We thank Yumiko Iwamoto for supporting the production of the video. We are also grateful to Akihiro Isomura for discussion and supports of image analysis, Hitoshi Miyachi for technical supports for generation of transgenic animals, Yuji Shinjo (Olympus Medical Science), Masatoshi Egawa (Olympus Medical Science), Takuya Ishizu (Olympus Medical Science) and Ouin Kunitaki (Andor Japan) for the technical support and discussions of the bioluminescence imaging system. This work was supported by Core Research for Evolutional Science and Technology (JPMJCR12W2) (R.K.), Grant-in-Aid for Scientific Research on Innovative Areas (MEXT 24116705 for H.S. and MEXT 16H06480 for R.K.), Grant-in-Aid for Scientific Research (C) (JSPS 18K06254) (H.S.), Takeda Foundation (R.K. and H.S.), and Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

All the procedure including animal subjects have been approved by Institutional Animal Care and Use Committee at the institute for Frontier Life and Medical Sciences, Kyoto University.
1. Bioluminescence reporters
NOTE: The luciferase reporter is suitable for measuring the rapid dynamics of promoter activity by fusing the degradation signal. Moreover, the luciferase fusion reporter enables monitoring of the protein dynamics in the single cell. Both types of reporters are available for mono-layer culture (dissociation culture) and tissue culture (slice culture) experiment.
<ol>
	<li>Reporter for monitoring Dll1 promoter activity17 
	NOTE: To monitor the rapid changes in gene expression, the rapid-response and unstable reporter is essential. Firefly luciferase (Fluc) shows rapid maturation compared with fluorescein reporters. Because ubiquitin fused luciferase reporter (Ub-Fluc) shows rapid degradation and rapid-turnover, it is very useful to monitor the dynamic gene expression in living cells20.
	<ol>
		<li>Use Dll1 promoter-driven destabilized luciferase reporter, ubiquitinated luciferase (pDll1-Ub-Fluc, Figure 1A upper panel), to monitor rapid changes in Dll1 expression at a transcriptional level17.</li>
		<li>Generate a transgenic mouse line carrying pDll1-Ub-Fluc for the stable expression of this reporter.</li>
	</ol>
	</li>
	<li>Reporter for monitoring Dll1 protein dynamics17.
	<ol>
		<li>For generation of knock-in mice, insert the luciferase cDNA into the 3&#180; termini of the Dll1 coding region so that Dll1-luciferase fusion protein is expressed (Dll1-Fluc reporter, Figure 1A, lower panel)17.</li>
		<li>Monitor the expression dynamics of Dll1 at protein levels by measuring the luciferase activity.</li>
		<li>For monitoring Dll1 protein dynamics at tissue levels, introduce the Dll1-Fluc reporter into NPCs in the embryonic telencephalon by in utero electroporation.</li>
	</ol>
	</li>
</ol>
2. Bioluminescence imaging system
<ol>
	<li>Construct a bioluminescence live-imaging system using an inverted microscope installed with a highly sensitivity, water-cooled CCD camera. In order to reduce the noise and obtain high sensitivity, cool the camera with chilled water provided by the water circulator at an optimized temperature of -90 &#176;C.</li>
	<li>For the live cell/tissue imaging, install the incubation system, controlling the temperature and mixed gas, to the microscope stage.</li>
	<li>For imaging the expression of destabilized luciferase reporter, use high numerical angle (NA: 1.30) 40x oil-immersion objective lens. The working distance of this lens is 0.2 mm, it is available for not only mono-layer cell culture but also slice culture.</li>
	<li>For dual monitoring of luciferase and fluorescein reporter expression, install the LED illumination device to the light path of the microscope.</li>
	<li>Control the time-lapse imaging with a software associated with the microscope (e.g., Multidimensional acquisition program of MetaMorph software).</li>
	<li>Prepare the entire system in a dark room, because the signal of the destabilized luciferase reporter is very faint, and to avoid the extraneous light prevent the acquisition.</li>
</ol>
3. Neural Stem/Progenitor Cell (NPC) dissociation cultures
<ol>
	<li>Preparation of culture media, dishes, and reagents for NPC dissociation culture
	<ol>
		<li>Prepare N2/B27 media for culturing neural stem/progenitor cells with a final concentration of 1x N2, 1x B27, 1 mM N-acetylcysteine, 10 ng/mL bFGF and 50 U/mL Penicillin/Streptomycin in DMEM/F12 media.</li>
		<li>Prepare papain solution for dissociation containing 7 U/mL papain, 0.006% DNase and 1 mM N-acetylcysteine in EBSS media.</li>
		<li>Prepare 100 mM of luciferin sodium solution for luminescence imaging. Dilute 100 mM luciferin solution in N2/B27 media to a final concentration of 1 mM. 
