No abstract

Cell transplantation therapy provides a regenerative strategy for neural repair. We tested the hypothesis that selective excitation of transplanted induced pluripotent stem cell-derived neural progenitor cells (iPS-NPCs) could recapitulate an activity-enriched microenvironment that confers regenerative benefits for the treatment of stroke. Mouse iPS-NPCs were transduced with a novel optochemogenetics fusion protein, luminopsin 3 (LMO3), which consisted of a bioluminescent luciferase, Gaussia luciferase, and an opsin, Volvox Channelrhodopsin 1. These LMO3-iPS-NPCs can be activated by either photostimulation using light or by the luciferase substrate coelenterazine (CTZ). In vitro stimulations of LMO3-iPS-NPCs increased expression of synapsin-1, postsynaptic density 95, brain derived neurotrophic factor (BDNF), and stromal cell-derived factor 1 and promoted neurite outgrowth. After transplantation into the ischemic cortex of mice, LMO3-iPS-NPCs differentiated into mature neurons. Synapse formation between implanted and host neurons was identified using immunogold electron microscopy and patch-clamp recordings. Stimulation of transplanted cells with daily intranasal administration of CTZ enhanced axonal myelination, synaptic transmission, improved thalamocortical connectivity, and functional recovery. Patch-clamp and multielectrode array recordings in brain slices showed that CTZ or light stimulation facilitated synaptic transmission and induced neuroplasticity mimicking the LTP of EPSPs. Stroke mice received the combined LMO3-iPS-NPC/CTZ treatment, but not cell or CTZ alone, showed enhanced neural network connections in the peri-infarct region, promoted optimal functional recoveries after stroke in male and female, young and aged mice. Thus, excitation of transplanted cells via the noninvasive optochemogenetics treatment provides a novel integrative cell therapy with comprehensive regenerative benefits after stroke.

Optogenetic techniques provide an unprecedented ability to precisely manipulate neural activity in the context of complex neural circuitry. Although the toolbox of optogenetic probes continues to expand at a rapid pace with more efficient and responsive reagents, hardware-based light delivery is still a major hurdle that limits its practical use in vivo. We have bypassed the challenges of external light delivery by directly coupling a bioluminescent light source (a genetically encoded luciferase) to an inhibitory opsin, which we term an inhibitory luminopsin (iLMO). iLMO was shown to suppress action potential firing and synchronous bursting activity in vitro in response to both external light and luciferase substrate. iLMO was further shown to suppress single-unit firing rate and local field potentials in the hippocampus of anesthetized rats. Finally, expression of iLMO was scaled up to multiple structures of the basal ganglia to modulate rotational behavior of freely moving animals in a hardware-independent fashion. This novel class of optogenetic probes demonstrates how non-invasive inhibition of neural activity can be achieved, which adds to the versatility, scalability, and practicality of optogenetic applications in freely behaving animals.

Abstract. 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 Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Background: Several point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use. Objectives: The aim of this study was to assess test performance of the Accula SARS-CoV-2 test. Study design: The performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa coefficient. Results: Overall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%). Conclusion: Compared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.

Optogenetics provides an array of elements for specific biophysical control, while designer chemogenetic receptors provide a minimally invasive method to control circuits in vivo by peripheral injection. We developed a strategy for selective regulation of activity in specific cells that integrates opto- and chemogenetic approaches, and thus allows manipulation of neuronal activity over a range of spatial and temporal scales in the same experimental animal. Light-sensing molecules (opsins) are activated by biologically produced light through luciferases upon peripheral injection of a small molecule substrate. Such luminescent opsins, luminopsins, allow conventional fiber optic use of optogenetic sensors, while at the same time providing chemogenetic access to the same sensors. We describe applications of this approach in cultured neurons in vitro, in brain slices ex vivo, and in awake and anesthetized animals in vivo.

25 Background: Several point-of-care (POC) molecular tests have received emergency use 26

Although optogenetic techniques have proven to be invaluable for manipulating and understanding complex neural dynamics over the past decade, they still face practical and translational challenges in targeting networks involving multiple, large, or difficult-to-illuminate areas of the brain. We utilized inhibitory luminopsins to simultaneously inhibit the dentate gyrus and anterior nucleus of the thalamus of the rat brain in a hardware-independent and cell-type specific manner. This approach was more effective at suppressing behavioral seizures than inhibition of the individual structures in a rat model of epilepsy. In addition to elucidating mechanisms of seizure suppression never directly demonstrated before, this work also illustrates how precise multi-focal control of pathological circuits can be advantageous for the treatment and understanding of disorders involving broad neural circuits such as epilepsy.