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Neurotransmitters and receptors in the motion vision pathway of Drosophila
Neurotransmitters and receptors in the motion vision pathway of Drosophila
The fruit fly Drosophila melanogaster is one of the most popular model organisms being used in the life sciences. Due to its small number of neurons compared to other vertebrate species and its genetic access, it has also proven itself to be an optimal model organism for neuroscience. Especially, the field of systems neuroscience has made quick advances in its study of neural circuits of the fly brain underlying sensory perception such as olfaction and vision, as well as complex behaviors such as mating, learning, and memory. The motion vision pathway in the optic lobe of the fruit fly is a prominent example of a computation-performing neural circuit that researchers have been trying to understand for decades. While the wiring of the main circuit elements has been described via EM-reconstructions and their response properties have been characterized comprehensively, the molecular mechanisms of direction-selectivity still remain elusive. However, subcellular components such as neurotransmitter receptors and ion channels are important since they define the sign and the temporal dynamics of synaptic connections within a circuit. Hence, the main focus of my thesis was the investigation of neurotransmitter receptors in the primary motion sensing T4/T5 neurons of the fly brain, including the development of required genetic tools. First, we developed a protocol for super-resolution STED imaging in Drosophila brain slices which allowed us to resolve fine dendritic structures of individual T4/T5 neurons deep inside the brain (Manuscript 1). Second, we used the glutamate sensor iGluSnFR to characterize the temporal dynamics of the three glutamatergic cell types of the motion vision pathway L1, Mi9 and LPi (Manuscript 2). We validated the usability of iGluSnFR for measuring glutamate signaling in adult Drosophila brains and found that responses recorded with iGluSnFR are faster than GCaMP signals of the same cells. In Manuscript 3, we developed new genetic strategies for conditional protein labeling. Specifically, we introduced FlpTag, a tool for endogenous, conditional labeling of proteins by means of a flippase-dependent, invertible GFP cassette integrated in the endogenous gene locus. Using these methods, we explored the subcellular localizations of neurotransmitter receptors for glutamate, GABA, acetylcholine and voltage-gated ion channels in T4/T5 neurons in Drosophila melanogaster. Within the dendrite, receptor subunits localize to different regions and in a spatial order that exactly matches the EM-reconstructed synapse numbers and distributions of the different input neurons described in previous studies. Further, we discovered a strictly segregated subcellular distribution of two voltage-gated ion channels in dendrite vs. axonal fibers in T4/T5 neurons. These findings lay the foundation for future functional investigations of receptors and ion channels in T4/T5 neurons and will be used by biophysically realistic model simulations of the motion-detecting circuit. In summary, we employed new methods to investigate neurotransmitters, their corresponding receptors, and voltage-gated ion channels in the motion vision pathway of the fruit fly. This work advanced our understanding of the biophysical mechanisms of motion-vision. Future studies can build on it to investigate the full molecular repertoire of T4/T5 neurons. Potentially, the strategies presented in this thesis can be expanded to different circuits or even different species in the future.
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Fendl, Sandra
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Fendl, Sandra (2021): Neurotransmitters and receptors in the motion vision pathway of Drosophila. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)
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Abstract

The fruit fly Drosophila melanogaster is one of the most popular model organisms being used in the life sciences. Due to its small number of neurons compared to other vertebrate species and its genetic access, it has also proven itself to be an optimal model organism for neuroscience. Especially, the field of systems neuroscience has made quick advances in its study of neural circuits of the fly brain underlying sensory perception such as olfaction and vision, as well as complex behaviors such as mating, learning, and memory. The motion vision pathway in the optic lobe of the fruit fly is a prominent example of a computation-performing neural circuit that researchers have been trying to understand for decades. While the wiring of the main circuit elements has been described via EM-reconstructions and their response properties have been characterized comprehensively, the molecular mechanisms of direction-selectivity still remain elusive. However, subcellular components such as neurotransmitter receptors and ion channels are important since they define the sign and the temporal dynamics of synaptic connections within a circuit. Hence, the main focus of my thesis was the investigation of neurotransmitter receptors in the primary motion sensing T4/T5 neurons of the fly brain, including the development of required genetic tools. First, we developed a protocol for super-resolution STED imaging in Drosophila brain slices which allowed us to resolve fine dendritic structures of individual T4/T5 neurons deep inside the brain (Manuscript 1). Second, we used the glutamate sensor iGluSnFR to characterize the temporal dynamics of the three glutamatergic cell types of the motion vision pathway L1, Mi9 and LPi (Manuscript 2). We validated the usability of iGluSnFR for measuring glutamate signaling in adult Drosophila brains and found that responses recorded with iGluSnFR are faster than GCaMP signals of the same cells. In Manuscript 3, we developed new genetic strategies for conditional protein labeling. Specifically, we introduced FlpTag, a tool for endogenous, conditional labeling of proteins by means of a flippase-dependent, invertible GFP cassette integrated in the endogenous gene locus. Using these methods, we explored the subcellular localizations of neurotransmitter receptors for glutamate, GABA, acetylcholine and voltage-gated ion channels in T4/T5 neurons in Drosophila melanogaster. Within the dendrite, receptor subunits localize to different regions and in a spatial order that exactly matches the EM-reconstructed synapse numbers and distributions of the different input neurons described in previous studies. Further, we discovered a strictly segregated subcellular distribution of two voltage-gated ion channels in dendrite vs. axonal fibers in T4/T5 neurons. These findings lay the foundation for future functional investigations of receptors and ion channels in T4/T5 neurons and will be used by biophysically realistic model simulations of the motion-detecting circuit. In summary, we employed new methods to investigate neurotransmitters, their corresponding receptors, and voltage-gated ion channels in the motion vision pathway of the fruit fly. This work advanced our understanding of the biophysical mechanisms of motion-vision. Future studies can build on it to investigate the full molecular repertoire of T4/T5 neurons. Potentially, the strategies presented in this thesis can be expanded to different circuits or even different species in the future.