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Fast confocal fluorescence imaging in freely behaving mice.

ABSTRACT: Fluorescence imaging in the brain of freely behaving mice is challenging due to severe miniaturization constraints. In particular, the ability to image a large field of view at high temporal resolution and with efficient out-of-focus background rejection still raises technical difficulties. Here, we present a novel fiberscope system that provides fast (up to 200 Hz) background-free fluorescence imaging in freely behaving mice over a field of view of diameter 230 μm. The fiberscope is composed of a custom-made multipoint-scanning confocal microscope coupled to the animal with an image guide and a micro-objective. By simultaneously registering a multipoint-scanning confocal image and a conventional widefield image, we subtracted the residual out-of-focus background and provided a background-free confocal image. Illumination and detection pinholes were created using a digital micromirror device, providing high adaptability to the sample structure and imaging conditions. Using this novel imaging tool, we demonstrated fast fluorescence imaging of microvasculature up to 120 μm deep in the mouse cortex, with an out-of-focus background reduced by two orders of magnitude compared with widefield microscopy. Taking advantage of the high acquisition rate (200 Hz), we measured red blood cell velocity in the cortical microvasculature and showed an increase in awake, unrestrained mice compared with anaesthetized animals.

SUBMITTER: Dussaux C

PROVIDER: S-EPMC6214968 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Fast confocal fluorescence imaging in freely behaving mice.

Dussaux Clara C Szabo Vivien V Chastagnier Yan Y Fodor Jozsua J Léger Jean-François JF Bourdieu Laurent L Perroy Julie J Ventalon Cathie C

Scientific reports 20181102 1

Fluorescence imaging in the brain of freely behaving mice is challenging due to severe miniaturization constraints. In particular, the ability to image a large field of view at high temporal resolution and with efficient out-of-focus background rejection still raises technical difficulties. Here, we present a novel fiberscope system that provides fast (up to 200 Hz) background-free fluorescence imaging in freely behaving mice over a field of view of diameter 230 μm. The fiberscope is composed of ...[more]

PMID: 30389966

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Project description:Prolonged exposure to abnormally high calcium concentrations is thought to be a core mechanism underlying hippocampal damage in epileptic patients; however, no prior study has characterized calcium activity during seizures in the live, intact hippocampus. We have directly investigated this possibility by combining whole-brain electroencephalographic (EEG) measurements with microendoscopic calcium imaging of pyramidal cells in the CA1 hippocampal region of freely behaving mice treated with the pro-convulsant kainic acid (KA). We observed that KA administration led to systematic patterns of epileptiform calcium activity: a series of large-scale, intensifying flashes of increased calcium fluorescence concurrent with a cluster of low-amplitude EEG waveforms. This was accompanied by a steady increase in cellular calcium levels (>5 fold increase relative to the baseline), followed by an intense spreading calcium wave characterized by a 218% increase in global mean intensity of calcium fluorescence (n = 8, range [114-349%], p < 10(-4); t-test). The wave had no consistent EEG phenotype and occurred before the onset of motor convulsions. Similar changes in calcium activity were also observed in animals treated with 2 different proconvulsant agents, N-methyl-D-aspartate (NMDA) and pentylenetetrazol (PTZ), suggesting the measured changes in calcium dynamics are a signature of seizure activity rather than a KA-specific pathology. Additionally, despite reducing the behavioral severity of KA-induced seizures, the anticonvulsant drug valproate (VA, 300 mg/kg) did not modify the observed abnormalities in calcium dynamics. These results confirm the presence of pathological calcium activity preceding convulsive motor seizures and support calcium as a candidate signaling molecule in a pathway connecting seizures to subsequent cellular damage. Integrating in vivo calcium imaging with traditional assessment of seizures could potentially increase translatability of pharmacological intervention, leading to novel drug screening paradigms and therapeutics designed to target and abolish abnormal patterns of both electrical and calcium excitation.

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Fast confocal fluorescence imaging in freely behaving mice.

Publications

Fast confocal fluorescence imaging in freely behaving mice.

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