		NOTE: Luciferin shows auto-fluorescein itself. In the case of luciferase-fluorescence dual imaging, especially EGFP, lowering the concentration of luciferin to 0.5 mM is better.</li>
		<li>Coat a 35-mm glass bottom dishes (glass diameter 27-mm) with 40 &#956;g/mL of poly-L-lysine (PLL) solution for 1 h at room temperature. After incubation, wash the plates 3x with PBS and let it dry.</li>
	</ol>
	</li>
	<li>Dissection of embryos and dissociation of NPCs
	<ol>
		<li>After euthanizing a pregnant pDll1-Ub-luc transgenic mouse (embryonic day 12.5 (E12.5)) by CO2 asphyxiation and cervical dislocation, cut through the abdomen and take out the uterus. 
		NOTE: Researchers must follow the regulations and guidelines of animal research committee of their institution. If anesthesia or pain relief drugs are used, use them properly.</li>
		<li>Transfer the uterus in a 10 cm Petri dish containing 25 mL of ice-cold PBS. Take out the embryos from the uterus in ice-cold PBS, using micro scissors and fine forceps.</li>
		<li>Cut off the head of each embryo using scissors and transfer it to 10 cm Petri dishes containing ice-cold DMEM/F12. Remove the epidermis and cartilage surrounding the brain and transfer the brain to 35 mm Petri dish containing ice-cold 3 mL of N2/B27 media.</li>
		<li>Cut off the right and left telencephalon from diencephalon (Figure 1Ba-f, C,D). Remove the meninges covering the surface of telencephalon using fine forceps (Figure 1Bi). Using fine forceps again, take out the dorso-lateral part of cortex from the telencephalon (Figure 1Bg,h,j-l E).</li>
		<li>Transfer the tissues to new 1.5 mL tubes using a P1000 pipette and remove any extra medium with a P200 pipette. Add 0.1 mL of papain solution per brain tissue (a pair of the cortex). After 15 min of incubation at 24 &#176;C, gently pipette the samples 10 times with P1000 pipette. Incubate the samples again for 15 min at 24 &#176;C.</li>
		<li>Gently pipette the samples 10 times with P1000 pipette. Centrifuge the samples for 3 min at 400 x g and room temperature (RT). Discard the supernatant.</li>
		<li>Add 1 mL of DMEM/F12 media to the pellet. Gently pipette 10 times with P1000 pipette. Centrifuge the samples for 3 min at 400 x g and RT. Discard the supernatant. Repeat this at least 2 more times.</li>
		<li>To the pellet, add 0.5 mL of N2/B27 media containing 1 mM luciferin and mix well. Seed the cells (1 x 106 cells) to a PLL-coated glass bottom dish. Culture the cells in the CO2 incubator for 1 h. Once the cells adhere to the dish, add 2 mL of N2/B27 media, containing 1 mM luciferin.</li>
	</ol>
	</li>
	<li>Visualization of luciferase reporter expression in NPC dissociation culture
	<ol>
		<li>Start up the luminescence live-imaging system before performing the dissection. For NPC culture, set the temperature of the stage incubator at 37 &#176;C and set the setting of the gas mixture 20% O2, 5% CO2.</li>
		<li>Put the immersion oil to the objective lens. Place the sample dish on the microscope stage. View the field manually and choose the best position and the focus of the cells of interest. Click live to acquire the test image.</li>
		<li>Run the time-lapse acquisition by 2-dimensional (luminescence and bright field) acquisitions for 24 h with Multi-Dimensional Acquisition program. Choose Multi-Dimensional Acquisition program and set the acquisition setting as follows (step 3.3.6).</li>
		<li>For luminescence image acquisition, use the following camera settings: low-transfer rate (50 kHz), 2 x 2/4 x 4 binning, 10 min/5 min exposure time. For bright-field image acquisition, use the following settings: intermediate-transfer rate (1 MHz), 1 x 1 binning and 100 ms exposure time.</li>
	</ol>
	</li>
</ol>
4. In utero electroporation
NOTE: This is performed for the introduction of Dll1-Fluc reporter into the neural progenitor cells.
