A double microscope/endoscope for the holographic manipulation of deep visual circuits – 2MEnHoloMD

Vision is the primary sense humans use to evaluate their surroundings and guide their behaviour. All forms of visual impairments have therefore a major negative impact on the life of affected subjects. Indeed, loss of eyesight is perceived by many people as one of the worst illness that could happen in their lives. According to recent studies, more than 10 million people are affected by moderate to severe vision impairments in Europe, with an associated total cost of at least € 50 billions per year. Studying how visual information is processed to give rise to the experience of sight is therefore of fundamental importance, with a potential high social and economic benefit.

In the mouse visual pathways, which can serve as a less complex model to study vision, the visual information is processed by different brain areas including the dorsal lateral geniculate nucleus (dLGN) of the thalamus and the primary visual cortex (V1). In particular, the dual connection between dLGN and deep layers of V1, such as layer 4 (L4) and layer 6 (L6) plays a fundamental role in correclty elaborating the visual information. However, if a lot is known about the anatomic organization and macroscopic wiring at the basis of this connection, it is in the fine details of the communication among individual neurons that perception is generated. A more profound understanding of visual circuits connecting dLGN and V1, including the possibility of finding cures to visual impairments, necessarily requires a precise study, with single cell resolution and large volumes capability, of both brain areas simultaneously, which is today experimentally out of reach.

Optical techniques, which in the last ten years have become one of the most prominent tools for the study of the brain, could provide such level of precision but suffer from two main limitations. (i) They can only see through few hundreds µm of biological tissue, whereas the mouse dLGN is located at the depth of ~ 3 mm, L4 at around 400 µm and L6 at > 600 µm. (ii) Studying large areas, as one would need to simultaneously investigate V1 and dLGN, separated by few mm in the mouse brain, while maintaining single cell spatial resolution is currently out of reach.

In this project we will overcome these limitations and develop a novel and ambitious optical system capable to read and drive activity in hundreds of neurons simultaneously in the same structure (within the dLGN or the same layer of V1) and across different but interconnected brain regions (dLGN to L4; across different layers of V1; L6 to dLGN) with single neuron resolution. To this end we will extend optical techniques that the Scientific Coordinator and the Emiliani’s group already fully master, such as computer-generated holography and wavefront shaping, to a completely new realm, i.e. that of micro-endoscopy and deep tissue microscopy to enable the precise study of deep brain structures.

The project is organized in 3 phases to maximize its impact: (1) the development of a minimally invasive optical micro-endoscope to image and manipulate specific neurons in the mouse dLGN; (2) the parallel development of an optimized two-photon microscope to image and manipulate neurons in deep cortical layers, such as L4 and L6; (3) a final phase in which we will couple the two previous techniques to gain simultaneous access to dLGN and V1.

The proposed research will contribute to shine new light on how vision and brain circuits in general function, by giving precise access to previously inaccessible regions. With the combined setup of phase (3) we will have, for the first time, the possibility of studying, with single neuron precision, large brain circuits that develop across different brain areas. The current project in therefore expected to have a great positive impact in neuroscience, neuro-photonics and microscopy in general.