Two-photon mapping of neocortical circuits

The central hypothesis in cortical research envisages a modular, or “canonical” cortical microcircuit, built out of many cell types which are connected in specific ways to implement a defined circuit function. However, there is no unified theory of how the cortex works because exact connectivity diagrams do not exist. Although “connectomes” of whole brains are being sought by large anatomical projects, the actual operational connections and their relative weights will not be revealed until living tissue is studied with a systematic high-throughput approach. This was recently done on inhibitory interneurons which were revealed to be densely connected in the circuit. The aim of this proposal is to find out how cortical excitatory cells (the pyramidal neurons, which constitute approximately 80% of cortical cells) are connected to each other by combining electrophysiological recordings with an optical method that will sample hundreds of possible synaptic connections in 10 minutes. Brain slices from transgenic mice will be used to genetically target specific cells. Two-photon stimulation of the new opsin C1V1 with spatial light modulator optics as well as uncaging of RuBi-caged glutamate will be used to find cells presynaptic to simultaneously patch clamp recorded neurons. I will also reveal synaptic weight matrices for pyramidal neurons in different layers of the cortex and chains of synaptically connected neurons. Morphology and immunohistochemistry of recorded neurons will be characterised to obtain further information of the circuits. The results will reveal the level of specificity in cortical connectivity and substantially facilitate understanding cortical function.