Precise multimodal optical control of neural ensemble activity.

Understanding brain function requires technologies that can control the activity of large populations of neurons with high fidelity in space and time. We developed a multiphoton holographic approach to activate or suppress the activity of ensembles of cortical neurons with cellular resolution and sub-millisecond precision. Since existing opsins were inadequate, we engineered new soma-targeted (ST) optogenetic tools, ST-ChroME and IRES-ST-eGtACR1, optimized for multiphoton activation and suppression. Employing a three-dimensional all-optical read-write interface, we demonstrate the ability to simultaneously photostimulate up to 50 neurons distributed in three dimensions in a 550 × 550 × 100-µm3 volume of brain tissue. This approach allows the synthesis and editing of complex neural activity patterns needed to gain insight into the principles of neural codes

Two-photon bidirectional control and imaging of neuronal excitability with cellular resolution in vivo

Two-photon bidirectional control and imaging of neuronal excitability with cellular resolution in viv

Multiphoton minimal inertia scanning for fast acquisition of neural activity signals

Objective: Multi-photon laser scanning microscopy provides a powerful tool for monitoring the spatiotemporal dynamics of neural circuit activity. It is, however, intrinsically a point scanning technique. Standard raster scanning enables imaging at subcellular resolution; however, acquisition rates are limited by the size of the field of view to be scanned. Recently developed scanning strategies such as Travelling Salesman Scanning (TSS) have been developed to maximize cellular sampling rate by scanning only select regions in the field of view corresponding to locations of interest such as somata. However, such strategies are not optimized for the mechanical properties of galvanometric scanners. We thus aimed to develop a new scanning algorithm which produces minimal inertia trajectories, and compare its performance with existing scanning algorithms. Approach: We describe here the Adaptive Spiral Scanning (SSA) algorithm, which fits a set of near-circular trajectories to the cellular distribution to avoid inertial drifts of galvanometer position. We compare its performance to raster scanning and TSS in terms of cellular sampling frequency and signal-to-noise ratio (SNR). Main Results: Using surrogate neuron spatial position data, we show that SSA acquisition rates are an order of magnitude higher than those for raster scanning and generally exceed those achieved by TSS for neural densities comparable with those found in the cortex. We show that this result also holds true for in vitro hippocampal mouse brain slices bath loaded with the synthetic calcium dye Cal-520 AM. The ability of TSS to "park" the laser on each neuron along the scanning trajectory, however, enables higher SNR than SSA when all targets are precisely scanned. Raster scanning has the highest SNR but at a substantial cost in number of cells scanned. To understand the impact of sampling rate and SNR on functional calcium imaging, we used the Crame ́r-Rao Bound on evoked calcium traces recorded simultaneously with electrophysiology traces to calculate the lower bound estimate of the spike timing occurrence. Significance: The results show that TSS and SSA achieve comparable accuracy in spike time estimates compared to raster scanning, despite lower SNR. SSA is an easily implementable way for standard multi-photon laser scanning systems to gain temporal precision in the detection of action potentials while scanning hundreds of active cells

Closed-loop experiments and brain machine interfaces with multiphoton microscopy

In the field of neuroscience, the importance of constructing closed-loop experimental systems has increased in conjunction with technological advances in measuring and controlling neural activity in live animals. This paper provides an overview of recent technological advances in the field, focusing on closed-loop experimental systems where multiphoton microscopy (the only method capable of recording and controlling targeted population activity of neurons at a single-cell resolution in vivo) works through real-time feedback. Specifically, we present some examples of brain machine interfaces (BMIs) using in vivo two-photon calcium imaging and discuss applications of two-photon optogenetic stimulation and adaptive optics to real-time BMIs. We also consider conditions for realizing future optical BMIs at the synaptic level, and their possible roles in understanding the computational principles of the brain

Two-photon minimal inertia scanning patterns for fast acquisition of calcium dynamics

Development of optical technologies aiming to reverse engineer neural circuits has been flourishing over the past two decades. Multi-Photon Laser Scanning Microscopy (MPLMS) together with the development of fast kinetic fluorescent calcium dyes has revolutionised the world of modern neuroscience. This technology enables mesoscale functional imaging in deep scattering brain tissues of large two (2D) and three dimensional (3D) neural networks. With single cell sensitivity in vitro as well as in vivo, it is one of the main contenders for deciphering higher brain functions. My approach in this thesis is to develop and test new scanning techniques for fast functional calcium imaging aiming to enhance the temporal precision of the acquisition. To avoid the slow and sequential "point" raster scanning nature of these Galvanometric Scanners (GSs) based microscopes, I developed new 2D and 3D scanning algorithms. These algorithms were developed in MATLAB with a simulation platform that models the main mechanical elements of the MPLSM. Both my 2D Adaptive Spiral Scanning (SSA) algorithm and my 3D Orbital Scanning Trajectory (OST) algorithm were developed to minimize the inertial slowdowns of the GSs and Electrical Tunable Lens (ETL) and therefore increase the temporal resolution of the acquisition. In 2D, I tested the SSA algorithm on in vitro hippocampal brain slices loaded with the synthetic calcium dye Cal520. To assess the performance of the scanning technique, I used the Cramer-Rao Bound (CRB) as a metric for signal quality. The CRB estimates the time of occurrence of an Action Potential (AP) from the calcium imaging data, taking into account the sampling frequency and the SNR of the acquisition. In this thesis, I show that the use of scanning strategies enables sampling rates one order of magnitude higher than traditional frame scanning in functional calcium imaging. I also show that frame scanning needs considerably higher SNR values than scanning strategies to reach the same temporal precision. In 3D, I implemented the scanning algorithms into the software and hardware of the MPLSM and recorded the trajectory of the focal point with a high-speed camera as a proof of principle. More analyses regarding the precision of the paths needs to be carried out in 3D for functional calcium imaging in vitro or in vivo. These software-based scanning strategies are attractive as they are inexpensive, easily transferable from one setup to another and enable fast functional calcium imaging with standard commercial MPSLMs. Finally, through this implementation of scanning strategies, I recorded multiple data sets of spontaneous and evoked activity in populations of Dentate Granular Cells (DGCs). This lead to the new beginning of a larger in vitro investigation at the microcircuit level on the functionality of the DG.Open Acces

