All-optical electrophysiology combines optics and genetics to control and record neuronal activity in living tissues in a non-invasive way.
The study of brain connections is one of the main challenges of modern neuroscience. Investigating how different brain areas communicate with each other, without altering their function, necessitates the use of increasingly sophisticated technologies. While they offer great temporal precision, techniques such as microelectrode electrophysiology are limited in spatial resolution, making it challenging to observe large neuronal populations in intact tissue.
To overcome these limitations, LENS researchers employ innovative approaches based on the integration of genetics and optics, assisted by artificial intelligence. All-optical electrophysiology which combines optogenetic stimulation with functional imaging, allowing scientists to selectively manipulate neuronal activity and observe their response in real time.
Optogenetics was first theorized in 1979 but only technologically mature in the past 20 years. It combines genetic manipulation techniques in animal models with highly precise microscopy. “The main idea is to introduce, through genetic techniques, light-sensitive proteins to stimulate specific neurons,” explains Lapo Turrini, researcher at the National Institute of Optics (CNR-INO) and affiliated with LENS. “Fluorescent proteins sensitive to calcium or voltage are also introduced at the same time, in order to measure the activity of neurons and the circuits they belong to.” This combination opens up the possibility of experimenting on live tissue with extremely high precision, while preserving tissue integrity. Brain activity is observed in vivo, with enough spatial and temporal resolution to study neural circuit function. The protein reacts to light stimulation: when the neuron activates, calcium ions bind to the indicator protein, increasing fluorescence intensity, which can be visualized in real time. Because the specimens are genetically modified to express a specific marker, many combinations of neuronal populations and observable activities are possible.
As with any microscopy technique, the analyzed samples must be sufficiently transparent for light to pass through. LENS researchers focus on two animal models: the zebrafish larva and the mouse. In zebrafish larvae are completely transparent during the first days of life, so it is possible to observe their entire brain in vivo. Using light-sheet microscopes, researchers can obtain three-dimensional images of overall brain activity, with a resolution that goes down to the single cells. “The specimen is immobilized in an agarose gel and kept in water at a constant temperature, while a thin sheet of light illuminates horizontal brain sections,” Turrini explains. “With this method we were able to reconstruct, for example, the effective connectivity of the left habenula, a structure deep within the brain shared across all vertebrates that is involved in many behavioral functions.”
In mice, natural transparency is lower – however, thanks to the low thickness of the mouse skull, it is still possible to stimulate and observe brain activity directly through it. “Even though we don’t directly observe the brain’s surface, the activation of specific areas and the light that passes through the skull give us enough information. We can analyze brain regions about one square centimeter in size, with a resolution of tens of micrometers,” clarifies Francesco Resta, member of the University of Florence Physics Department. “Although we cannot see individual neurons, we can observe the activation of selected groups of cells, such as excitatory neurons, and monitor how stimulation in one area spreads to other regions. We can directly observe and manipulate the ‘conversations’ between brain areas in the mouse, revealing the fundamental rules by which the brain processes and transmits information.” Using this system, for example, LENS researchers discovered that two areas of the mouse motor cortex, whose connection had been unclear, activate in a segregated manner.
This methodology requires extensive development of technology and instrumentation. Laser systems, optics, and animal models must be finely tailored to the experimental needs. “Collaboration between physicists, engineers, and biologists is essential,” explain Resta and Turrini. “The stimulation and detection arrays are custom-made to address our research questions.”
All-optical electrophysiology offers us an unprecedented window into the brain in action: a technology that combines manipulation and observation, allowing direct testing of how activity in one brain region influences others—and, ultimately, how neural networks generate perceptions, decisions, and behaviors.
Selected References
Turrini, L., Ricci, P., Sorelli, M. et al. Two-photon all-optical neurophysiology for the dissection of larval zebrafish brain functional and effective connectivity. Commun Biol 7, 1261 (2024). https://doi.org/10.1038/s42003-024-06731-3
Resta, F., Montagni, E., de Vito G., et al. Large-scale all-optical dissection of motor cortex connectivity shows a segregated organization of mouse forelimb representations. Cell Reports 41, 111627 (2022). https://doi.org/10.1016/j.celrep.2022.111627
