The striatum is a critical brain area in the control and initiation of movement. Recently, the activity in striatal spiny projection neurons (SPNs) has also been shown to be different for different gross body movements, suggesting that activity in striatum is movement-specific and can bias which movements animals will perform. However, these larger actions were performed with different body parts, and may reflect somatotopy more than action specificity, and therefore it is unclear how fine the granularity of movement-specificity is. To probe this, we have developed an isometric force task that requires no overt movement, and imaged and analyzed the activity in striatum during learning and execution. From the optically recorded neural activity combined with simultaneous behavior, we trained classifiers that could predict action from activity across days, revealing stable ensembles encoded specific actions. Finally, using targeted two-photon stimulation, we perturbed small sub-populations of these ensembles in closed-loop during behavior, seemingly leading to small, but perceptible, changes in action.
This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions.
Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics’ agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, in this article we review an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion article, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed and provide an outlook for the future directions.
KEYWORDS: 3D acquisition, Optogenetics, Holography, In vivo imaging, Spatial light modulators, 3D image processing, Optical manipulation, Neurons, Fiber lasers, Laser systems engineering
Optical manipulation of in vivo neural circuits with cellular resolution could be important for understanding cortical function. Despite recent progress, simultaneous optogenetic activation with cellular precision has either been limited to 2D planes, or a very small numbers of neurons over a limited volume. Here we demonstrate a novel paradigm for simultaneous 3D activation using a low repetition rate pulse-amplified fiber laser system and a spatial light modulator (SLM) to project 3D holographic excitation patterns on the cortex of mice in vivo for targeted volumetric 3D photoactivation. This method is compatible with two-photon imaging, and enables the simultaneous activation of multiple cells in 3D, using red-shifted opsins, such as C1V1 or ReaChR, while simultaneously imaging GFP-based sensors such as GCaMP6. This all-optical imaging and 3D manipulation approach achieves simultaneous reading and writing of cortical activity, and should be a powerful tool for the study of neuronal circuits.
Imaging the neuronal activity throughout the brain with high temporal and spatial resolution is an important step in understanding how the brain works. Two-photon laser scanning microscopy with fluorescent calcium indicators has enabled this type of experiments in vivo. Most of these microscopes acquire images serially, with a single laser beam, limiting the overall imaging speed. To overcome this limit, multiple beamlets can be used to image in parallel multiple regions. Here, we demonstrate a novel scheme of a two-photon laser-scanning microscope that can simultaneously record neuronal activity at multiple planes of the sample with a single photomultiplier tube. A spatial light modulator is used to generate the designated multiple beamlets, and a constrained non-negative matrix factorization algorithm is used to demix the signals from multiple scanned planes. We simultaneously record neuronal activity of multiple layers of a mouse cortex at 10 fps in vivo. This novel imaging scheme provides a powerful tool for mapping the brain activity.
Microscopy incorporating spatial light modulators (SLMs) enables three dimensional (3D) excitation and monitoring of the activity of neuronal ensembles, enabling studies of neuronal circuit activity both in vitro and in vivo. In this paper we present a portable (22 cm x 42.5 cm x 30 cm), SLM-based epi-fluorescence upright microscope (“Pocketscope”) that enables 3D calcium imaging and photoactivation of neurons in brain slices. Here we describe the implementation of the instrument; quantify the volume over which neural activity can be excited; and demonstrate the use of the system for mapping neural circuits in brain slices.
We have developed a spatial light modulator (SLM) based microscope that uses diffraction to shape the incoming two-photon
laser source to any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different
regions of a sample with three-dimensional precision at high frame rates. Additionally, we have combined this
microscope with a new class of two photon active neuromodulators with Ruthenium BiPyridine (RuBi) based cages that
offer great flexibility for neuronal control.
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