2,599 research outputs found

    In vivo two-photon imaging of the embryonic cortex reveals spontaneous ketamine-sensitive calcium activity

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    Prior to sensory experience spontaneous activity appears to play a fundamental role in the correct formation of prominent functional features of different cortical regions. The use of anaesthesia during pregnancy such as ketamine is largely considered to negatively affect neuronal development by interfering with synaptic transmission. Interestingly, the characteristics of spontaneous activity as well as the acute functional effects of maternal anaesthesia remain largely untested in the embryonic cortex in vivo. In the present work, we performed in vivo imaging of spontaneous calcium activity and cell motility in the marginal zone of the cortex of E14-15 embryos connected to the mother. We made use of a preparation where the blood circulation from the mother through the umbilical cord is preserved and fluctuations in intracellular calcium in the embryonic frontal cortex are acquired using two-photon imaging. We found that spontaneous transients were either sporadic or correlated in clusters of neuronal ensembles at this age. These events were not sensitive to maternal isoflurane anaesthesia but were strongly inhibited by acute in situ or maternal application of low concentration of the anaesthetic ketamine (a non-competitive antagonist of NMDA receptors). Moreover, simultaneous imaging of cell motility revealed a correlated strong sensitivity to ketamine. These results show that anaesthetic compounds can differ significantly in their impact on spontaneous early cortical activity as well as motility of cells in the marginal zone. The effects found in this study may be relevant in the etiology of heightened vulnerability to cerebral dysfunction associated with the use of ketamine during pregnancy.Peer reviewe

    Differential sensitivity of brainstem vs cortical astrocytes to changes in pH reveals functional regional specialization of astroglia

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    Astrocytes might function as brain interoceptors capable of detecting different (chemo)sensory modalities and transmitting sensory information to the relevant neural networks controlling vital functions. For example, astrocytes which reside near the ventral surface of the brainstem (central respiratory chemosensitive area) respond to physiological decreases in pH with vigorous elevations in intracellular Ca(2+) and release of ATP. ATP transmits astroglial excitation to the brainstem respiratory network and contributes to adaptive changes in lung ventilation. Here we show that in terms of pH-sensitivity ventral brainstem astrocytes are clearly distinct from astrocytes residing in the cerebral cortex. We monitored vesicular fusion in cultured rat brainstem astrocytes using total internal reflection fluorescence microscopy and found that approximately 35% of them respond to acidification with an increased rate of exocytosis of ATP-containing vesicular compartments. These fusion events require intracellular Ca(2+) signaling and are independent of autocrine ATP actions. In contrast, the rate of vesicular fusion in cultured cortical astrocytes is not affected by changes in pH. Compared to cortical astrocytes, ventral brainstem astrocytes display higher levels of expression of genes encoding proteins associated with ATP vesicular transport and fusion, including vesicle-associated membrane protein-3 and vesicular nucleotide transporter. These results suggest that astrocytes residing in different parts of the rat brain are functionally specialized. In contrast to cortical astrocytes, astrocytes of the brainstem chemosensitive area(s) possess signaling properties which are functionally relevant โ€“ they are able to sense changes in pH and respond to acidification with enhanced vesicular release of ATP

    Visualization and Correction of Automated Segmentation, Tracking and Lineaging from 5-D Stem Cell Image Sequences

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    Results: We present an application that enables the quantitative analysis of multichannel 5-D (x, y, z, t, channel) and large montage confocal fluorescence microscopy images. The image sequences show stem cells together with blood vessels, enabling quantification of the dynamic behaviors of stem cells in relation to their vascular niche, with applications in developmental and cancer biology. Our application automatically segments, tracks, and lineages the image sequence data and then allows the user to view and edit the results of automated algorithms in a stereoscopic 3-D window while simultaneously viewing the stem cell lineage tree in a 2-D window. Using the GPU to store and render the image sequence data enables a hybrid computational approach. An inference-based approach utilizing user-provided edits to automatically correct related mistakes executes interactively on the system CPU while the GPU handles 3-D visualization tasks. Conclusions: By exploiting commodity computer gaming hardware, we have developed an application that can be run in the laboratory to facilitate rapid iteration through biological experiments. There is a pressing need for visualization and analysis tools for 5-D live cell image data. We combine accurate unsupervised processes with an intuitive visualization of the results. Our validation interface allows for each data set to be corrected to 100% accuracy, ensuring that downstream data analysis is accurate and verifiable. Our tool is the first to combine all of these aspects, leveraging the synergies obtained by utilizing validation information from stereo visualization to improve the low level image processing tasks.Comment: BioVis 2014 conferenc

