We have worked both on the technological development (Aim A) and on using the developed systems for the investigation of visual circuits (Aim B).
Main achievements for the technological development have been the finalization of a two-photon (2P) flexible fiber bundle-based microscope (named 2P-FENDO). The capabilities of 2P-FENDO have been validated in L2/3 visual and barrel cortex of freely moving animals. This was the first demonstration of an all-optical neuronal manipulation in freely moving mice at cellular resolution (N. Accanto, et al. Neuron, 2023).
To achieve precise optical control of neuronal circuits with high spatial temporal precision, we have worked on testing and identifying new optogenetics constructs that optimize the use of all-optical optogenetics (A1.3). Specifically, we have been able to demonstrate 2P bi-directional neuronal control of neuronal activity using the opsin BiPOLE developed by the Peter Hegemann labs (J. Vierock, et al. Nature communications, 2021) and efficient 2P inhibition using the newly developed a new K+ conducting opsin (WichR) also developed by the Hegemann lab (J. Vierock, et al. Science Advance 2022).
We have designed and tested two possible configurations for volumetric imaging (A2), one called SPAM, enables enhanced volumetric imaging combining patterned illumination and scan imaging (H. Massilia, et al. in preparation). The second one uses a galvanometric mirror and a spatial light modulator for ultra-fast sequential patterned illumination (named FLiT). We used FLiT to demonstrate two new illumination methods, termed hybrid- and cyclic-illumination and achieve sub-millisecond control of sequential neuronal activation and high throughput multicell illumination while minimizing light-induced thermal rises (G. Faini, et al. Nature Communications 2022)
Finally, we have refined the necessary technology for fast probing of functional connectivity (A3). This includes the development of an optical microscope for fast voltage imaging that we used for the first demonstration of simultaneous multitarget voltage imaging using the new 2P voltage indicator JEDI (developed by the F. St Pierre lab; R. Sims et al. under review in Nature communications; A3.1). Also, we have optimized an experimental and theoretical strategy for high throughput connectivity mapping using sequential and parallel stimulation, the latter in combination with compressed sensing analysis ( A3.2).
Main achievement for Aim B: has been the use of the approach developed in A.3.2 for 2D and 3D high throughput connectivity mapping to probe horizontal connectivity in layer L2/3 of the visual cortex (Aim B1; IW Chen, et al. in preparation), we could probe for the first time, in vivo, more than 100 connections with single cell resolution.
