAMs) in regulating R7 targeting and filopodial dynamics and their effect on synapse formation. The analyses presented here include the protein tyrosine phosphatases (PTPs), Lar and PTP69D, and the fly homologue of the amyloid precursor protein (APP), APP-like (APPL). I also report the novel function of Neurexin-1 (Nrx) in R7 targeting and synapse formation. Lar interaction with Liprin- during synapse formation is essential to stabilize R7 terminals in their target layer. Their loss of function causes filopodial dynamics changes and affects bulbous tip stability and synapse formation. Lar and Liprin- regulate bulbous tip stability by stabilizing microtubules in the formed bulbous tips to provide mechanical support for pre-postsynaptic contacts to facilitate synapse formation. To test this hypothesis, I reduced actin depolymerization by knocking-down the cofilin phosphatase, Chronophin (dCIN), which increased filopodia stability and caused an increase in synapse count in R7s. Loss of ptp69d caused very mild R7 retractions and a filopodia formation defect with no effect on bulbous tip dynamics and synapse formation, showing the distinct functions of PTP69D that are not redundant to Lar in R7s as was proposed previously. nrx mutant R7s did not show a targeting defect but rather an elevated synaptic transmission. Mutant R7s fail to stabilize bulbous tips, yet they formed synapses with more and aberrant postsynaptic partners, suggesting that Nrx is a negative regulator of synapse formation in R7s. Finally, the reported subtle retraction phenotype in appl mutant R7s was found to be independent of APPL. Additionally, studying the APPL proteolytic cleavage products showed that the extracellular and the intracellular fragments were differentially trafficked in different stages of developing neurons. The intracellular fragment localized to the axon terminals of R7s, while the extracellular fragment was secreted from photoreceptors and picked up by cortical glia where it eventually affects endolysosomal trafficking in a dose-dependent manner. This study challenges the proposed function of the tested CAMs as guidance molecules. Live imaging revealed that none of the tested proteins instructed R7 targeting to their correct layer, but rather stabilize their terminals in the correct layer. Changes of filopodial dynamics associated with their loss of function and the corresponding synaptic changes imply that synapse formation relies on stabilizing pre-postsynaptic contacts and not on a molecular match-making mechanism.Gehirne von höheren Tieren bestehen aus einem komplexen Netzwerk aus vielen verschalteten Neuronen. Die Entwicklungsprozesse, die an der Verschaltung eines funktionalen Gehirns beteiligt sind, werden durch eine Vielzahl von Proteinfamilien kontrolliert. Diese steuern neuronales Pathfinding und Targeting, die Spezifizierung synaptischer Partner, und Synapsenbildung. Ich untersuchte die Rolle von Zelladhäsionmolekülen (CAMs) im regulierenden R7-Photorezeptorterminal und die Dynamik von Filopodien und deren Effekt auf die Synapsenbildung. Dazu verwendete ich das visuelle System der Fruchtfliege, sowie Live Imaging von sich entwickelnden Photorezeptoren in intakten Drosophila-Gehirnen in ex vivo Kulturen. Ich untersuchte die zwei Tyrosinphosphatasen (PTPs), Lar und PTP69D, und das Fliegenhomolog des Amyloid-Precursor-Protein (APP) APP-like (APPL). Außerdem untersuchte ich die Funktion von Neurexin-1 (Nrx), wessen Rolle in Photorezeptor-Targeting und -Synapsenbildung bisher nicht bekannt war. Lar und das damit interagierende Protein, Liprin-α, stabilisierten R7-Terminale in ihrer Zielschicht. Ihr Funktionsverlust in R7 führte zu Veränderungen in der Filopodiendynamik und beeinflusste die Stabilität der Bulbous Tips und Synapsenformation. Lar und Liprin-α steigerten die Stabilität der Bulbous Tips, indem sie die Mikrotubuli stabilisierten, und somit eine mechanische Stütze für prä-postsynaptische Kontakte bot, wodurch die Synapsenbildung erleichtert wurde. Um diese Hypothese zu testen reduzierte ich due Aktin Polymerisation durch Knock-Down von Chronophin (dCIN) was die Filopodienstabilität erhöhte und dadurch zu mehr Synapsen führte. Der Verlust von ptp69d führte zu einer sehr milden R7-Rücknahme und einem Filopodiendefekt ohne Effekt auf die Bulbous Tip-Dynamik und Synapsenbildung. Das bestätigt die frühere Vermutung, dass die ausgeprägten Funktionen von PTP69D in R7 nicht zu Lar redundant sind. In nrx-mutierten Photorezeptoren wurde kein R7 targeting Defekt bemerkt, dafür aber erhöhte synaptische Transmission. nrx-mutierte R7 bildeten keine stabilen Bulbous Tips aus, bildeten jedoch Synapsen mit mehr und abnormen postsynaptischen Partnern. Dies suggeriert, dass Nrx als ein negativer Regulator für Synapsenbildung in Photorezeptoren wirkt. Schließlich konnte gezeigt werden, dass der subtile Retraktion-Phänotyp in appl-mutierten R7 unabhängig von APPL-Funktion in Photorezeptoren ist. Außerdem zeigten Experimente an den intra- und extrazellulären Fragmenten der proteolytischen Spaltungsprodukte von APPL, dass sie in unterschiedlichen Stadien der sich entwickelnden Neuronen verschieden transportiert werden. Das intrazelluläre Fragment lokalisierte sich in den Axonterminalien von R7, während das extrazelluläre Fragment aus den Photorezeptoren ausgeschieden und von Gliazellen in Regionen der Hirnrinde aufgenommen wurde, wo es schließlich den endolysosomalen Transport spezifisch beeinflusste. Diese Studie stellt die vorgeschlagene Funktion der getesteten CAMs als ´guidance` Moleküle in Frage. Live Imaging zeigte, dass keine der getesteten Proteine das Targeting zur richtigen Schicht von R7 kontrolliert, sondern stattdessen für eine Stabilisierung der Terminale in der richtigen Schicht sorgt. Veränderungen der Filopodiendynamik, die mit deren Funktionsverlust und daraus resultierenden Veränderungen in den Synapsen in Zusammenhang gebracht werden, implizieren, dass die Synapsenbildung auf eine Stabilisierung der prä-postsynaptischen Kontakte angewiesen ist, statt auf einen molekularen Match-Making-Mechanismus

