The relationship between the wiring and function of neuronal network has always been the central theme in neuroscience1. Understanding the circuit mechanisms of brain function at cellular level is fundamental for tackling brain dysfunction. With the increasing incidences in modern society, neuropsychiatric diseases have become a heavy burden. Hence, knowledge of how neuronal network is causally linked to brain function will contribute to the remedy of its diseases.
This project aims at investigating the relationship between neuronal wiring and visual selectivity in the living mouse visual cortex. Cortical neurons integrate presynaptic inputs from connecting partner cells and generate action potentials (AP) to code for features of sensory stimuli. Convergent evidences point towards stronger horizontal connectivity between co-tuned neurons2. Important questions such as whether this function-wiring correlation can be generalized to vertical connections remain however unresolved. These studies are thus far conducted in a correlated manner, i.e. correlating the functional selectivity measured in vivo with the connectivity mapped in vitro. Mapping connections in vivo allows to compare the horizontal and vertical wiring in the same animal. To this end, the ability to selectively control the excitability of one or several neurons distributed in a 3D cortical volume is necessary.
Optogenetics makes possible to control neuronal firing by shining light onto neurons expressing light-sensitive ion channels, opsins. One main challenge of single brain cell manipulation is to focus light onto individual opsin-positive neuron through the scattering brain. Providing excellent optical sectioning, two-photon (2P) excitation of opsin is a promising solution to tissue scattering. Innovative optical methods of scanning or parallel approaches, in combination with the new-generation opsins and amplified laser sources, have permitted 2P optogenetic activation in a spatiotemporally precise manner in the scattering brain tissue. The scanning method swiftly deflects the laser beam in a trajectory covering the target neuronal soma for actuating opsins sequentially. On the other hand, cell-shape light-pattern is created via the parallel approach. The host laboratory is at the forefront for 2P optogenetic activation by developing parallel methods including computer-generated holography (CGH) and temporal focusing (TF)3. The advantage of parallel methods is simultaneous actuation of opsins under the illumination area4, thus allowing fast and precise neuronal activation for opsins of a wide range of channel kinetics5. In addition, advance in soma-targeted expression of opsins prevents neurite activation from non-target cells, thus ensuring unbiased single-cell activation. Finally, recently developed fiber lasers of amplified pulse-energy provide plentiful power reserve for parallel stimulation of up to hundreds of cells simultaneously.
In this project 3DMUCHO, two research objectives are proposed to achieve the goal of uncovering the functional connectivity by applying the cutting-edge 3D light-shaping technology. First, in combination with 2P-guided whole-cell recording from a postsynaptic cell in lightly anesthetized mouse visual cortex, its presynaptic partners can be identified by holographic stimulation of neighboring cells horizontally and vertically with illumination conditions inducing a train of APs. Second, functional connectivity can be revealed by probing connections from functionally determined cells, e.g. sharing visual selectivity, in vivo.
1. Chen, I.-W. Papagiakoumou, E. & Emiliani, V. Towards circuit optogenetics. Curr. Opin. Neurobiol. 50, 179–189 (2018).
2. Ko, H. et al. Functional specificity of local synaptic connections in neocortical networks. Nature 473, 87–91 (2011).
3. Papagiakoumou, E. et al. Two-photon optogenetics by computer-generated holography. in Optogenetics: A Roadmap (ed. Stroh, A.) 133, (Springer, 2018).
4. Ronzitti, E., Ventalon, C., Canepari, M. & Forget, B. C. Recent advances in patterned photostimulation for optogenetics. J. Opt. 19, 113001 (2017).
5. Chen, I.-W. et al. In vivo sub-millisecond two-photon optogenetics with temporally focused patterned light . J. Neurosci. 39, 1785–18 (2019).