    A New Method to Address Unmet Needs for Extracting Individual Cell Migration Features from a Large Number of Cells Embedded in 3D Volumes

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    Background: In vitro cell observation has been widely used by biologists and pharmacologists for screening molecule-induced effects on cancer cells. Computer-assisted time-lapse microscopy enables automated live cell imaging in vitro, enabling cell behavior characterization through image analysis, in particular regarding cell migration. In this context, 3D cell assays in transparent matrix gels have been developed to provide more realistic in vitro 3D environments for monitoring cell migration (fundamentally different from cell motility behavior observed in 2D), which is related to the spread of cancer and metastases. Methodology/Principal Findings: In this paper we propose an improved automated tracking method that is designed to robustly and individually follow a large number of unlabeled cells observed under phase-contrast microscopy in 3D gels. The method automatically detects and tracks individual cells across a sequence of acquired volumes, using a template matching filtering method that in turn allows for robust detection and mean-shift tracking. The robustness of the method results from detecting and managing the cases where two cell (mean-shift) trackers converge to the same point. The resulting trajectories quantify cell migration through statistical analysis of 3D trajectory descriptors. We manually validated the method and observed efficient cell detection and a low tracking error rate (6%). We also applied the method in a real biological experiment where the pro-migratory effects of hyaluronic acid (HA) were analyzed on brain cancer cells. Using collagen gels with increased HA proportions, we were able to evidence a dose-response effect on cell migration abilities. Conclusions/Significance: The developed method enables biomedical researchers to automatically and robustly quantify the pro- or anti-migratory effects of different experimental conditions on unlabeled cell cultures in a 3D environment. ยฉ 2011 Adanja et al.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

    Intravital imaging of the kidney

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    Two-photon intravital microscopy is a powerful tool that allows the examination of dynamic cellular processes in the live animal with unprecedented resolution. Indeed, it offers the ability to address unique biological questions that may not be solved by other means. While two-photon intravital microscopy has been successfully applied to study many organs, the kidney presents its own unique challenges that need to be overcome in order to optimize and validate imaging data. For kidney imaging, the complexity of renal architecture and salient autofluorescence merit special considerations as these elements directly impact image acquisition and data interpretation. Here, using illustrative cases, we provide practical guides and discuss issues that may arise during two-photon live imaging of the rodent kidney

    A biophysical evaluation of cell-substrate interactions during spreading, migration and neuron differentiation