Augmenting Wiring Diagrams of Neural Circuits with Activity in Larval Drosophila

Neural circuit models explain an animal's behavior as evoked activity of different circuit elements of its nervous system. Synaptic wiring diagrams mapped from structural imaging of nervous systems guide modeling of neural circuits on the basis of connectivity. However, connectivity alone may not sufficiently constrain the set of plausible circuit hypotheses for empirical study. Combining structural imaging of synaptic connectivity with functional information from activity imaging can further constrain these hypotheses to create unequivocal neural circuit models. This thesis develops computational methods and tools to cross-reference structural and activity imaging of explant larval Drosophila central nervous systems at cellular resolution. Augmenting synaptic wiring diagrams with activity maps via these methods relates circuit structure and function at the neuronal level on a per-behavior basis. Neuronal activity of larval central nervous systems expressing pan-neuronal calcium indicators is imaged in a light sheet microscope, which are then structurally imaged with high throughput electron microscopy. Methods and tools are provided for the assembly of these image volumes, spatial registration between imaging modalities, automated detection of relevant tissue and cellular structures in each, extraction of activity time series, and morphological identification of neurons in structural imaging using reference wiring diagrams mapped from other larvae. Using these methods, existing wiring diagrams mapped from a reference first instar larva were identified with neurons in a larva augmented with activity information for a neural circuit involved in peristaltic motor control. This demonstrates the feasibility of the contributed methods to associate the wiring diagrams of arbitrary circuits of interest with activity timeseries across multiple individuals, behaviors, and behavioral bouts. To demonstrate capability to augment wiring diagrams with information besides activity, these methods are also applied to multiple larvae each expressing specific neurotransmitter labels rather than calcium indicators in the light sheet microscopy. This work scaffolds future modeling of circuits underlying behavior that can only be mechanistically understood in the context of multi-modal observation of synaptic connectivity, functional activity and molecular markers. The methods developed also enable comparative connectomics between multiple individuals, which is necessary to study inter-individual variability in circuits and to observe experimental intervention in the development, structure, and function of neural circuits.Howard Hughes Medical Institute Janelia Research Campu

