Monitoring norepinephrine release in vivo using next-generation GRABNE sensors.

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter. The first-generation NE sensor makes in vivo, real-time, cell-type-specific and region-specific NE detection possible, but its low NE sensitivity limits its utility. Here, we developed the second-generation GPCR-activation-based NE sensors (GRABNE2m and GRABNE2h) with a superior response and high sensitivity and selectivity to NE both in vitro and in vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell-type-specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRABNE sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo, providing new insights into the physiological and pathophysiological roles of NE.

Improved green and red GRAB sensors for monitoring spatiotemporal serotonin release in vivo.

The serotonergic system plays important roles in both physiological and pathological processes, and is a therapeutic target for many psychiatric disorders. Although several genetically encoded GFP-based serotonin (5-HT) sensors were recently developed, their sensitivities and spectral profiles are relatively limited. To overcome these limitations, we optimized green fluorescent G-protein-coupled receptor (GPCR)-activation-based 5-HT (GRAB5-HT) sensors and developed a red fluorescent GRAB5-HT sensor. These sensors exhibit excellent cell surface trafficking and high specificity, sensitivity and spatiotemporal resolution, making them suitable for monitoring 5-HT dynamics in vivo. Besides recording subcortical 5-HT release in freely moving mice, we observed both uniform and gradient 5-HT release in the mouse dorsal cortex with mesoscopic imaging. Finally, we performed dual-color imaging and observed seizure-induced waves of 5-HT release throughout the cortex following calcium and endocannabinoid waves. In summary, these 5-HT sensors can offer valuable insights regarding the serotonergic system in both health and disease.

Cerebral cortex and hippocampus neural interaction during vagus nerve stimulation under in vivo large-scale imaging.

Objective: The purpose of this study was to study mechanisms of VNS modulation from a single neuron perspective utilizing a practical observation platform with single neuron resolution and widefield, real-time imaging coupled with an animal model simultaneously exposing the cerebral cortex and the hippocampus. Methods: We utilized the observation platform characterized of widefield of view, real-time imaging, and high spatiotemporal resolution to obtain the neuronal activities in the cerebral cortex and the hippocampus during VNS in awake states and under anesthesia. Results: Some neurons in the hippocampus were tightly related to VNS modulation, and varied types of neurons showed distinct responses to VNS modulation. Conclusion: We utilized such an observation platform coupled with a novel animal model to obtain more information on neuron activities in the cerebral cortex and the hippocampus, providing an effective method to further study the mechanisms of therapeutic effects modulated by VNS.

Simultaneous Observation of Mouse Cortical and Hippocampal Neural Dynamics under Anesthesia through a Cranial Microprism Window.

The fluorescence microscope has been widely used to explore dynamic processes in vivo in mouse brains, with advantages of a large field-of-view and high spatiotemporal resolution. However, owing to background light and tissue scattering, the single-photon wide-field microscope fails to record dynamic neural activities in the deep brain. To achieve simultaneous imaging of deep-brain regions and the superficial cortex, we combined the extended-field-of-view microscopy previously proposed with a novel prism-based cranial window to provide a longitudinal view. As well as a right-angle microprism for imaging above 1 mm, we also designed a new rectangular-trapezoidal microprism cranial window to extend the depth of observation to 1.5 mm and to reduce brain injury. We validated our method with structural imaging of microglia cells in the superficial cortex and deep-brain regions. We also recorded neuronal activity from the mouse brains in awake and anesthesitized states. The results highlight the great potential of our methods for simultaneous dynamic imaging in the superficial and deep layers of mouse brains.

Real-time brain-wide multi-planar microscopy for simultaneous cortex and hippocampus imaging at the cellular resolution in mice.

Interactions between the cerebral cortex and the deep cerebellar nuclei play important roles in cognitive processes. However, conventional microscopes fail to dynamically record cellular structures in distinct brain regions and at different depths, which requires high resolution, large field of view (FOV), and depth of field (DOF). Here we propose a single-photon excited fluorescence microscopy technique that performs simultaneous cortex and hippocampus imaging, enabled by a customized microscope and a chronic optical window. After we implant a glass microwindow above the hippocampus, the surface of the hippocampus is shifted to the superficial plane. We demonstrate that the proposed technique is able to image cellular structures and blood vessel dynamics in the cortex and the hippocampus in in vivo experiments, and is compatible with various mesoscopic systems.

Photobleaching Imprinting Enhanced Background Rejection in Line-Scanning Temporal Focusing Microscopy.

Compared with two-photon point-scanning microscopy, two-photon temporal focusing microscopy (2pTFM) provides a parallel high-speed imaging strategy with optical sectioning capability. Owing to out-of-focus fluorescence induced by scattering, 2pTFM suffers deteriorated signal-to-background ratio (SBR) for deep imaging in turbid tissue, Here, we utilized the photobleaching property of fluorophore to eliminate out-of-focus fluorescence. According to different decay rates in different focal depth, we extract the in-focus signals out of backgrounds through time-lapse images. We analyzed the theoretical foundations of photobleaching imprinting of the line-scanning temporal focusing microscopy, simulated implementation for background rejection, and demonstrated the contrast enhancement in MCF-10A human mammary epithelial cells and cleared Thy1-YFP mouse brains. More than 50% of total background light rejection was achieved, providing higher SBR images of the MCF-10A samples and mouse brains. The photobleaching imprinting method can be easily adapted to other fluorescence dyes or proteins, which may have application in studies involving relatively large and nontransparent organisms.