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    The development of engineered scaffolds has become a popular current avenue to treat numerous traumas and disease. In order to optimize the efficiency of these treatments, it is necessary to have a more thorough understanding of how cells interact with their substrate and how these interactions directly affect cellular behavior. Cell spreading is a critical component of numerous biological phenomena, including embryonic development, cancer metastasis, immune response, and wound healing. Along with spreading, cell adhesion and migration are all strongly dependent on the interactions between the cell and its substrate. Cell-substrate interactions can affect critical cellular mechanisms including internal cellular signaling, protein synthesis, differentiation, and replication and also influence the magnitude of adherence and motility. In an effort to better understand cell-substrate interactions we characterize the initial stages of cell spreading and blebbing using cell-substrate specific microscopy techniques, and identify the effects of cytoskeletal disruption and membrane modification on surface interactions and spreading. We identify that blebs appear after a sharp change in cellular tension, such as following rapid cell-substrate detachment with trypsin. An increased lag phase of spreading appears with increased blebbing; however, blebbing can be tuned by supplying the cell with more time to perform plasma membrane recycling. We developed software algorithms to detect individual bleb dynamics from TIRF and IRM images, and characterize three types of bleb-adhesion behaviors. Overall, we show that blebs initially create the first adhesion sites for the cell during spreading; however, their continuous protrusion and retraction events contribute to the slow spreading period prior to fast growth. In addition, we identify the elastic modulus of the rat cortex and characterize a polyacrylamide gel system that evaluates the effects of substrate stiffness on cortical outgrowth. Remarkably, we illustrate that cortical neuron differentiation and outgrowth are insensitive to substrate stiffness, and observe only morphological differences between laminin versus PDL-coated substrates. Together, this research identifies cell-specific behaviors critical to spreading and migration. The thorough evaluations of spreading and migration behavior presented here contribute to the understanding of critical cellular phenomena and suggest potential therapeutic targets for treatment of cardiovascular disease and neurological disorders

