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Dissertation Defense: Functional Labeling of Individualized Post-Synaptic Neurons using Optogenetics and trans-Tango

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Allison Castenda

Dissertation Defense: Functional Labeling of Individualized Post-Synaptic Neurons using Optogenetics and trans-Tango

Allison Castenda

Graduate Student, Translational Biology, Medicine, and Health
Graduate Research Assistant, Ni Lab
April 27, 2023 at 8:30 a.m.,  Room 101, Life Sciences 1

About this Dissertation

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 tagged with fluorescent reporters. 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.

More About the Candidate and Project


Virginia Tech, Translational Biology, Medicine, and Health, Ph.D. Candidate

Virginia Tech, B.S., Biological Sciences


Graduate Research Assistant, Ni Lab



Lina Ni, Ph.D., Assistant Professor, School of Neuroscience

Committee Members

  • Shihoko Kojima, Associate Professor, Biological Sciences
  • Igor Sharakhov, Department of Entomology
  • Julia Gohlke, Associate Professor, Environmental Health
  • Alexis Taylor Breaux International Study Fellowship
  • Girls Launch! Kindergarten Videos Grant

Peer-reviewed publications

Castaneda, A. N., Huda, A., Whitaker, I. B.M., Reilly, J. E., Shelby, G. S., & Ni, L. (2023). Functional Labeling of Individualized Post-Synaptic Neurons using Optogenetics and trans-Tango. Manuscript in preparation.

Huda, A., Omelchenko, A. A., Vaden, T. J., Castaneda, A. N., & Ni, L. (2022). Responses of different Drosophila species to temperature changes. Journal of Experimental Biology, 225(11), jeb243708.

Mosig, R. A., Castaneda, A. N., Deslauriers, J. C., Frazier, L. P., He, K. L., Maghzian, N., Pokharel, A., Schrier, C. T., Zhu, L., Koike, N., Tyson, J. J., Green, C. B., Takahashi, J. S., & Kojima, S. (2021). Natural antisense transcript of Period2, Per2AS, regulates the amplitude of the mouse circadian clock. Genes & development, 35(11-12), 899–913.


Allison Castaneda and Lina Ni. Investigating the Mechanisms of High Temperature-Mediated Unconsciousness in a Drosophila TrpA1 and Gr28b Double Knock-out Mutant. Poster presented at: TBMH Annual Banquet and Research Symposium; 2018 Oct 12; Blacksburg, VA.

MCB Interview Weekend Poster Session; 2019 Jan 18; Blacksburg, VA.

Oral presentation, August 14th 2020, School of Neuroscience Virtual Retreat.

  • Japanese Cultural Association Dance Instructor – 2017-2021
  • Exodus at Virginia Tech Dance Instructor – 2021-present