We developed the new technical approaches to monitor and manipulate neural activity patterns in the intact mouse brain. As a necessary step towards the development of this technology, we first applied two-photon holographic illumination to map the activity of cortical cells with millisecond temporal resolution and subcellular spatial resolution in the mouse cortex in vivo (Bovetti et al. Sci. Reports 2017) and we validated this approach in GRIN lens-based endoscopes for fast imaging in deep brain regions (Moretti et al. Biom. Optics Express 2016). We also precisely evaluated the limitation of traditional raster scanning approaches to monitor spiking activity using fluorescent calcium activity reporters and we proposed a smart line scanning method to improve the inference of action potential from fluorescence measurements in population imaging (Brondi et al. Cell Reports 2020). We further designed, developed, and validated a new methods to correct optical aberrations in GRIN lens-based endoscopes using 3D microprinting (Antonini et al. eLife 2020). We then combined these advanced optical methods to monitor the activity of superficial and deep neuronal circuits with wide-field single-photon optogenetic stimulation of the inhibitory opsin Archaerhodopsin (Bovetti et al. Sci. Reports 2017). This new experimental approach allowed mapping the response of neuronal circuits in the intact mammalian brain with unprecedented temporal resolution and no stimulation artefacts during inhibitory optogenetic manipulations. More recently, we combined two-photon holography to stimulate neurons expressing blue light-sensitive opsins (ChR2 and GtACR2) with two-photon imaging of the red-shifted indicator jRCaMP1a in vivo. We demonstrated efficient control of neural excitability across cells types and layers with holographic stimulation and improved spatial resolution by opsin somatic targeting. Moreover, we performed simultaneous two-photon imaging of jRCaMP1a and bidirectional two-photon manipulation of cellular activity with negligible effect of the imaging beam on opsin excitation (Forli et al. Cell Reports 2018). This methods was recently improved using a new opsin variant which allowed high-efficiency stimulation of neurons with down to < 1 mW average power per cell (Forli et al. eLife 2021). Importantly, we contributed to develop the conceptual framework for the application of two-photon optogenetics to understand the brain code underlying behavioral perception (Panzeri et al. Neuron 2017) and we built models of cortical activity during different network states (Zerlaut et al. 2019). We started applying these advanced optical and theoretical approaches to dissect out the cellular and network mechanisms underlying the generation of spontaneous and sensory-evoked circuit activity in the neocortex (De Stasi et al. Cer. Cortex 2016, Zucca et al. eLife 2017, Mariotti et al. Nat. Comm. 2018, Zucca et al. Curr. Biol. 2019, Vecchia et al. Curr. Biol. 2020). The experimental activities related to the development of aberration correction in endoscopes using microfabricated lenses resulted in the foundation of one spin-off company (SmartMicroOptics S.r.l.).
Dissemination of scientific findings and achievements proceeded in parallel with experimental activities throughout the entire project. Results have been published in international peer-reviewed journals in open access modality and regularly presented to national and international conferences, invited seminars, and specialized schools. In collaboration with the Communication and Foreign Press Office at the IIT, we participated to many initiative to disseminate the results of the project to various audiences. Among many, dissemination activities included open day events targeted to middle- and high-school students, one-to-one video interviews, contribution to ERC-related events, presentations to the general public, articles in newspaper, and a video celebration for the 10,000th ERC awardee.