    ์‚ด์•„์žˆ๋Š” ๋‰ด๋Ÿฐ๊ณผ ๋™๋ฌผ์—์„œ mRNA ๊ด€์ฐฐ์— ๋Œ€ํ•œ ์—ฐ๊ตฌ

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    ํ•™์œ„๋…ผ๋ฌธ(๋ฐ•์‚ฌ) -- ์„œ์šธ๋Œ€ํ•™๊ต๋Œ€ํ•™์› : ์ž์—ฐ๊ณผํ•™๋Œ€ํ•™ ๋ฌผ๋ฆฌยท์ฒœ๋ฌธํ•™๋ถ€(๋ฌผ๋ฆฌํ•™์ „๊ณต), 2021.8. ์ด๋ณ‘ํ›ˆ.mRNA๋Š” ์œ ์ „์ž ๋ฐœํ˜„์˜ ์ฒซ๋ฒˆ์งธ ์‚ฐ๋ฌผ์ด๋ฉด์„œ, ๋ฆฌ๋ณด์†œ๊ณผ ํ•จ๊ป˜ ๋‹จ๋ฐฑ์งˆ์„ ํ•ฉ์„ฑํ•œ๋‹ค. ํŠนํžˆ ๋‰ด๋Ÿฐ์—์„œ, ๋ช‡๋ช‡ RNA๋“ค์€ ์ž๊ทน์— ์˜ํ•ด ๋งŒ๋“ค์–ด์ง€๊ณ , ๋‰ด๋Ÿฐ์˜ ํŠน์ • ๋ถ€๋ถ„์œผ๋กœ ์ˆ˜์†ก๋˜์–ด ๊ตญ์†Œ์ ์œผ๋กœ ๋‹จ๋ฐฑ์งˆ ์–‘์„ ์กฐ์ ˆํ•  ์ˆ˜ ์žˆ๊ฒŒ ํ•œ๋‹ค. ์ตœ๊ทผ mRNA ํ‘œ์ง€ ๊ธฐ์ˆ ์˜ ๋ฐœ์ „์œผ๋กœ ์‚ด์•„์žˆ๋Š” ์„ธํฌ์—์„œ ๋‹จ์ผ mRNA๋ฅผ ๊ด€์ฐฐํ•˜๋Š” ๊ฒƒ์ด ๊ฐ€๋Šฅํ•ด์กŒ๋‹ค. ์ด ์—ฐ๊ตฌ์—์„œ, ์šฐ๋ฆฌ๋Š” RNA ์ด๋ฏธ์ง• ๊ธฐ์ˆ ์„ ์ด์šฉํ•ด, ๊ธฐ์–ต ํ˜•์„ฑ๊ณผ ์ƒ๊ธฐํ•  ๋•Œ ํ™œ์„ฑํ™”๋œ ๋‰ด๋Ÿฐ์˜ ์ง‘ํ•ฉ์„ ์ฐพ๋Š” ๊ฒƒ ๋ฟ ์•„๋‹ˆ๋ผ, ๋‰ด๋Ÿฐ์˜ ์ถ•์‚ญ๋Œ๊ธฐ์—์„œ mRNA๊ฐ€ ์–ด๋–ป๊ฒŒ ์ˆ˜์†ก๋˜๋Š”์ง€๋ฅผ ๊ด€์ฐฐํ–ˆ๋‹ค. ์ด ๋…ผ๋ฌธ์˜ ์ฒซ ๋ถ€๋ถ„์—์„œ ์šฐ๋ฆฌ๋Š” ์‹ ๊ฒฝ ์ž๊ทน์— ๋ฐ˜์‘ํ•ด์„œ ๋งŒ๋“ค์–ด์ง€๋Š” ๊ฒƒ์œผ๋กœ ์•Œ๋ ค์ง„, Arc ์œ ์ „์ž์˜ ์ „์‚ฌ๋ฅผ ๊ด€์ฐฐํ•˜์˜€๋‹ค. ๊ธฐ์–ต์€ engram ํ˜น์€ ๊ธฐ์–ต ํ”์  (memory trace)๋ผ๊ณ  ๋ถˆ๋ฆฌ๋Š” ๋‰ด๋Ÿฐ๋“ค์˜ ์ง‘ํ•ฉ์— ์ €์žฅ๋˜์–ด ์žˆ๋‹ค๊ณ  ์ƒ๊ฐ๋œ๋‹ค. ๊ทธ๋Ÿฌ๋‚˜, ์‹œ๊ฐ„์— ๋”ฐ๋ผ์„œ ์ด๋Ÿฐ ๊ธฐ์–ต ํ”์ ์„ธํฌ๋“ค์˜ ์ง‘ํ•ฉ์ด ์–ด๋–ป๊ฒŒ ๋ณ€ํ•˜๊ณ , ๋ณ€ํ™”ํ•˜๋ฉด์„œ๋„ ์–ด๋–ป๊ฒŒ ์ •๋ณด๋ฅผ ์œ ์ง€ํ•  ์ˆ˜ ์žˆ๋Š”์ง€ ์ž˜ ์•Œ๋ ค์ ธ ์žˆ์ง€ ์•Š๋‹ค. ๋˜ํ•œ, ์‚ด์•„์žˆ๋Š” ๋™๋ฌผ์—์„œ, ๊ธฐ์–ต ํ”์ ์„ธํฌ๋ฅผ ๊ธด ์‹œ๊ฐ„ ๋™์•ˆ ์—ฌ๋Ÿฌ ๋ฒˆ ์ฐพ์•„๋‚ด๋Š” ๊ฒƒ์€ ์–ด๋ ค์šด ์ผ์ด์—ˆ๋‹ค. ์ด ์—ฐ๊ตฌ์—์„œ๋Š” genetically-encoded RNA indicator (GERI) ๊ธฐ์ˆ ์„ ์‚ฌ์šฉํ•ด, ๊ธฐ์–ต ํ”์ ์„ธํฌ์˜ ํ‘œ์‹์œผ๋กœ ๋„๋ฆฌ ์‚ฌ์šฉ๋˜๋Š” Arc mRNA์˜ ์ „์‚ฌ๊ณผ์ •์„ ์‚ด์•„์žˆ๋Š” ์ฅ์—์„œ ๊ด€์ฐฐํ•˜์˜€๋‹ค. GERI๋ฅผ ์ด์šฉํ•จ์œผ๋กœ์จ, ๊ธฐ์กด ๋ฐฉ๋ฒ•๋“ค์˜ ํ•œ๊ณ„์ ์ด์—ˆ๋˜ ์‹œ๊ฐ„ ์ œ์•ฝ ์—†์ด, ์‹ค์‹œ๊ฐ„์œผ๋กœ Arc๋ฅผ ๋ฐœํ˜„ํ•˜๋Š” ๋‰ด๋Ÿฐ๋“ค์„ ์ฐพ์•„๋‚ผ ์ˆ˜ ์žˆ์—ˆ๋‹ค. ์ฅ์—๊ฒŒ ๊ณต๊ฐ„ ๊ณตํฌ ๊ธฐ์–ต์„ ์ฃผ๊ณ  ๋‚˜์„œ ์—ฌ๋Ÿฌ ๋ฒˆ ๊ธฐ์–ต์„ ์ƒ๊ธฐ์‹œํ‚ค๋Š” ํ–‰๋™์‹คํ—˜ ํ›„์— Arc๋ฅผ ๋ฐœํ˜„ํ•˜๋Š” ์„ธํฌ๋ฅผ ์‹๋ณ„ํ–ˆ์„ ๋•Œ, CA1์—์„œ๋Š” Arc๋ฅผ ๋ฐœํ˜„ํ•˜๋Š” ์„ธํฌ๊ฐ€ ์ดํ‹€ ํ›„์—๋Š” ๋” ์ด์ƒ ํ™œ์„ฑํ™”๋˜์ง€ ์•Š์•˜์œผ๋‚˜, RSC์˜ ๊ฒฝ์šฐ 4ํผ์„ผํŠธ์˜ ๋‰ด๋Ÿฐ๋“ค์ด ๊ณ„์†ํ•ด์„œ ํ™œ์„ฑํ™”ํ•˜๋Š” ๊ฒƒ์„ ๊ด€์ฐฐํ–ˆ๋‹ค. ์‹ ๊ฒฝํ™œ๋™๊ณผ ์œ ์ „์ž ๋ฐœํ˜„์„ ๊ฐ™์ด ์กฐ์‚ฌํ•˜๊ธฐ ์œ„ํ•ด, ์ฅ๊ฐ€ ๊ฐ€์ƒ ํ™˜๊ฒฝ์„ ํƒํ—˜ํ•˜๊ณ  ์žˆ์„ ๋•Œ GERI์™€ ์นผ์Š˜ ์ด๋ฏธ์ง•์„ ๋™์‹œ์— ์ง„ํ–‰ํ•˜์˜€๋‹ค. ๊ทธ ๊ฒฐ๊ณผ, ๊ธฐ์–ต์„ ํ˜•์„ฑํ•  ๋•Œ์™€ ์ƒ๊ธฐ์‹œํ‚ฌ ๋•Œ Arc๋ฅผ ๋ฐœํ˜„ํ–ˆ๋˜ ๋‰ด๋Ÿฐ๋“ค์ด ๊ธฐ์–ต์„ ํ‘œ์ƒํ•˜๋Š” ๊ฒƒ์„ ์•Œ์•„๋‚ผ ์ˆ˜ ์žˆ์—ˆ๋‹ค. ์ด์ฒ˜๋Ÿผ GERI ๊ธฐ์ˆ ์„ ์ด์šฉํ•ด ์‚ด์•„์žˆ๋Š” ๋™๋ฌผ์—์„œ ์œ ์ „์ž ๋ฐœํ˜„๋œ ์„ธํฌ๋ฅผ ์ฐพ์•„๋‚ด๋Š” ๋ฐฉ์‹์€ ๋‹ค์–‘ํ•œ ํ•™์Šต ๋ฐ ๊ธฐ์–ต ๊ณผ์ •์—์„œ ๊ธฐ์–ต ํ”์ ์„ธํฌ์˜ dynamics์— ๋Œ€ํ•ด ์•Œ์•„๋‚ผ ์ˆ˜ ์žˆ์„ ๊ฒƒ์œผ๋กœ ๊ธฐ๋Œ€๋œ๋‹ค. ์ด ๋…ผ๋ฌธ์˜ ๋‘๋ฒˆ์งธ ๋ถ€๋ถ„์—์„œ, ์šฐ๋ฆฌ๋Š” ์„ธํฌ ๊ณจ๊ฒฉ์˜ ๊ธฐ๋ณธ ๊ตฌ์„ฑ ๋‹จ์œ„๊ฐ€ ๋˜๋Š” ฮฒ-actin์˜ mRNA๋ฅผ ์ถ•์‚ญ๋Œ๊ธฐ์—์„œ ๊ด€์ฐฐํ•˜์˜€๋‹ค. mRNA์˜ ๊ตญ์†Œํ™” (localization)๋ฅผ ํ†ตํ•œ ๊ตญ์†Œ ๋‹จ๋ฐฑ์งˆ ํ•ฉ์„ฑ์€ ์ถ•์‚ญ๋Œ๊ธฐ (axon)์˜ ์„ฑ์žฅ๊ณผ ์žฌ์ƒ์— ์ค‘์š”ํ•œ ์—ญํ• ์ด ์žˆ๋‹ค๊ณ  ์•Œ๋ ค์ ธ ์žˆ๋‹ค. ํ•˜์ง€๋งŒ, ์•„์ง mRNA์˜ ๊ตญ์†Œํ™”๊ฐ€ ์ถ•์‚ญ๋Œ๊ธฐ์—์„œ ์–ด๋–ป๊ฒŒ ์กฐ์ ˆ๋˜๊ณ  ์žˆ๋Š”์ง€ ์ž˜ ์•Œ๋ ค์ ธ ์žˆ์ง€ ์•Š๋‹ค. ์ด ๋ฌธ์ œ๋ฅผ ํ•ด๊ฒฐํ•˜๊ธฐ ์œ„ํ•ด์„œ, ์šฐ๋ฆฌ๋Š” ๋ชจ๋“  ฮฒ-actin mRNA๊ฐ€ ํ˜•๊ด‘์œผ๋กœ ํ‘œ์ง€๋œ ์œ ์ „์ž ๋ณ€ํ˜• ์ฅ๋ฅผ ์ด์šฉํ•ด, ์‚ด์•„์žˆ๋Š” ์ถ•์‚ญ๋Œ๊ธฐ์—์„œ ฮฒ-actin mRNA๋ฅผ ๊ด€์ฐฐํ•˜์˜€๋‹ค. ์ด ์ฅ์˜ ๋‰ด๋Ÿฐ์„ ์ถ•์‚ญ์„ ๊ตฌ๋ถ„ํ•ด ์ค„ ์ˆ˜ ์žˆ๋Š” ๋ฏธ์„ธ์œ ์ฒด ์žฅ์น˜ (microfluidic device)์— ๋ฐฐ์–‘ํ•œ ๋’ค์—, ฮฒ-actin mRNA๋ฅผ ๊ด€์ฐฐํ•˜๊ณ  ์ถ”์ ์„ ์ง„ํ–‰ํ–ˆ๋‹ค. ์ถ•์‚ญ์€ ์„ธํฌ ๋ชธํ†ต์œผ๋กœ๋ถ€ํ„ฐ ๊ธธ๊ฒŒ ์ž๋ผ๊ธฐ ๋•Œ๋ฌธ์— mRNA๊ฐ€ ๋จผ ๊ฑฐ๋ฆฌ๋ฅผ ์ˆ˜์†ก๋˜์–ด์•ผ ํ•จ์—๋„ ๋ถˆ๊ตฌํ•˜๊ณ , ๋Œ€๋ถ€๋ถ„์˜ mRNA๊ฐ€ ์ˆ˜์ƒ๋Œ๊ธฐ์— ๋น„ํ•ด ๋œ ์›€์ง์ด๊ณ  ์ž‘์€ ์˜์—ญ์—์„œ ์›€์ง์ด๋Š” ๊ฒƒ์„ ๋ณด์•˜๋‹ค. ์šฐ๋ฆฌ๋Š” ฮฒ-actin mRNA๊ฐ€ ์ฃผ๋กœ ์ถ•์‚ญ๋Œ๊ธฐ์˜ ๊ฐ€์ง€๊ฐ€ ๋  ์ˆ˜ ์žˆ๋Š” filopodia ๊ทผ์ฒ˜์™€, ์‹œ๋ƒ…์Šค๊ฐ€ ๋งŒ๋“ค์–ด์ง€๋Š” bouton ๊ทผ์ฒ˜์— ๊ตญ์†Œํ™”๋˜๋Š” ๊ฒƒ์„ ๊ด€์ฐฐํ–ˆ๋‹ค. Filopodia์™€ bouton์ด actin์ด ํ’๋ถ€ํ•œ ๋ถ€๋ถ„์œผ๋กœ ์•Œ๋ ค์ ธ ์žˆ๊ธฐ ๋•Œ๋ฌธ์—, ์šฐ๋ฆฌ๋Š” ์•กํ‹ด ํ•„๋ผ๋ฉ˜ํŠธ์™€ ฮฒ-actin mRNA์˜ ์›€์ง์ž„๊ฐ„์— ์—ฐ๊ด€์„ฑ์„ ์กฐ์‚ฌํ–ˆ๋‹ค. ํฅ๋ฏธ๋กญ๊ฒŒ๋„, ์šฐ๋ฆฌ๋Š” ฮฒ-actin mRNA๊ฐ€ ์•กํ‹ด ํ•„๋ผ๋ฉ˜ํŠธ์™€ ๊ฐ™์ด ๊ตญ์†Œํ™” ๋˜๊ณ , ฮฒ-actin mRNA๊ฐ€ ์•กํ‹ด ํ•„๋ผ๋ฉ˜ํŠธ ์•ˆ์—์„œ sub-diffusiveํ•œ ์›€์ง์ž„์„ ๋ณด์˜€์œผ๋ฉฐ, ๋จผ ๊ฑฐ๋ฆฌ๋ฅผ ์›€์ง์ด๋˜ mRNA๋„ ์•กํ‹ด ํ•„๋ผ๋ฉ˜ํŠธ์— ๊ณ ์ •๋˜๋Š” ๋ชจ์Šต๋„ ํ™•์ธํ•  ์ˆ˜ ์žˆ์—ˆ๋‹ค. ์ถ•์‚ญ์—์„œ ฮฒ-actin mRNA ์›€์ง์ž„์„ ๋ณธ ์ด๋ฒˆ ๊ด€์ฐฐ์€ mRNA ์ˆ˜์†ก ๋ฐ ๊ตญ์†Œํ™”์— ๋Œ€ํ•œ ์ƒ๋ฌผ๋ฌผ๋ฆฌํ•™ ์  ๋ฉ”์ปค๋‹ˆ์ฆ˜์˜ ๊ธฐ๋ฐ˜์ด ๋  ์ˆ˜ ์žˆ์„ ๊ฒƒ์ด๋‹ค.mRNA is the first product of the gene expression and facilitates the protein synthesis. Especially in neurons, some RNAs are transcribed in response to stimuli and transported to the specific region, altering local proteome for neurons to function normally. Recent advances of mRNA labeling techniques allowed us to observe the single mRNAs in live cells. In this thesis, we applied RNA imaging technique not only to identify the neuronal ensemble that activated during memory formation and retrieval, but also to traffic mRNAs transported to the axon. In the first part of the thesis, we observed the transcription site of Arc gene, one of the immediate-early gene, which is rapidly transcribed upon the neural stimuli. Because of the characteristic of expressing in response to stimuli, Arc is widely used as a marker for memory trace cells thought to store memories. However, little is known about the ensemble dynamics of these cells because it has been challenging to observe them repeatedly over long periods of time in vivo. To overcome this limitation, we present a genetically-encoded RNA indicator (GERI) technique for intravital chronic imaging of endogenous Arc mRNA. We used our GERI to identify Arc-positive neurons in real time without the time lag associated with reporter protein expression in conventional approaches. We found that Arc-positive neuronal populations rapidly turned over within two days in CA1, whereas ~4% of neurons in the retrosplenial cortex consistently expressed Arc upon contextual fear conditioning and repeated memory retrievals. Dual imaging of GERI and calcium indicator in CA1 of mice navigating a virtual reality