<meta charset="utf8"></head><body>小鼠感觉皮层发育中的神经元活动映射

<ol><li>Rao, M. S., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elucidating+mechanisms+of+neuronal+circuit+formation+in+layer+4+of+the+somatosensory+cortex+via+intravital+imaging%5BTitle%5D)+AND+%22Neuroscience+Research%22%5BJournal%5D)">Elucidating mechanisms of neuronal circuit formation in layer 4 of the somatosensory cortex via intravital imaging.</a> Neuroscience Research. 167, 47-53 (2021).</li>
<li>Iwasato, T., Erzurumlu, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+tactile+sensory+circuits+in+the+CNS%5BTitle%5D)+AND+%22Current+Opinion+in+Neurobiology%22%5BJournal%5D)">Development of tactile sensory circuits in the CNS.</a> Current Opinion in Neurobiology. 53, 66-75 (2018).</li>
<li>Martini, F. J., Guillamón-Vivancos, T., Moreno-Juan, V., Valdeolmillos, M., López-Bendito, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+activity+in+developing+thalamic+and+cortical+sensory+networks%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Spontaneous activity in developing thalamic and cortical sensory networks.</a> Neuron. 109 (16), 2519-2534 (2021).</li>
<li>Ackman, J. B., Burbridge, T. J., Crair, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+waves+coordinate+patterned+activity+throughout+the+developing+visual+system%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Retinal waves coordinate patterned activity throughout the developing visual system.</a> Nature. 490 (7419), 219-225 (2012).</li>
<li>Tritsch, N. X., Yi, E., Gale, J. E., Glowatzki, E., Bergles, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+origin+of+spontaneous+activity+in+the+developing+auditory+system%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The origin of spontaneous activity in the developing auditory system.</a> Nature. 450 (7166), 50-55 (2007).</li>
<li>Babola, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Homeostatic+control+of+spontaneous+activity+in+the+developing+auditory+system%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Homeostatic control of spontaneous activity in the developing auditory system.</a> Neuron. 99 (3), 511-524.e5 (2018).</li>
<li>Mizuno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patchwork-type+spontaneous+activity+in+neonatal+barrel+cortex+layer+4+transmitted+via+thalamocortical+projections%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Patchwork-type spontaneous activity in neonatal barrel cortex layer 4 transmitted via thalamocortical projections.</a> Cell Reports. 22 (1), 123-135 (2018).</li>
<li>Mizuno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NMDAR-regulated+dynamics+of+layer+4+neuronal+dendrites+during+thalamocortical+reorganization+in+neonates%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">NMDAR-regulated dynamics of layer 4 neuronal dendrites during thalamocortical reorganization in neonates.</a> Neuron. 82 (2), 365-379 (2014).</li>
<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation%3A+visualization+of+neuronal+migration+in+the+developing+cortex%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li>
<li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294 (5544), 1071-1074 (2001).</li>
<li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240 (1), 237-246 (2001).</li>
<li>Holtmaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term%2C+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4 (8), 1128-1144 (2009).</li>
<li>Mizuno, H., Nakazawa, S., Iwasato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+two-photon+imaging+of+cortical+neurons+in+neonatal+mice%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">In vivo two-photon imaging of cortical neurons in neonatal mice.</a> Journal of Visualized Experiments. 140, e58340(2018).</li>
<li>Egashira, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+two-photon+calcium+imaging+of+cortical+neurons+in+neonatal+mice%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">In vivo two-photon calcium imaging of cortical neurons in neonatal mice.</a> STAR Protocols. 4 (2), 102245(2023).</li>
<li>Cantu, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EZcalcium%3A+Open-source+toolbox+for+analysis+of+calcium+imaging+data%5BTitle%5D)+AND+%22Frontiers+in+Neural+Circuits%22%5BJournal%5D)">EZcalcium: Open-source toolbox for analysis of calcium imaging data.</a> Frontiers in Neural Circuits. 14, 25(2020).</li>
<li>Maruoka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lattice+system+of+functionally+distinct+cell+types+in+the+neocortex%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Lattice system of functionally distinct cell types in the neocortex.</a> Science. 358 (6363), 610-615 (2017).</li>
<li>Antón-Bolaños, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prenatal+activity+from+thalamic+neurons+governs+the+emergence+of+functional+cortical+maps+in+mice%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Prenatal activity from thalamic neurons governs the emergence of functional cortical maps in mice.</a> Science. 364 (6444), 987-990 (2019).</li>
<li>Guillamón-Vivancos, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Input-dependent+segregation+of+visual+and+somatosensory+circuits+in+the+mouse+superior+colliculus%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Input-dependent segregation of visual and somatosensory circuits in the mouse superior colliculus.</a> Science. 377 (6608), 845-850 (2022).</li>
<li>Cardin, J. A., Crair, M. C., Higley, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesoscopic+imaging%3A+Shining+a+wide+light+on+large-scale+neural+dynamics%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Mesoscopic imaging: Shining a wide light on large-scale neural dynamics.</a> Neuron. 108 (1), 33-43 (2020).</li>
<li>Chen, J. L., Voigt, F. F., Javadzadeh, M., Krueppel, R., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-range+population+dynamics+of+anatomically+defined+neocortical+networks%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Long-range population dynamics of anatomically defined neocortical networks.</a> eLife. 5, e14679(2016).</li>