<ol>
	<li>Preparation of tools and reagents for electroporation
	<ol>
		<li>Prepare micro capillaries for injection of DNA into the ventricle of the embryonic telencephalon. Stretch a grass capillary with heat and cut and polish the tip of it with the polishing machine.</li>
		<li>Prepare a mixture of DNA with dye (e.g., Trypan blue). The concentration of each DNA is 1 &#956;g/&#956;L in PBS and add the dye to a final concentration of 10%. 
		NOTE: Use the mixture of DNA for visualization of Dll1 protein dynamics as follows: Dll1-Fluc (Dll1 protein reporter, Figure 2E upper) and pEF-EGFP (EF promoter driven EGFP expression vector, Figure 2E lower), monitoring the location and morphology of transfected cells.</li>
		<li>Prepare two types of anesthetics: 3.88 mg/mL of pentobarbital and 0.12% of xylazine, in sterile 1x PBS.</li>
		<li>Use the sterile instruments and surgical gloves during operation.</li>
	</ol>
	</li>
	<li>In utero electroporation
	<ol>
		<li>Anesthetize a time pregnant ICR mouse (E12.5) by intraperitoneal administration of anesthetics with 500 &#956;L of 3.88 mg/mL pentobarbital and 500 &#956;L of 0.12% xylazine. 
		NOTE: Researchers must follow the regulation of animal experiments. If one needs to use anesthesia or pain relief drugs, use them properly.</li>
		<li>Clip the hair and disinfect the skin with surgical scrub.</li>
		<li>Check for the lack of toe pinch response. Once pedal reflex is not observed, make a 2-3 cm cut through the abdomen along the midline. Put gauzes around the dissected part and wet the gauzes with warm PBS not to dry up the incision. Take out the right uterine horn gently with ring-shaped forceps and count the number of embryos.</li>
		<li>Inject 1-2 &#956;L of the mixed DNA into the ventricle of the telencephalon of embryos gently with micro capillary. 
		NOTE: When the injection is successful, one can see the blue color of the dye over the surface of the uterus.</li>
		<li>Wet the uterus and the electrode before providing the voltage pulses, and then hold the head of an embryo gently. Set the positively charged electrode to the side of the hemisphere, in which the DNA was injected. Ensure that the condition of the pulse of electroporation is 30-50 V for 50 ms, the interval of the pulses is 1 s (50 ms charge and 950 ms non-charge). Provide 5 pulses to one embryo and check that bubbles are generated from the negatively charged electrode. 
		NOTE: Make sure the direction of the electrode: DNA is incorporated into cells on the side of positive electrode. During this procedure, do not move the position of the electrode.</li>
		<li>Repeat the steps 4.2.3-4.2.4 for other embryos and return the uterine horn to the abdominal cavity. Carry out the same procedure to the embryos in the left uterine horn.</li>
		<li>After putting back the left side, suture the incision by a silk suture (4-0) with a needle (17 mm). Before closing the incision completely, put warm PBS into the abdominal cavity 
		NOTE: The interval between sutures is around 2 mm.</li>
		<li>After finishing the surgery, place the mouse on a heating pad for post anesthesia recovery. House the mouse individually. 
		NOTE: Researchers must follow their local instiutional regulation of animal experiments. If one needs to use pain relief drugs, use them properly.</li>
	</ol>
	</li>
</ol>
5. Preparation of slice cultures of the developing cortex and visualization of luciferase reporter expression in the cortical slices
<ol>
	<li>Preparation of culture media and reagents for slice cultures of the cortex
	<ol>
		<li>Prepare enriched media for culturing cortical slices containing 5% fetal bovine serum and 5% horse serum in N2/B27 media.</li>
		<li>Bubble 100% O2 gas through DMEM/F12 media for dissection and making cortical slices. 
		NOTE: To use oxygen gas safely, store the O2 cylinder in the cylinder cabinet and monitor the oxygen concentration in the air by an oxygen concentration meter.</li>
		<li>Dilute 100 mM luciferin sodium solution in enriched media to a final concentration of 1 mM. 
		NOTE: Luciferin shows auto-fluorescein itself. In the case of luciferase-fluorescence dual imaging, especially EGFP, lower concentration of luciferin (0.5 mM) is better.</li>
		<li>Set the multi-gas incubator at 37 &#176;C by 40% O2 and 5% CO2.</li>
	</ol>
	</li>
	<li>Dissection of embryos and examination of the expression of fluorescent reporter
	<ol>
		<li>After euthanizing the pregnant mouse to which the reporters (E13.5) are introduced by in utero electroporation (step 4.2), cut through the abdomen and take out the uterus. 
