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.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/115738 |
| Date | 11 July 2023 |
| Creators | Castaneda, Allison Nicole |
| Contributors | Graduate School, Ni, Lina, Kojima, Shihoko, Sharakhov, Igor V., Gohlke, Julia M. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/vnd.openxmlformats-officedocument.wordprocessingml.document |
| Rights | Creative Commons Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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