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Investigation of 3D Functional Connectivity in Mouse Visual Cortex using Holography and Optogenetics

Periodic Reporting for period 1 - 3DMUCHO (Investigation of 3D Functional Connectivity in Mouse Visual Cortex using Holography and Optogenetics)

Reporting period: 2017-04-01 to 2019-03-31

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).
During the two-year period of this project, several milestones have been achieved:
- In vivo single-cell holographic activation for opsins of diverse channel kinetics
We applied the custom-built TF-CGH for spike generation for opsins of channel closing time constants ranging 10-100 ms: the slow ReaChR, the intermediate CoChR, and the fast ChrimsonR. Using 2P-guided patch recordings at the layer 2/3 of anesthetized mouse visual cortex to measure the spiking in target cell, we found the illumination conditions of brief duration <10 ms and low power of 10-50 mW at 1030 nm that enable single AP induction of <10 ms latency and <1 ms jitter for the above opsins. Repetitive holographic illuminations onto the target cell induce regular spike trains or irregular ones that mimic its spontaneous firing.

- All-optical single-/multi- cell holographic activation
The main advantage of parallel holographic stimulation is simultaneous targeting of one or multiple cells. In preparation co-expressing ReaChR/GCaMP6 or somatic CoChR/GCaMP6, we were able to control the activity of one or many layer 2/3 neurons all-optically by reading out increased GCaMP6 signal in the target neurons upon holographic illuminations in lightly anesthetized or awake mice. Using 2P-guided patch recordings upon 2P scanning at different power, we quantified the crosstalk spiking for opsins ReaChR, CoChR and ChrimsonR. Notably, the slow channel kinetics of ReaChR renders it more prone to spurious firing for scanning power >60 mW.

- Functional mapping of orientation selectivity
To be able to probe connections between functionally identified neurons, we sought to determine the tuning properties of a neuronal network. Co-tuned cells of layer 2/3 neurons were identified according to the orientation selectivity measured by performing GCaMP6 calcium imaging upon presenting drifting gratings.

The above results, providing the first step to in vivo functional connectivity mapping, are mostly summarized in a research article published in Journal of Neuroscience (2019) and a review published in Current Opinion in Neurobiology (2018).
Results from this project add a piece of valuable information about the relevant parameters of holographic illumination for fast and precise in vivo 2P optogenetic neuronal activation. To complete this on-going project of in vivo functional connectivity mapping, the Fellow will keep optimizing the opsin/indicator combination for minimum crosstalk activation, design strategies for efficiently probing neuronal connections using somatic opsin, and implement the 3D light-shaping pioneered in the host group.

Successful realization of the proposed project will not only contribute to decipher a fundamental question of the relationship between connectivity and visual selectivity in visual cortex, but also allow this approach being transferred to other brain regions. This knowledge will be particularly important for the study and remedy of brain functions. In addition, the proposed action has a strong interdisciplinary characteristic, bringing together neurophysiology, molecular engineering of opsins, and wavefront light-shaping, and further reinforcing each aspect.