Two-photon imaging with fluorescent biosensors to study neuronal activity in vivo in physiological and pathological conditions

The development of tools to allow in vivo measurement in intact neural circuitry represents a dramatic improvement in our understanding of brain activity. Understanding brain function is not only a challenging scientific quest aiming to disentangle the functional relationship among the electro-chemical dynamics in the brain with the cognitive and behavioral outputs, but it’s also a clinically crucial endeavor toward the study, diagnostics, treatment, and intervention of neurological diseases. Since the advent of laser scanning microscope has allowed for biological structures to be imaged at the theoretical limits of resolution predicted by Abbe, in vivo imaging poses significant and unique optical challenges beyond simply providing an enlarged view of a specimen. Two-photon laser scanning fluorescent microscopy has become the imaging standard for in vivo imaging due to its ability to obtain high signal-to-noise dynamic images while inducing minimal damage to the sample of interest. At the same time the development of fluorescent protein-based biosensors has been crucial for life science research. The information obtained through brain imaging facilitate both functional interpretation and medical advancements toward addressing neurological diseases. While this method provides unique merits in studying brain activities, it also accompanies certain pitfalls that prevent the technique to dominate. Complementary to optical neuroimaging is electrophysiology, through which electric signals in the brain can be detected and related to neuronal and cortical functions. Different approaches often requires appropriate data analysis to reveal the complexity of the mechanisms we are dealing with. Here I present the development of two techniques that allow to explore different aspects of neuronal computation. In Chapter 1, “Does High GABA always mean High Inhibition?” I present a tool for in vivo measurement of intracellular chloride concentration as a proxy for the understanding of inhibitory capability of the neural circuits during development and during day cycles in mice. In Chapter 2, “Statistically-based approach: a new way to study epilepsy in Zebrafish” I introduce a novel approach to exploit the complementarity of calcium imaging and electrophysiology to better understand conditions associated to hyperexcitability such as epilepsy.