<li>Ota, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((cell-resolution%2C+contiguous-wide+two-photon+imaging+to+reveal+functional+network+architectures+across+multi-modal+cortical+areas%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas.</a> Neuron. 109 (11), 1810-1824 (2021).</li>
<li>Zariwala, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Cre-dependent+GCaMP3+reporter+mouse+for+neuronal+imaging+in+vivo%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.</a> The Journal of Neuroscience. 32 (9), 3131-3141 (2012).</li>
<li>Murakami, T., Matsui, T., Uemura, M., Ohki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modular+strategy+for+development+of+the+hierarchical+visual+network+in+mice%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Modular strategy for development of the hierarchical visual network in mice.</a> Nature. 608 (7923), 578-585 (2022).</li>
<li>Pnevmatikakis, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+denoising%2C+deconvolution%2C+and+demixing+of+Calcium+imaging+data%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Simultaneous denoising, deconvolution, and demixing of Calcium imaging data.</a> Neuron. 89 (2), 285-299 (2016).</li>
<li>Shemesh, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precision+calcium+imaging+of+dense+neural+populations+via+a+cell-body-targeted+calcium+indicator%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Precision calcium imaging of dense neural populations via a cell-body-targeted calcium indicator.</a> Neuron. 107 (3), 470-486 (2020).</li>
<li>Giovannucci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CaImAn+an+open+source+tool+for+scalable+calcium+imaging+data+analysis%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">CaImAn an open source tool for scalable calcium imaging data analysis.</a> Elife. 8, e38173(2019).</li>
<li>Pachitariu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suite2p%3A+beyond+10%2C000+neurons+with+standard+two-photon+microscopy%5BTitle%5D)+AND+%22BioRxiv%22%5BJournal%5D)">Suite2p: beyond 10,000 neurons with standard two-photon microscopy.</a> BioRxiv. , (2017).</li>
<li>Sitdikova, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isoflurane+suppresses+early+cortical+activity%5BTitle%5D)+AND+%22Annals+of+Clinical+and+Translational+Neurology%22%5BJournal%5D)">Isoflurane suppresses early cortical activity.</a> Annals of Clinical and Translational Neurology. 1 (1), 15-26 (2014).</li>
<li>Marques-Smith, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Transient+translaminar+GABAergic+interneuron+circuit+connects+thalamocortical+recipient+layers+in+neonatal+somatosensory+cortex%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A Transient translaminar GABAergic interneuron circuit connects thalamocortical recipient layers in neonatal somatosensory cortex.</a> Neuron. 89 (3), 536-549 (2016).</li>
<li>Tuncdemir, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+somatostatin+interneuron+connectivity+mediates+the+maturation+of+deep+layer+cortical+circuits%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Early somatostatin interneuron connectivity mediates the maturation of deep layer cortical circuits.</a> Neuron. 89 (3), 521-535 (2016).</li>
<li>Nakazawa, S., Yoshimura, Y., Takagi, M., Mizuno, H., Iwasato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+phase+transitions+in+spatial+organization+of+spontaneous+activity+in+postnatal+barrel+cortex+layer+4%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Developmental phase transitions in spatial organization of spontaneous activity in postnatal barrel cortex layer 4.</a> The Journal of Neuroscience. 40 (40), 7637-7650 (2020).</li>
<li>Yu, Y. -C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preferential+electrical+coupling+regulates+neocortical+lineage-dependent+microcircuit+assembly%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly.</a> Nature. 486 (7401), 113-117 (2012).</li>
<li>Siegel, F., Heimel, J. A., Peters, J., Lohmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+and+central+inputs+shape+network+dynamics+in+the+developing+visual+cortex+in+vivo%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Peripheral and central inputs shape network dynamics in the developing visual cortex in vivo.</a> Current Biology. 22 (3), 253-258 (2012).</li>
<li>Nakagawa, N., Hosoya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slow+dynamics+in+microcolumnar+gap+junction+network+of+developing+neocortical+pyramidal+neurons%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Slow dynamics in microcolumnar gap junction network of developing neocortical pyramidal neurons.</a> Neuroscience. 406, 554-554 (2019).</li>
<li>Valiullina, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+changes+in+electrophysiological+properties+and+a+transition+from+electrical+to+chemical+coupling+between+excitatory+layer+4+neurons+in+the+rat+barrel+cortex%5BTitle%5D)+AND+%22Frontiers+in+Neural+Circuits%22%5BJournal%5D)">Developmental changes in electrophysiological properties and a transition from electrical to chemical coupling between excitatory layer 4 neurons in the rat barrel cortex.</a> Frontiers in Neural Circuits. 10, 1(2016).</li>
<li>Avitan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+and+evoked+activity+patterns+diverge+over+development%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Spontaneous and evoked activity patterns diverge over development.</a> Elife. 10, e61942(2021).</li>
<li>Mölter, J., Avitan, L., Goodhill, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detecting+neural+assemblies+in+calcium+imaging+data%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">Detecting neural assemblies in calcium imaging data.</a> BMC Biology. 16 (1), 143(2018).</li>
<li>Nakazawa, S., Mizuno, H., Iwasato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+dynamics+of+cortical+neuron+dendritic+trees+revealed+by+long-term+in+vivo+imaging+in+neonates%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Differential dynamics of cortical neuron dendritic trees revealed by long-term in vivo imaging in neonates.</a> Nature Communications. 9 (1), 3106(2018).</li>
</ol>

