Selective Recording and Stimulation of Neurons in the Mouse Hippocampus and Cortex: Two-Photon Imaging, Uncaging, and Behaviour

Two-photon imaging is becoming one of the most widely used technique in Neuroscience. Combined with selective neuronal stimulation (or inhibition) techniques, it offers an infinite variety of experiments aimed to better understand the brain at different scales. However, the optimal conditions for applying each of these techniques are not usually the same, and even though there are ways of overcoming this limitation, these solutions require expensive resources. This PhD thesis aimed to optimise the combination of two-photon imaging and two-photon uncaging by finding an excitation wavelength suitable for both processes, which is the main limitation when the two methods are used together. This method was designed in order to be suitable for both in vitro and in vivo experiments. First, in vitro experiments were performed in order to maximise the quality of brain slices. Secondly, the physicochemical characteristics of the protein used for Calcium imaging, GCaMP6s, were studied with the objective of finding a range of excitation wavelengths suitable for doing imaging but, at the same time, closer to the optimal uncaging wavelength. Two-photon uncaging of Glutamate was achieved using light at 850 nm, a wavelength that also permitted monitoring the neuronal response to the stimulation. It is expected that this technique will soon be applied in vivo with the objective of developing an animal model for concept representation in the hippocampus. This project consists of training mice in a virtual reality set-up that allows testing them in a two-forced choice paradigm. In the case of finding neurons firing selectively to one of these objects, independently of low-level features, the uncaging technique will be useful for manipulating the normal activity of those neurons and study how this manipulation a affects the animal's behaviour.