New optogenetics method minimizes thermal harm to neurons | Spectrum
Two proteins: Equipping cortical neurons with a calcium sensor (red) and an opsin protein (green) enables researchers to study neural circuits in mice.
A newly engineered protein for optogenetic experiments requires less light than traditional techniques to excite or inactivate neurons, thereby reducing thermal damage to brain tissue.
The protein, described in eLife in May, improves on a method called two-photon optogenetics, which allows researchers to study neural circuits by stimulating them while monitoring their activity, including in live mice. It could help researchers map the neural circuits that connect different regions of the brain associated with autism, such as the amygdala, prefrontal cortex, and cerebellum.
To apply two-photon optogenetics, scientists first develop cells in an animal’s brain to express two proteins: an opsin, which activates or silences neurons in response to pulses of blue light, and a calcium sensor, which fluoresces red light when it binds to calcium ions; These ions flow into a neuron just before it fires. Researchers can then use two separate lasers to stimulate a programmed series of individual neurons and then monitor their levels of activity. In contrast, traditional optogenetics relies on LED lights to turn on thousands of neurons at once.
“By stimulating individual neurons, we can begin to ask questions about information coding in the brain in ways that wide-field single-photon optogenetics cannot,” says Ofer Yizhar, professor of neurobiology at the Weizmann Institute of Science, Israel. who developed the novel protein.
Previous proteins that have been used in two-photon optogenetics often have a spectral overlap, which means that the red laser can inadvertently activate the light-sensitive opsins. And some of the opsin proteins present a further challenge: To activate them, they need strong light pulses that can heat up and damage brain tissue.
Yizhar and colleagues made a modified version of an existing opsin protein by attaching a short peptide sequence that attached it to the soma or cell body of neurons and a fluorescent tag that helps the team visualize engineered cells. The original protein, CoChR, is expressed in the dendrites and axons of a neuron, making it difficult to spot and target. They call the new protein stCoChR or “soma-targeted channel rhodopsin”.
The team constructed mice to express stCoChR in pyramidal neurons in the cerebral cortex, with or without an existing calcium sensor called jRCaMP1a.
The researchers also developed and tested a laser system that can deliver more energy to neurons per pulse than typical lasers, but with a lower repetition rate. They found that the energy required to stimulate neurons fell well below the threshold that damages brain tissue. The engineered opsin could also be stimulated more efficiently than previous versions and had no spectral overlap with the calcium sensor.
“Now that we have this very sensitive duct rhodopsin, we can reduce the light output,” says Yizhar. “That means we can split the laser into multiple beamlets and now we have 100 times more beamlets to stimulate 100 times more neurons, which gives us much more flexibility to create pretty much any pattern we stimulate want. ”
The technology has some limitations: calcium sensors are an indirect measure of neural activity, so the laser cannot detect every action potential. Yizhar hopes to use voltage indicators in future experiments – proteins that directly sense electrical charges in neurons.
With the improved opsin protein, he and his colleagues also want to analyze the neural circuits that underlie learning and autism.
“We’re interested in the prefrontal cortex and autism models of neural connectivity,” says Yizhar. “The kind of questions we are interested in are how the prefrontal network is wired, how it changes in the learning process and also how it changes due to autism-related mutations.”
Quote this article: https://doi.org/10.53053/IGVE9122