environment revealed that only the overlapping population of neurons expressing Arc during encoding and retrieval exhibited relatively high calcium activity in a context-specific manner. This in vivo RNA imaging approach has potential to unravel the dynamics of engram cells underlying various learning and memory processes. In the second part of this thesis, we imaged ฮฒ-actin mRNAs, which can generate a cytoskeletal protein, ฮฒ-actin, through translation. Local protein synthesis has a critical role in axonal guidance and regeneration. Yet it is not clearly understood how the mRNA localization is regulated in axons. To address these questions, we investigated mRNA motion in live axons using a transgenic mouse that expresses fluorescently labeled endogenous ฮฒ-actin mRNA. By culturing hippocampal neurons in a microfluidic device that allows separation of axons from dendrites, we performed single particle tracking of ฮฒ-actin mRNA selectively in axons. Although axonal mRNAs need to travel a long distance, we observed that most axonal mRNAs show much less directed motion than dendritic mRNAs. We found that ฮฒ-actin mRNAs frequently localize at the neck of filopodia which can grow as axon collateral branches and at varicosities where synapses typically occur. Since both filopodia and varicosities are known as actin-rich areas, we investigated the dynamics of actin filaments and ฮฒ-actin mRNAs simultaneously by using high-speed dual-color imaging. We found that axonal mRNAs colocalize with actin filaments and show sub-diffusive motion within the actin-rich regions. The novel findings on the dynamics of ฮฒ-actin mRNA will shed important light on the biophysical mechanisms of mRNA transport and localization in axons.1. INTRODUCTION, 1 1.1. Neuronal ensemble, 1 1.2. Immediate-early Gene (IEG), 3 1.3. Methods for IEG-positive neurons, 3 1.4. Two-photon microscope, 5 1.5. References, 7 2. IMAGING ARC mRNA TRANSCRIPTION SITES IN LIVE MICE, 9 2.1. Introduction, 9 2.2. Materials