<table><tbody><tr><td>20&amp;times; objective lens (water immersion)</td><td></td><td></td><td></td></tr><tr><td>250 mL Vacuum Filter/Storage Bottle System</td><td>Corning</td><td>431096</td><td></td></tr><tr><td>4%-paraformaldehyde phosphate buffer solution (4% PFA)</td><td>Nacalai</td><td>09154-85</td><td></td></tr><tr><td>Acrylic resin (UNIFAST II)</td><td>GC</td><td>N/A</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9793</td><td></td></tr><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Nacalai</td><td>06731-05</td><td></td></tr><tr><td>Electroporator</td><td>BEX</td><td>GEB14</td><td></td></tr><tr><td>Eye drop (Scopisol)</td><td>Senju Pharmaceutical</td><td>N/A</td><td></td></tr><tr><td>Fluorescence stereo microscope</td><td>Leica</td><td>M165FC</td><td></td></tr><tr><td>Glucose</td><td>Nacalai</td><td>16806-25</td><td></td></tr><tr><td>Heating pad</td><td>Muromachi Kikai</td><td>FHC-HPS</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Isoflurane</td><td>Pfizer</td><td>N/A</td><td></td></tr><tr><td>KCl</td><td>Nacalai</td><td>28514-75</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>131-00405</td><td></td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>PC-100</td><td></td></tr><tr><td>Multiphoton laser</td><td>Spectra-Physics</td><td>Mai Tai eHP DeepSee</td><td></td></tr><tr><td>Multiphoton microscope</td><td>Zeiss</td><td>LSM 7MP</td><td></td></tr><tr><td>NaCl</td><td>Nacalai</td><td>31320-05</td><td></td></tr><tr><td>Non-woven fabric (Kimwipe)</td><td>Kimberly Clark</td><td>S-200</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Nacalai</td><td>27575-31</td><td></td></tr><tr><td>Plasmid: CAG-loxP-STOP-loxP-GCaMP6s-ires-tTA-WPRE</td><td>Addgene</td><td>pK175</td><td></td></tr><tr><td>Plasmid: TRE-nCre</td><td>Addgene</td><td>pK031</td><td></td></tr><tr><td>Precision calibrated micropipets</td><td>Drummond</td><td>2-000-050</td><td></td></tr><tr><td>Razor blade</td><td>Feather</td><td>FA-10</td><td></td></tr><tr><td>Rimadyl (50 mg/mL Carprofen)</td><td>Zoetis JP</td><td>N/A</td><td></td></tr><tr><td>Round cover glass, 3-mm-diameter&amp;nbsp;</td><td>Matsunami</td><td>CS01078</td><td></td></tr><tr><td>Saline</td><td>Otsuka</td><td>035175315</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Nacalai</td><td>26427-72</td><td></td></tr><tr><td>Stage for imaging living pup (two single-axis translation stage for XY positioning, two-axis goniometer, base plate, adjustable pillar for z positioning)</td><td>ThorLabs</td><td>LT1/M, GN2/M, BM2060/M, MLP01/M</td><td></td></tr><tr><td>TCA-RFP mouse</td><td>N/A</td><td>N/A</td><td>Mizuno et al., 2018a</td></tr><tr><td>Tissue adhesive (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Titanium bar</td><td>Endo Scientific Instrument</td><td>N/A</td><td>Custom made (Mizuno et al., 2018b)</td></tr><tr><td>Titanium bar fixing plate</td><td></td><td>N/A</td><td>Custom made (Mizuno et al., 2018b)</td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Tweezers with platinum plate electrode, 5 mm diameter</td><td>BEX</td><td>CUY650P5</td><td></td></tr><tr><td>Wild-type ICR mouse</td><td>Nihon SLC</td><td>Slc:ICR</td><td></td></tr></tbody></table>

