Dissertations, Theses, and Capstone Projects

Date of Degree

9-2022

Document Type

Thesis

Degree Name

M.S.

Program

Cognitive Neuroscience

Advisor

Andreas Kottmann

Subject Categories

Cognitive Neuroscience | Molecular and Cellular Neuroscience | Other Neuroscience and Neurobiology

Keywords

GRAB, neurotransmitter sensors, Sonic Hedgehog, Smoothened, GPCR

Abstract

Dynamic neurotransmitter and neuromodulator signaling in functional circuits is the neural substrate of animal behavior and cognition. The study of said circuits requires tools with sufficient spatiotemporal dynamics that can probe complex signaling patterns and decode their functional relevance by coupling the signal to behavioral output, ideally in awake, behaving animals. Much is known about the role of classical neurotransmitters such as dopamine in behavior, but a wide variety of peptides and small molecules also regulate neuronal transmission. One of these is Shh, whose presence has been observed in a variety of brain regions known to modulate movement, perception, and cognition. However, elucidating the relevance of Shh signaling in behavior is hampered by the lack of reagents that can be used to detect and quantify dynamic Shh signaling in awake, behaving animals. Here I describe the development and initial characterization of the first genetically- encoded activation-based sensor to probe Shh signaling dynamics with high spatiotemporal resolution (SmoGRAB). This indicator was engineered by inserting a cpGFP moiety into the third intracellular loop of Smothened (Smo), the GPCR that translates the Shh signal. I observe concentration dependent changes in fluorescence in response to Smo pharmacology with sub-second kinetics in transfected HEK293FT cells.

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