and Methods, 10 2.3. Results and Discussion, 18 2.4. References, 26 3. NEURONS EXPRESSING ARC mRNA DURING REPEATED MEMORY RETRIEVALS, 28 3.1. Introduction, 28 3.2. Results and Discussion, 28 3.3. References, 35 4. NEURAL ACTIVITIES OF ARC+ NEURONS, 36 4.1. Introduction, 36 4.2. Materials and Methods, 37 4.3. Results and Discussion, 38 4.4. References, 52 5. AXONAL mRNA DYNAMICS IN LIVE NEURONS, 54 5.1. Introduction, 54 5.2. Materials and Methods, 55 5.3. Results and Discussion, 59 5.4. References, 70 6. CONCLUSION AND OUTLOOK, 72 ABSTRACT IN KOREAN (๊ตญ๋ฌธ์ดˆ๋ก), 76๋ฐ•

    FGF Signaling Mediates Regeneration of the Differentiating Cerebellum through Repatterning of the Anterior Hindbrain and Reinitiation of Neuronal Migration

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    To address the regenerative capability of the differentiating hindbrain, we ablated the cerebellum in wild-type and transgenic zebrafish embryos. These larvae showed no obvious locomotive malfunction several days after the ablation. Expression analysis and in vivo time-lapse recording in GFP (green fluorescent protein)-transgenic embryos indicate that cerebellar neuronal cells can regenerate from the remaining anterior hindbrain. The onset of regeneration is accompanied by repatterning within the anterior hindbrain. Inhibition of FGF signaling immediately after cerebellar ablation results in the lack of regenerating cerebellar neuronal cells and the absence of cerebellar structures several days later. Moreover, impaired FGF signaling inhibits the repatterning of the anterior hindbrain and the reexpression of rhombic lip marker genes soon after cerebellar ablation. This demonstrates that the hindbrain is highly plastic in recapitulating early embryonic differentiation mechanisms during regeneration. Moreover, the regenerating system offers a means to uncouple cerebellar differentiation from complex morphogenetic tissue rearrangements
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