MU scientists expand toolbox to study the brain
March 20, 2018
Thermogenetics is a powerful new tool for studying the brain. The technique allows scientists to use temperature to control the electrical firing of a genetically modified neuron using special temperature-sensing proteins. By being able to turn on a set of neurons at will, the technique gives scientists a precise tool to examine how they contribute to a particular behavior.
Now, a team of faculty and students from the University of Missouri report the biophysical characterization of a recently discovered temperature-sensing protein and demonstrate its ability to control behavior in fruit flies. As the first such protein to be discovered from a family of genes encoding gustatory receptors, the protein represents a novel thermogenetic tool.
“Thermogenetics is expanding the horizons of brain research by allowing us to precisely control specific neurons in the brain and measure behavioral changes,” said Zars. “Currently, there are a relatively small number of proteins that do respond to temperature in a way that is useful for work in flies. The addition of a new thermogenetic tool — one with different thermal and biophysical properties — increases the complexity of questions we can address and how we can use the technology.”
The team first used biophysical techniques to confirm that the protein, called Gr28bD, can generate an inward temperature-dependent current, capable of increasing electrical activity in neurons. The team then expressed the protein in fruit fly neurons and, using live imaging techniques, demonstrated that the generated current is enough to drive neural activity in a temperature-dependent manner.
Autoosa Salari, Ph.D. ’17 (now a postdoctoral fellow at U.C. Berkeley); Marzie Amirshenava, a doctoral student in biological sciences; Ben Zars, a senior majoring in biological sciences; and Kayla Miguel, a former NSF-REU student from the University of Miami and now a Ph.D. student at Northwestern University, completed the biophysical studies in the lab of Dr. Mirela Milescu. Benton Berigan, a doctoral student in biological sciences, and Jenna Lin, a senior majoring in biological engineering, performed the cellular imaging studies with Dr. Lorin Milescu.
“The live imaging is incredible, because we can watch as neurons fire electrical signals,” said Berigan, who is one of three first-authors of the study. “We first put a fly under the microscope, into a special chamber designed and 3D-printed by my colleague Jenna. Then, we use our lab-developed software platform to visualize in real-time neurons going from a baseline florescence to a high-intensity signal when the protein is activated with temperature. Then, when we lower the temperature, we can observe them return to baseline.”
To test if the protein could be used as a thermogenetic tool to control behavior in flies, the scientists tested fruit flies in a specially designed heat box that allows them to precisely control environmental temperature.
Aditi Mishra, a doctoral candidate in biological sciences, and Abbey Robinson, a senior majoring in biological sciences, performed the behavioral experiments in the lab of Dr. Troy Zars.
“When the temperature of the heat box got to 34 degrees Celsius, the proteins were activated, which caused all the neurons to fire and the fly to become paralyzed. When the temperature was returned to normal, the flies behaved normally. That’s how we were able to show that the protein has a behavioral effect,” explained Mishra, who is also a lead author of the study.
The protein, which is activated at a different temperature than other thermogenetic proteins currently in use, is the first temperature-sensitive protein to be culled from a large and previously unexplored family of gustatory receptors from fruit flies. The researchers said that differences in biophysical properties within this family offer both immediate and future advantages to neuroscientists studying the brain.
“Immediately, this protein expands the toolbox for researchers by allowing them to pick proteins that might be most advantageous for their organism or question,” said Berigan. “Gr28bD could become a powerful tool in controlling neuronal activity and studying how neuronal circuits function. Since this protein is not found in any mammal, it is a good candidate for the development of thermogenetic tools to be used for basic research, and potentially one day in humans.”
Zars said the study represents proof-of-concept. “We picked a very simple, straightforward behavior to show that it would work. Now, we can go to smaller sets of neurons and test for specific functions,” he said.
Thermogenetics builds on ongoing advances in the field of optogenetics, which uses light-sensitive proteins to control neural activity. Aside from the source by which they control neural firing (temperature versus light), the complementary approaches differ in the neural response: light induces a fast response in neurons, while the neural response from temperature is more gradual. When paired together, these temporal differences, said Zars, could be a powerful approach to unraveling the intricacies of the brain.
“To have the ability to modify modulatory neurons in a circuit that you can watch with a microscope and see how individual neurons react to stimuli and then modulation at the same time using not just light but light and temperature is powerful,” he said.
Like optogenetics, Zars said the thermogenetic proteins could eventually be used in humans where they could provide a variety of potential diagnostic and therapeutic benefits.
The article, titled “The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool,” appeared in the January issue of the journal Scientific Reports.
The study was supported by an Early Concept Grant for Exploratory Research (EAGER) from the National Science Foundation, as part of the federal Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative.
Written by: Melody Kroll
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