SignificanceLuminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research.AimWe investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs.ApproachWe quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons.ResultsLuciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical.ConclusionsWhole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.
SignificanceBioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO).AimTo further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation.ApproachWe developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1).ResultsA new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard) and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior.ConclusionsWe report a robust new option for achieving multiple modes of control in a single actuator and a promising engineering strategy for continued improvement of BL-OG.
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: Magnetic resonance imaging, 3D printing, Brain, Xenon, Neuroimaging, Signal to noise ratio, Additive manufacturing, 3D acquisition, Tumors, Copper
Three-dimensional (3D) printing has significantly impacted the quality, efficiency, and reproducibility of preclinical magnetic resonance imaging. It has vastly expanded the ability to produce MR-compatible parts that readily permit customization of animal handling, achieve consistent positioning of anatomy and RF coils promptly, and accelerate throughput. It permits the rapid and cost-effective creation of parts customized to a specific imaging study, animal species, animal weight, or even one unique animal, not routinely used in preclinical research. We illustrate the power of this technology by describing five preclinical studies and specific solutions enabled by different 3D printing processes and materials. We describe fixtures, assemblies, and devices that were created to ensure the safety of anesthetized lemurs during an MR examination of their brain or to facilitate localized, contrast-enhanced measurements of white blood cell concentration in a mouse model of pancreatitis. We illustrate expansive use of 3D printing to build a customized birdcage coil and components of a ventilator to enable imaging of pulmonary gas exchange in rats using hyperpolarized Xe129. Finally, we present applications of 3D printing to create high-quality, dual RF coils to accelerate brain connectivity mapping in mouse brain specimens and to increase the throughput of brain tumor examinations in a mouse model of pituitary adenoma.
KEYWORDS: Bioluminescence, Cameras, Live cell imaging, Luminescence, In vivo imaging, Proteins, Microscopes, Signal detection, Electron multiplying charge coupled devices, Objectives
The use of bioluminescent reporters in neuroscience research continues to grow at a rapid pace as their applications and unique advantages over conventional fluorescent reporters become more appreciated. Here, we describe practical methods and principles for detecting and imaging bioluminescence from live cells and animals. We systematically tested various components of our conventional fluorescence microscope to optimize it for long-term bioluminescence imaging. High-resolution bioluminescence images from live neurons were obtained with our microscope setup, which could be continuously captured for several hours with no signs of phototoxicity. Bioluminescence from the mouse brain was also imaged noninvasively through the intact skull with a conventional luminescence imager. These methods demonstrate how bioluminescence can be routinely detected and measured from live cells and animals in a cost-effective way with common reagents and equipment.
The manipulation of genetically targeted neurons with light (optogenetics) continues to provide unprecedented avenues
into studying the function of the mammalian brain. However, potential translation into the clinical arena faces a number
of significant hurdles, foremost among them the need for insertion of optical fibers into the brain to deliver light to
opsins expressed on neuronal membranes. In order to overcome these hardware-related problems, we have developed an
alternative strategy for delivering light to opsins which does not involve fiber implants. Rather, the light is produced by a
protein, luciferase, which oxidizes intravenously applied substrate, thereby emitting bioluminescence. In proof-ofprinciple
studies employing a fusion protein of a light-generating luciferase to a light-sensing opsin (luminopsin), we
showed that light emitted by Gaussia luciferase is indeed able to activate channelrhodopsin, allowing modulation of
neuronal activity when expressed in cultured neurons. Here we assessed applicability of the concept in vivo in mice
expressing luminopsins from viral vectors and from genetically engineered transgenes. The experiments demonstrate
that intravenously applied substrate reaches neurons in the brain, causing the luciferase to produce bioluminescence
which can be imaged in vivo, and that activation of channelrhodopsin by bioluminescence is sufficient to affect behavior.
Further developments of such technology based on combining optogenetics with bioluminescence - i.e. combining lightsensing molecules with biologically produced light through luciferases - should bring optogenetics closer to clinical
applications.
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