Neural circuitry, or how neurons connect across brain regions to form functional units, is the fundamental basis of all brain processing and behavior. There are several neural circuit analysis tools available across different model organisms, but currently the field lacks a comprehensive method that can 1) target post-synaptic neurons using a pre-synaptic driver line, 2) assess post-synaptic neuron morphology, and 3) test behavioral response of the post-synaptic neurons in an isolated manner. This work will present FLIPSOT, or Functional Labeling of Individualized Post-Synaptic Neurons using Optogenetics and trans-Tango, which is a method developed to fulfill all three of these conditions. FLIPSOT uses a pre-synaptic driver line to drive trans-Tango, triggering heat-shock-dependent expression of post-synaptic optogenetic receptors. When heat shocked for a suitable duration of time, optogenetic activation or inhibition is made possible in a randomized selection of post-synaptic cells, allowing testing and comparison of function. Finally, imaging of each brain confirms which neurons were targeted per animal, and analysis across trials can reveal which post-synaptic neurons are necessary and/or sufficient for the relevant behavior. FLIPSOT is then tested within Drosophila melanogaster to evaluate the necessity and sufficiency of post-synaptic neurons in the Drosophila Heating Cell circuit, which is a circuit that functions to drive warmth avoidance behavior. FLIPSOT presents a new combinatory tool for evaluation of behavioral necessity and sufficiency of post-synaptic cells. The tool can easily be utilized to test many different behaviors and circuits through modification of the pre-synaptic driver line. Lastly, the success of this tool within flies paves the way for possible future adaptation in other model organisms, including mammals. / Doctor of Philosophy / The human brain is made up of billions of neurons, each of which are interconnected in various ways to allow communication. When a group of connected neurons work together to carry out a specific function, that group is known as a neural circuit. Neural circuits are the physical basis of brain activity, and different circuits are necessary for all bodily functions, including breathing, movement, regulation of sleep, memory, and all senses. Disruptions in neural circuits can be found in many brain-related diseases and disorders such as depression, anxiety, and Alzheimer's disease. One example of a neural circuit is that of temperature sensation. When someone holds a cube of ice, temperature-sensing neurons in the hand pass signals along neurons in the spine until they reach the brain. There, the signals are carried to various brain regions to be processed and recognized as cold, and eventually, pain. When the sensory signals of cold and pain grow too prominent to ignore, the person may move to avoid the feeling. In this case, the brain will send signals back down to neurons responsible for movement in the arm, allowing the person to drop the ice cube. Avoidance of temperatures that are too warm or cold is an evolutionary trait that is important in preventing the body from harm. Even in a relatively simple system like temperature sensation, neural circuits can be complex and difficult to study, especially in higher order organisms such as mammals. For this reason, it can be beneficial to use simpler animals such as Drosophila melanogaster, or the common fruit fly. Flies have far fewer neurons than humans, meaning their neuronal connections are also significantly less complicated, and there are many genetic tools available in flies that aren't available in mammalian models such as mice. Additionally, flies are inexpensive, easy to raise, and grow quickly, making them ideal for troubleshooting new tools and replicating experiments. Though somewhat different in anatomy, fly brain function is similar enough to humans and other mammals that findings can often be applied across species. Studies in flies can also be applied in other insects, such as mosquitoes, which are notorious for carrying deadly diseases. Though there are several available tools in flies to study neural circuits, many tools are better for usage in sensory neurons themselves than in the neurons that carry signals in the brain afterward. This work presents a new tool, abbreviated as FLIPSOT, that modifies and combines several existing genetic methods in order to help examine those higher order neurons. FLIPSOT allows users to determine which higher order neurons are important in leading to behavioral responses, as opposed to carrying the signal to other brain regions, such as those associated with memory. Then, FLIPSOT is implemented in a warmth-sensing neural circuit known as the Heating Cell (HC) circuit and used to identify the higher order neurons needed for fly warmth avoidance. Development of tools such as FLIPSOT helps to expand our knowledge in the fields of neural circuits and behavior. Genetic tools can also be more easily tested in flies prior to attempting to implement them in other organisms, such as mice. Finally, studying temperature in flies can help create a deeper understanding of how temperature sensation works in all animals, including humans.

Network dynamics are thought to be the substrate of brain information processing and of mental representations. Moreover, network-wide dysfunctions are recognized to be at the core of several psychiatric and neurodegenerative disorders. Yet, our ability to target specific networks for functional or genetic manipulations remains limited. The development of monosynaptically-restricted Rabies virus, G-deleted Rabies virus (ΔG-Rabies), has greatly facilitated the anatomical investigation of neural circuits, revealing the network synaptic structure upstream of defined neuronal populations. However, the inherent cytotoxicity of the Rabies virus largely restrains its use to the mere structural characterisation of neural networks. To overcome this limitation, I generated novel tools that allow the manipulation of neural networks for the entire life of the animal, without affecting neuronal and circuit properties. I first developed a viral system obtained by engineering the Rabies virus genome to eliminate its cytotoxicity. This led to the generation of a Self-inactivating Rabies virus (SiR) that transcriptionally disappears from the infected neurons while leaving permanent genetic access to the traced network. I showed that SiR provides a virtually unlimited temporal window for the functional manipulation of neural circuits in vivo without adverse effects on neuronal physiology. To further expand our ways of intervening on neural networks function I then developed a completely virus-free system, named Genetically-Encoded TransSynaptic Shuttle (GETSS), which is the only specific genetically-encoded transsynaptic tracer to date. In this thesis, I established novel approaches that provide, for the first time, the functional and genetic access to traced network elements in vivo for the lifetime of the animal, with no cytotoxic effects, no changes in the electrophysiological properties of the traced neurons and no adverse effects on network function. This opens new horizons in the functional investigation of neural circuits and potentially represent the first approaches to experimentally monitor neural circuit remodelling in vivo.

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