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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - VISONby3DSTIM (Restoration of visual perception by artificial stimulation performed by 3D EAO microscopy)

Teaser

Summary

Visual function is the most important of the five senses for orientation in the everyday environment and social life, therefore visual impairment leads to restrictions in all aspects of the daily life. Visual impairment diseases sum up multiple level of impairment and different areas of the visual pathway involved. For retinal degeneration multiple therapies started the emerge in the past decade, including retinal transplantation, different implant and even virus vector technology targeting the retina directly, however there is no silhouette for solution yet when other areas of the brain are involved.
Our endeavor is to provide theoretical and practical basis for the creation of a possible visual prosthetics which would provide vision not through the eye, but the neural tissue of the brain itself, directly. Our strength what helps us to achieve what we undertook is the microscopes we design and build. These microscopes that operate on the basis of the acousto-optical and two-photon principles are capable of recording the activity of a large number of cells in the cortical part of the brain (in this case the so-called visual cortex) and also to stimulate those cells very precisely both in space and in time.

Work performed

In the first part of our ERC grant period we have designed, built, and amended such a 3D microscope, which may operate with multiple independent laser lines to record and simultaneously stimulate neurons in 3D in large cortical volumes. To reveal what the cellular networks in the visual cortex are exactly doing when computing the features of the surrounding world, it is vital that we image not only small regions but the entire V1 and also secondary visual areas in the entire depth of the cortex with high speed. To achieve high scanning depths and to extend z-scanning range and provide simultaneous photo-stimulation we also built a virus laboratory which provides us means to produce and deliver genetically coded sensors and opsins in many combinations.
We have also designed and produced a large objective (a mesoscopic objective) to image not only the entire depth of the cortex, but also to provide large field-of-view simultaneously. Currently we can provide 3300 Ã— 3000 Ã— 1000 Âµm3 scanning volume for fast 3D imaging. These are the prerequisites of revealing what happens in the brain when we dwell in the visual world.
We have built and still are working on a virtual reality environment which makes possible a reality-like visual stimulation under the very complex microscope, so we can study the activity of the visual cortex whilst the mouse is engaged in a given visual task with different visual cues.
We have also started recording activity of neuronal populations with the fast 3D acousto-optical microscope in mice navigating in virtual reality and revealed new principles driving the processes in visual computations. We have detected that temporal components of the visual information are represented in the V1 region of the cortex in overlapping spatiotemporal clusters of neurons.
Our eventual goal is to better characterize the coding and relevance of these novel spatio-temporal clusters in cortical networks activity in response to the visual features of the environment when the animal is navigating in it, and then replicate them by stimulating the neural tissue directly, skipping the information from the eyes but providing similar subjective perception for the animal.
We have already performed many recordings in the visual cortex of the animals running in virtual mazes. We have realized that the network activities have a precise temporal build up which have not been explored so far in the related literature. This temporal build up and changes of it may be described by novel mathematical tools. This insight is an important step towards solving the practical question of how to stimulate the network properly, how to activate clusters of neurons to elicit similar behavior than what would be initiated by real sight.
Our future plans are to find the appropriate so called â€œopsinsâ€ with which it will be possible to actually do the stimulation in a cell-type specific, temporally and spatially very precise manner (the opsins we work with currently have some limitations in these respects); finish the development of a flexible virtual reality system; complete the analysis of the principles guiding visual cortical network operations; and eventually test our results by guiding animal through the virtual maze by stimulating directly their brains.

Final results

Understanding neural computation requires methods that can simultaneously read out activity on both somatic and dendritic scales. As shown by Katona el al., AO point scanning can effectively record fluorescent signal from up to 1000 points from in vitro preparation or from anesthetized animal. But the maximal scanning rate is limited by the switching time of the AO deflectors. In the ERC grant we present a novel technology, 3D DRIFT AO scanning, which can extend each scanning point to small 3D lines, surfaces, or volume elements for flexible and fast imaging of complex structures simultaneously in multiple locations around our ROIs. Extending the ROIs with maintained good temporal resolution preserved fluorescence information during brain motion and allows off-line motion compensation and elimination of motion artefacts. The scanning abilities were demonstrated by fast 3D recording of over 150 dendritic spines with 3D lines, over 100 somata with squares and cubes, or multiple spiny dendritic segments with surface and volume elements in behaving mice. Also, when measuring from awake, behaving animals relatively big motion artifacts can occur caused by vessel pulsing, respiration or locomotion.
We would like to extend FOV to 5 mm and fast scanning volume up to 5000 Ã— 5000 Ã— 1000 Âµm3 with a preserved subcellular resolution in the center to perform dendritic imaging and to avoid neuropil contamination.