Periodic Reporting for period 3 - HOLOVIS (Holographic control of visual circuits)
Période du rapport: 2023-10-01 au 2025-03-31
The aim of this research program is to produce novel all-optical technologies to explore brain functions at the mesoscopic scale with cellular resolution opening a new phase in optogenetics that I named circuit optogenetics.
Revealing the neural codes supporting specific mammalian brain functions is a daunting task demanding to relate in vivo the individual activities of large numbers of neurons recorded jointly within collectives that form distinct nodes of a network and to perform precisely targeted and calibrated interventions in the spatiotemporal dynamics of neural circuits on the scale of naturalistic patterns of activity. Despite recent technical advances, these experiments remain out of reach because we lack a comprehensive approach for large-scale, multi-region, in depth, single cell and millisecond precise manipulation of neural circuits. HOLOVIS will tackle these limitations through the construction of an innovative paradigm combining optogenetics with cutting-edge technology of wave front shaping, compressed sensing, microendoscopy, wave-guide probes, laser developments and opsin engineering.
My lab has pioneered the use of wave front shaping for neuroscience and developed in the past years a number of new optical methods, for patterned optogenetic neuronal stimulation. Here, we will push forward this technology and first demonstrate the performances of these breakthrough systems to reveal how inter, intra-laminar and cortical/sub-cortical wiring construct and refine visual orientation selectivity in mice.
We will focus on the visual system of mice, whose input-output responses to controlled sensory stimulations have been characterized in decades of studies. However, we are persuaded that our approach can be used to reveal the connectivity rules that underlie specific patterns of activity of any neuronal circuit, thus defining the functional building blocks of distinct brain areas.
Revealing the neural codes supporting specific mammalian brain functions is a daunting task demanding to relate in vivo the individual activities of large numbers of neurons recorded jointly within collectives that form distinct nodes of a network and to perform precisely targeted and calibrated interventions in the spatiotemporal dynamics of neural circuits on the scale of naturalistic patterns of activity. Despite recent technical advances, these experiments remain out of reach because we lack a comprehensive approach for large-scale, multi-region, in depth, single cell and millisecond precise manipulation of neural circuits. HOLOVIS will tackle these limitations through the construction of an innovative paradigm combining optogenetics with cutting-edge technology of wave front shaping, compressed sensing, microendoscopy, wave-guide probes, laser developments and opsin engineering.
My lab has pioneered the use of wave front shaping for neuroscience and developed in the past years a number of new optical methods, for patterned optogenetic neuronal stimulation. Here, we will push forward this technology and first demonstrate the performances of these breakthrough systems to reveal how inter, intra-laminar and cortical/sub-cortical wiring construct and refine visual orientation selectivity in mice.
We will focus on the visual system of mice, whose input-output responses to controlled sensory stimulations have been characterized in decades of studies. However, we are persuaded that our approach can be used to reveal the connectivity rules that underlie specific patterns of activity of any neuronal circuit, thus defining the functional building blocks of distinct brain areas.
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.
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.
Overall the project has a strong dual technological and neuroscientific character with several innovative elements. We will produce novel « all optical » technologies to explore brain functions at the mesoscopic scale with cellular resolution. Coupled either with 2P or 3P excitation the system will enable imaging and patterned photostimulation of neuronal circuits at few hundred microns or mm deep. Using new schemes for fast patterned illumination, the system will enable simultaneous photostimulation at up to 1kHz frame rate within a mm3 volume. For greater depths, the system will use advanced schemes for patterned endoscopy, enabling imaging and patterned activation at depths of several mm. The development of the multi-site microscope will enable simultaneous manipulation of brain circuits across multiple brain areas. The use of novel laser sources will enable simultaneous targeting of hundreds of targets. Finally, the combination of patterned illumination with compressed sensing algorithms and multitarget voltage recording, will provide a new approach for fast connectivity probing of large (2D and 3D) cell population.
With these capabilities it ought to be possible to elucidate the rules of connectivity, the identity of specific key neuronal players as well as the synaptic mechanisms underlying sensory perception. More generally, simultaneous manipulation and recording of many neurons in distinct layers of connected neuronal networks in different brain areas will be instrumental for decoding several aspects of cognition, such as memory, attention, motor initiation, decision making, and cognitive flexibility.
The unique capability of this system will be used to investigate for the first time, at single cell, level in-vivo, cortical-subcortical circuitry involved in processing and refinement of visual orientation selectivity. The degree of precision allowed by the new optical techniques and their minimal invasiveness will represent a new paradigm for the investigation of intact circuitry in sensory cortex and thalamus.
With these capabilities it ought to be possible to elucidate the rules of connectivity, the identity of specific key neuronal players as well as the synaptic mechanisms underlying sensory perception. More generally, simultaneous manipulation and recording of many neurons in distinct layers of connected neuronal networks in different brain areas will be instrumental for decoding several aspects of cognition, such as memory, attention, motor initiation, decision making, and cognitive flexibility.
The unique capability of this system will be used to investigate for the first time, at single cell, level in-vivo, cortical-subcortical circuitry involved in processing and refinement of visual orientation selectivity. The degree of precision allowed by the new optical techniques and their minimal invasiveness will represent a new paradigm for the investigation of intact circuitry in sensory cortex and thalamus.