Dendritic spines and structural plasticity in Drosophila

The morphology of dendrites is important for neuronal function and for the proper connectivity within neuronal circuits. The often very complex shape of dendritic trees is brought about by the action of many different genes throughout development. Moreover, neuronal activity is often involved in refining synaptic connections and shaping dendrites. Aiming at a better understanding of the interplay between genes and neuronal activity during dendrite differentiation I was trying to identify suitable neurons in the Drosophila central nervous system. Describing the morphology and cytoskeletal organization of a group of visual interneurons involved in motion processing I provided evidence that the dendrites of these neurons do bear small protrusions that share essential characteristics with vertebrate spines. Vertebrate spines received a lot of recent attention because neuronal activity can induce lasting changes in their morphology even in the adult. These morphological changes are believed to be cellular correlates of learning and memory. The observation of similar structures in flies raised the possibility to study structural plasticity in a genetically accessible model organism. Experience-dependent alterations in the volume of a region in the insect brain, called mushroom body calyx, have been shown. The calyx is known to contain the dendrites of olfactory interneurons, Kenyon cells, which are known to be required for the retrieval of olfactory memories in flies. I wanted to address if morphological rearrangements of the dendrites of these cells could underlie the experience-dependent changes in calycal volume. Kenyon cell dendrites and their presynaptic partners are known to form synaptic complexes, called microglomeruli, throughout the calyx. My results help refining the anatomical description of these structures. These findings are important to understand how olfactory experience is represented in the fly brain and how olfactory memories might be formed. Moreover, I developed a computer algorithm to quantitatively describe the morphology of these microglomeruli in an automated manner. Thereby, I found indications for morphological rearrangements of calycal microglomeruli during the first days of the adult life of Drosophila. I could show that olfactory experience is not required for these morphological alterations. My findings provide the basis for ongoing attempts to study the influence of neuronal activity on the dendritic morphology of Kenyon cells in more detail

Illuminating cAMP dynamics at the synapse with multiphoton FLIM-FRET Imaging

The study of signalling pathways within mammalian physiology has long been hindered by the size of the players involved, being far beyond the realms of the conventional light microscope. The advent of advanced fluorescent imaging techniques has revolutionised our capabilities to probe biological processes. The work in this thesis particularly utilised Förster resonance energy transfer (FRET), a fluorescence-based technique that can provide functional readouts of the processes underlying cellular function. Specifically I worked to develop and optimise a fluorescence imaging system for investigating the dynamics and function of cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger. The neuroscientific study of how the brain can learn and recall memories is a rapidly advancing field. The current challenges of tackling dementias, such as Alzheimer’s disease, and preventing memory loss can only be addressed through better understanding of how memories can be stored. It is now believed that neurons retain memories within their synapses, the femtolitre structures that determine the strength of these connections. cAMP has been shown to play a distinctive role in orchestrating the retention of long term memory at the synaptic level. However, its spatial and temporal activation profiles are still not fully understood. To address this, my PhD project combined FRET readouts with cutting edge imaging techniques applied to synapses in neuronal cultures that provide reasonably convenient optical access. By examining the structure of these synapses, along with the measurement of cAMP concentration in different neuronal regions, this project uncovered the highly compartmentalised nature of this signalling molecule, seen to act directly at the sites of strengthening synapses. Through the optimisation of a FRET imaging system for studying activity in neuronal tissues, this project establishes a method for the future investigation of a plethora of pathways underlying the healthy functioning of the mammalian brain.Open Acces

Dopaminergic Modulation Shapes Sensorimotor Processing in the Drosophila Mushroom Body

To survive in a complex and dynamic environment, animals must adapt their behavior based on their current needs and prior experiences. This flexibility is often mediated by neuromodulation within neural circuits that link sensory representations to alternative behavioral responses depending on contextual cues and learned associations. In Drosophila, the mushroom body is a prominent neural structure essential for olfactory learning. Dopaminergic neurons convey salient information about reward and punishment to the mushroom body in order to adjust synaptic connectivity between Kenyon cells, the neurons representing olfactory stimuli, and the mushroom body output neurons that ultimately influence behavior. However, we still lack a mechanistic understanding of how the dopaminergic neurons represent the moment-tomoment experience of a fly and drive changes in this sensory-to-motor transformation. Furthermore, very little is known about how the output neuron pathways lead to the execution of appropriate odor-related behaviors. We took advantage of the mushroom body’s modular circuit organization to investigate how the dopaminergic neuron population encodes different contextual cues. In vivo functional imaging of the dopaminergic neurons reveals that they represent both external reinforcement stimuli, like sugar rewards or punitive electric shock, as well as the fly’s motor state, through coordinated and partially antagonistic activity patterns across the population. This multiplexing of motor and reward signals by the dopaminergic neurons parallels the dual roles of dopaminergic inputs to the vertebrate basal ganglia, thus demonstrating a conserved link between these distantly related neural circuits. We proceed to demonstrate that this dopaminergic signal in the mushroom body modifies neurotransmission with synaptic specificity and temporal precision to coordinately regulate the propagation of sensory signals through the output neurons. To explore how these output pathways ultimately influence olfactory navigation we have developed a closed loop olfactory paradigm in which we can monitor and manipulate the mushroom body output neurons as a fly navigates in a virtual olfactory environment. We have begun to probe the mushroom body circuitry in the context of olfactory navigation. These preliminary investigations have led to the identification of putative pathways for linking mushroom body output with the circuits that implement odor-tracking behavior and the characterization of the complex sensorimotor representations in the dopaminergic network. Our work reveals that the Drosophila dopaminergic system modulates mushroom body output at both acute and enduring timescales to guide immediate behaviors and learned responses