		NOTE: Researchers must follow the regulation of animal experiments. If using anesthesia or pain relief drugs, use them properly.</li>
		<li>Transfer the uterus to a 10 cm Petri dish containing PBS. Take out the embryos from uterus in PBS, using micro scissors and fine forceps. Cut off the head of each embryo and transfer to a 10 cm Petri dish containing DMEM/F12 media bubbled with 100% O2 gas. Remove epidermis and cartilage surrounding the brain in DMEM/F12 media.</li>
		<li>Put the brain on the lid of the Petri dish with ring-shaped forceps and set the lid on the fluorescence stereoscopic microscope stage. Check the region of the cortex expressing fluorescent protein under the excitation light (Figure 2A,B).</li>
		<li>Transfer the brain to a silicone rubber cutting board filled with 30 mL of DMEM/F12 media bubbled with 100% O2 gas. Remove meninges covering the surface of the telencephalon by fine forceps.</li>
		<li>Cut the border between medial and lateral part of the dorsal telencephalon and separate into two hemispheres using micro surgical knife or fine forceps. Using a micro surgical knife, cut the cortex like stripes and make cortical slices (Figure 2C,D). Pipette slices with medium using pipette and transfer them to enriched media in a 35 mm dish.</li>
		<li>Put the slices on the culture inserts in the glass bottom dishes with enriched media. Correct the direction of slices using fine forceps. Set up the cut surface of the slices to the surface of culture insert. Remove the extra media by a pipette. Incubate the slices in multi-gas incubator set by 40% O2 and 5% CO2 at 37 &#176;C for 30 min.</li>
		<li>Add 300 &#956;L of enriched media containing 1 mM luciferin to the outside of culture insert in the glass bottom dish.</li>
	</ol>
	</li>
	<li>Visualization of luciferase reporter expression in slice cultures
	<ol>
		<li>Start the luminescence live-imaging system before starting the dissection. Use the following condition of cortical slice cultures: 40% O2, 5% CO2 and 37 &#176;C.</li>
		<li>Put the immersion oil to 40x objective lens. Put the sample dish to the stage of microscope. Acquire the test image of fluorescent and set the position and the focus plane to the region of interest under the illumination of excitation light.</li>
		<li>Run the time-lapse acquisition by 3-dimentional (luminescence, fluorescence and bright field) acquisitions for 24 h (Figure 2F-H). For luminescence image acquisition, use the following camera settings: low-transfer rate (50 kHz), 2x2/4x4 binning, 10 min/5 min exposure time. For the fluorescence and bright field image acquisition, use the following settings: intermediate-transfer rate (1 MHz), 1x1 binning and 100 ms exposure time.</li>
	</ol>
	</li>
</ol>
6. Image processing and analysis
<ol>
	<li>Connect each image and make the stack images. Click Import Image Sequence from the File menu (File | Import | Image Sequence) and choose the folder, where the acquired images are saved. Put some words contained in file name to the column of File name contains.</li>
	<li>To remove the noise of cosmic ray on the bioluminescence images, apply the SpikeNoise Filter plug-in of ImageJ/Fiji. Open the stack images and click SpikeNoise Filter.</li>
	<li>Apply Savitzky Golay Temporal Filter plug-in to get clear dynamics of reporter expression. Open the stack images and click Savitzky Golay Temporal Filter.</li>
	<li>To measure the intensity of reporter expression, apply Z Axis Profile Plus plug-in to each single cell. Select the cells and set ROIs, region of interest and click Z Axis Profile Plus.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Gene+Expression+in+a+Single+Cell.">Stochastic Gene Expression in a Single Cell.</a> Science. 297, 1183-1186 (2002).</li><li>Nelson, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillations+in+NF-kappaB+signaling+control+the+dynamics+of+gene+expression.">Oscillations in NF-kappaB signaling control the dynamics of gene expression.</a> Science. 306, 704-708 (2004).</li><li>Purvis, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+dynamics+control+cell+fate.">p53 dynamics control cell fate.</a> Science. 336, 1440-1444 (2012).</li><li>Hansen, A. S., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter+decoding+of+transcription+factor+dynamics+involves+a+trade-off+between+noise+and+control+of+gene+expression.">Promoter decoding of transcription factor dynamics involves a trade-off between noise and control of gene expression.</a> Molecular Systems Biology. 9, 704 (2014).</li><li>Hansen, A. S., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cis+Determinants+of+Promoter+Threshold+and+Activation+Timescale.">cis Determinants of Promoter Threshold and Activation Timescale.</a> Cell Reports. 12, 1226-1233 (2015).</li><li>Johnson, H. E., Toettcher, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+Dynamics+Control+Cell+Fate+in+the+Early+Drosophila+Embryo.">Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo.