In this thesis, we sought to develop tools to simultaneously measure and manipulate neural activity using intact nervous systems with relevant behavior activity, ultimately broadening the use of these technologies and laying the groundwork for deploying these tools using other experimental systems that provide additional basic and translational relevance. 

In the first project, we explored the thermosensitive properties of Drosophila Gr28bD, a recently discovered thermosensitive protein involved in thermosensation. In summary, we show that Gr28bD can be expressed in a heterologous system, and it generates a temperature dependent cationic current that is not voltage sensitive. When expressed in Drosophila motor neurons, it can be used a thermogenetic tool to modulate neuronal activity (measured via calcium imaging  with GCaMP6f), which ultimately leads to behavioral changes in flies.

In a second project, we hypothesized that other naturally occurring thermosensitive Grs with different temperature properties are expressed in Drosophila species which inhabit diverse global climates. We tested Gr28bD orthologs from five species: D. simulans (DsimGr), D. yakuba (DyakGr), D. psuedoobscura (DpseGr), D. willistoni (DwilGr), and D. mojavensis (DmojGr) using thermogenetic strategies and calcium imaging via GCaMP6f in D. melanogaster flies. In summary, we found that DwilGr flies show a similar temperature response to Gr28bD flies (37.5 - 38°C), whereas DsimGr, DyakGr, and DpseGr flies are activated at lower temperatures (33 - 35°C). Additionally, we show that DyakGr and DpseGr flies have a greater effect on neuronal activity compared to Gr28bD. Interestingly, DmojGr is not temperature sensitive below 40°C. This work further validates Drosophila Grs as a family of thermosensors with thermogenetic applications, and it provides insight into the structure and function of thermosensitive GRs. 

In a third project, we aimed to implement tools for neural stimulation and recordings in a novel system that has not been well characterized yet has significant basic and translational relevance. In summary, we’ve laid the groundwork to record autonomic activity during the filling and emptying of mouse bladder using a combination of in vivo calcium imaging using GCaMP6s, electrophysiology recordings from multiple peripheral nerves, and bladder circuit output via a bladder catheter. This work provides insight on basic autonomic activity during the filling and voiding of the mouse bladder, and we are optimistic that we can use these tools to understand how spinal cord injury and neurological disease impact autonomic signals in the lower urinary tract.

Publications

Mishra, A.*, Salari, A.*, Berigan, B.R.*, Miguel, K.C., Amirshenava, M., Robinson, A., Zars, B.C., Lin, J.L., Milescu, L.S., Milescu, M., Zars, T. The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool (2018) Scientific Reports, 8 (1), art. no. 901. (*Equal contributions)

Committee

• Dr. David Schulz (Chair)
• Dr. Rex Cocroft
• Dr. Michael Garcia
• Dr. Ilker Ozden