The Enlightened Brain: Novel Imaging Methods Focus on Epileptic Networks at Multiple Scales

Epilepsy research is rapidly adopting novel fluorescence optical imaging methods to tackle unresolved questions on the cellular and circuit mechanisms of seizure generation and evolution. State of the art two-photon microscopy and wide-field fluorescence imaging can record the activity in epileptic networks at multiple scales, from neuronal microcircuits to brain-wide networks. These approaches exploit transgenic and viral technologies to target genetically encoded calcium and voltage sensitive indicators to subclasses of neurons, and achieve genetic specificity, spatial resolution and scalability that can complement electrophysiological recordings from awake animal models of epilepsy. Two-photon microscopy is well suited to study single neuron dynamics during interictal and ictal events, and highlight the differences between the activity of excitatory and inhibitory neuronal classes in the focus and propagation zone. In contrast, wide-field fluorescence imaging provides mesoscopic recordings from the entire cortical surface, necessary to investigate seizure propagation pathways, and how the unfolding of epileptic events depends on the topology of brain-wide functional connectivity. Answering these questions will inform pre-clinical studies attempting to suppress seizures with gene therapy, optogenetic or chemogenetic strategies. Dissecting which network nodes outside the seizure onset zone are important for seizure generation, propagation and termination can be used to optimize current and future evaluation methods to identify an optimal surgical strategy

Extended field-of-view ultrathin microendoscopes with built-in aberration correction for high-resolution functional imaging with minimal invasiveness

TB

Optogenetic Brain Interfaces

The brain is a large network of interconnected neurons where each cell functions as a nonlinear processing element. Unraveling the mysteries of information processing in the complex networks of the brain requires versatile neurostimulation and imaging techniques. Optogenetics is a new stimulation method which allows the activity of neurons to be modulated by light. For this purpose, the cell-types of interest are genetically targeted to produce light-sensitive proteins. Once these proteins are expressed, neural activity can be controlled by exposing the cells to light of appropriate wavelengths. Optogenetics provides a unique combination of features, including multimodal control over neural function and genetic targeting of specific cell-types. Together, these versatile features combine to a powerful experimental approach, suitable for the study of the circuitry of psychiatric and neurological disorders. The advent of optogenetics was followed by extensive research aimed to produce new lines of light-sensitive proteins and to develop new technologies: for example, to control the distribution of light inside the brain tissue or to combine optogenetics with other modalities including electrophysiology, electrocorticography, nonlinear microscopy, and functional magnetic resonance imaging. In this paper, the authors review some of the recent advances in the field of optogenetics and related technologies and provide their vision for the future of the field.United States. Defense Advanced Research Projects Agency (Space and Naval Warfare Systems Center, Pacific Grant/Contract No. N66001-12-C-4025)University of Wisconsin--Madison (Research growth initiative; grant 101X254)University of Wisconsin--Madison (Research growth initiative; grant 101X172)University of Wisconsin--Madison (Research growth initiative; grant 101X213)National Science Foundation (U.S.) (MRSEC DMR-0819762)National Science Foundation (U.S.) (NSF CAREER CBET-1253890)National Institutes of Health (U.S.) (NIH/NIBIB R00 Award (4R00EB008738)National Institutes of Health (U.S.) (NIH Director’s New Innovator award (1-DP2-OD002989))Okawa Foundation (Research Grant Award)National Institutes of Health (U.S.) (NIH Director’s New Innovator Award (1DP2OD007265))National Science Foundation (U.S.) (NSF CAREER Award (1056008)Alfred P. Sloan Foundation (Fellowship)Human Frontier Science Program (Strasbourg, France) (Grant No. 1351/12)Israeli Centers of Research Excellence (I-CORE grant, program 51/11)MINERVA Foundation (Germany

Simultaneous two-photon imaging and photo-stimulation with structured light illumination.

Holographic microscopy is increasingly recognized as a promising tool for the study of the central nervous system. Here we present a "holographic module", a simple optical path that can be combined with commercial scanheads for simultaneous imaging and uncaging with structured two-photon light. The present microscope is coupled to two independently tunable lasers and has two principal configurations: holographic imaging combined with galvo-steered uncaging and holographic uncaging combined with conventional scanning imaging. We applied this flexible system for simultaneous two-photon imaging and photostimulation of neuronal cells with complex light patterns, opening new perspectives for the study of brain function in situ and in vivo