</a> Developmental Cell. 48, 361-370 (2019).</li><li>Badr, C. E., Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging:+Progress+and+applications.">Bioluminescence imaging: Progress and applications.</a> Trends in Biotechnology. 29, 624-633 (2011).</li><li>Nakajima, Y., Ohmiya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+assays:+multicolor+luciferase+assay,+secreted+luciferase+assay+and+imaging+luciferase+assay.">Bioluminescence assays: multicolor luciferase assay, secreted luciferase assay and imaging luciferase assay.</a> Expert Opinion on Drug Discovery. 5, 835-849 (2010).</li><li>Nakajima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+beetle+luciferase+for+high-resolution+bioluminescence+imaging.">Enhanced beetle luciferase for high-resolution bioluminescence imaging.</a> PLoS One. 5, (2010).</li><li>Stacer, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoLuc+reporter+for+dual+luciferase+imaging+in+living+animals.">NanoLuc reporter for dual luciferase imaging in living animals.</a> Molecular Imaging. 12, (2013).</li></ol>

The authors have no conflicting financial interest.

The components of Notch signaling show oscillatory expressions in synchrony during somitogenesis but out of synchrony during neurogenesis, leading to the difficulties in capturing the expression dynamics by static analysis in the latter case. Thus, real-time monitoring is required to reveal the expression dynamics of Notch signaling components, such as Hes1 and Dll1. Because the periods of the expressions of Hes1 and Dll1 oscillations are extremely short, approximately 2-3 h, rapid res...

The mammalian brain is composed of a large number of various types of neurons and glial cells. All cells are generated from neural stem/progenitor cells (NPCs), which first proliferate to expand their numbers, then start to differentiate into neurons, and finally give rise to glial cells1,2,3,4,5. Once cells have differentiated into neurons, they cannot proliferate or increase their numbers, and, therefore, the maintenance of NPCs until later stages is important. Notch signaling via cell-cell interactions plays an important role in maintaining NPCs6,7. Notch ligands interact with the membrane protein, Notch, on the surface of neighboring cells and activates the Notch protein. After activation, proteolysis of Notch protein occurs, thereby releasing the intracellular domain of Notch (NICD) from the cell membrane into the nucleus8,9,10. In the nucleus, NICD binds to the promoter regions of Hes1 and Hes5 (Hes1/5) and activates the expression of these genes. Hes1/5 repress the expression of the proneural genes Ascl1 and Neurogenin1/2 (Neurog1/2)11,12,13,14. Because proneural genes induce neuronal differentiation, Hes1/5 play essential roles in maintaining NPCs. Furthermore, as proneural genes can activate the expression of the Notch ligand Delta-like1 (Dll1), Hes1/5 also repress the expression of Dll1. Therefore, the expression of Dll1 leads to neighboring cells being negative for Dll1 via Notch signaling. In this way, cells inhibit adjacent cells from following their same fate, a phenomenon known as the lateral inhibition8. In the developing brain, lateral inhibition plays a role in generating various different cell types.
Real-time imaging at the single cell level reveals dynamic expressions of the components of Notch signaling in NPCs15,16,17. Notch signaling activates the expression of Hes1, but Hes1 protein binds to its own promoter and represses its own expression. Furthermore, Hes1 is an extremely unstable protein, that is degraded by the ubiquitin-proteasome pathway; therefore, the repression of its own promoter is only short lived and then the transcription starts again. In this way, the expression of Hes1 oscillates at both the transcription and translational levels in a 2 h cycle18. The oscillatory expression of Hes1, in turn, induces the oscillatory expression of the downstream target genes, such as Ascl1, Neurog2, and Dll1, via periodic repression15,16,17,19. While proneural genes can induce neuronal differentiation, their oscillatory expression is not sufficient for neuronal differentiation; rather their sustained expression is essential for the neuronal differentiation. The oscillatory expression of proneural genes is important for maintaining NPCs rather than for inducing neuronal differentiation14,15,16. The expression of Dll1 oscillates at both the transcription and translational levels during various morphogenesis, such as neurogenesis and somitogenesis. The dynamic expression of Dll1 is important for the normal morphogenesis and steady expression of Dll1 induces defects in neurogenesis and somitogenesis17. These findings demonstrate the important function that the dynamics of gene expression and protein kinetics have on the regulation of various developmental events (i.e., different expression dynamics produce different outputs in cellular behaviors).