in vivo visualization of spontaneous activity in neonatal mouse sensory cortex at a single-neuron resolution

Read this Scientific Article Protocol about Author Spotlight: Deciphering Neural Circuit Formation from Two-Photon Microscopy and Single Neuron Imaging at JoVE.com

<meta charset="utf8"></head><body>作者聚焦:从双光子显微镜和单神经元成像破译神经回路形成

在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

in-vivo-visualization-spontaneous-activity-neonatal-mouse-sensory

Research

JoVE Journal

Neuroscience

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

772 次观看.  Kagoshima University. 我们研究的目的是研究发育中的大脑皮层中神经回路的形成。我们的目标是了解神经元活动在此过程中的作用。近年来，双光子显微镜使观察活体动物神经元回路形成的过程成为可能。我们开发了一种对新生儿大脑皮层中的单个神经元进行成像的方法。通过同时观察活动模式及其形态的变化，我们将能够阐明哪些活动模式与神经元回路的形成有...

视频: 作者聚焦:从双光子显微镜和单神经元成像破译神经回路形成

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

多层皮质Ca<sup&gt; 2+</sup&gt;使用棱镜探针和小型荧光显微镜成像自由移动的小鼠

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

科研

行为学

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

神经科学

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

植入颅窗以在清醒小鼠 中重复体内 成像

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化

副本

摘要

探索更多视频

多层皮质Ca

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

双光子

同时双光子

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

体内新生小鼠皮层神经元的双光子成像

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

植入颅窗以在清醒小鼠中重复体内成像

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化

副本

摘要

探索更多视频

多层皮质Ca

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

双光子

同时双光子

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

体内新生小鼠皮层神经元的双光子成像

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

植入颅窗以在清醒小鼠 中重复体内 成像

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

植入颅窗以在清醒小鼠中重复体内成像