To analyze the dynamics of Notch signaling, the static analysis of tissues and cells are insufficient because they are constantly changing. Real-time imaging of single cells is a powerful tool to reveal the dynamics in gene expression. The dynamic expression of Notch signaling molecules undergo rapid cyclic responses in the period of 2-3 h. This rapid periodic expression presents two difficult problems for the real-time monitoring: (1) the expression of the molecules is suppressed to low levels, and (2) rapid turnover requires fast-response reporters. To overcome these problems, we previously developed a bioluminescence real-time imaging method20. Because the bioluminescence reporter has a higher sensitivity and shorter maturation time than fluorescent reporters, this strategy enables us to monitor the rapid dynamics in living cells. Using real-time visualization, we found that more genes exhibited dynamic expression than we had previously thought. In addition, the number of reports showing expression and protein dynamics in living cells and the significance of these dynamics in various biological events has increased, suggesting a fundamental role of the dynamics in gene expressions21,22.
In this report, we describe a way to visualize the expression of the Notch ligand Dll1 in NPCs in both dissociated cultures and in cortical slice cultures. To monitor the dynamics of Dll1 transcription at single cell levels, we generated dissociated cultures of NPCs derived from the embryonic telencephalon of transgenic mice carrying pDll1-Ub-Fluc reporter, a Dll1 promoter-driven destabilized luciferase reporter. To monitor Dll1 protein dynamics in vivo, we introduced the Dll1-Fluc fusion reporter into NPCs in the cortex and visualized the expression of the reporter in NPCs in cortical slice cultures. Real-time imaging enabled us to capture the various features of gene expression and protein dynamics in living cells at high temporal resolution.

<table><tbody><tr><td>&lt;strong&gt;Bioluminescence Imaging System&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chilled water circulator (chiller)</td><td>Julabo</td><td>Model: F12-ED</td><td></td></tr><tr><td>Cooled CCD camera</td><td>Andor Technology</td><td>Model: iKon-M 934</td><td></td></tr><tr><td>Incubator system</td><td>TOKAI HIT</td><td>Model: INU-ONICS</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>Model: IX81</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>Model: IX83</td><td></td></tr><tr><td>LED illumination device</td><td>CoolLED</td><td>Model: pE1</td><td></td></tr><tr><td>MetaMorph</td><td>MOLECULAR DEVICES</td><td>Model: 40000</td><td></td></tr><tr><td>Mix gas controller</td><td>Tokken</td><td>Model: TK-MIGM OLO2</td><td></td></tr><tr><td>Objective lens</td><td>Olympus</td><td>Model: UPLFLN 40X O</td><td></td></tr><tr><td>&lt;strong&gt;Preparations for Dissection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dissection microscope</td><td>Nikon</td><td>Model: SMZ-2B</td><td></td></tr><tr><td>Fluorescence stereoscopic microscope</td><td>Leica</td><td>Model: MZ16FA</td><td></td></tr><tr><td>Fine forceps</td><td>DUMONT</td><td>INOX No.5</td><td></td></tr><tr><td>&lt;strong&gt;Scissors, Micro scissors&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Ring-shaped forceps</td><td></td><td></td><td></td></tr><tr><td>10-cm plastic petri dish</td><td>greiner</td><td>664160-013</td><td></td></tr><tr><td>35-mm plastic petri dish</td><td>greiner</td><td>627160</td><td></td></tr><tr><td>PBS</td><td>Nacalai Tesque</td><td>14249-24</td><td></td></tr><tr><td>DMEM/F12</td><td>invitrogen</td><td>11039-021</td><td></td></tr><tr><td>&lt;strong&gt;Reagents for NPC dissociation culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B27 supplement</td><td>invitrogen</td><td>12587-010</td><td></td></tr><tr><td>bFGF</td><td>invitrogen</td><td>13256-029</td><td>Stock solution: 1 &amp;mu;g/ml in 0.1% BSA/PBS</td></tr><tr><td>D-luciferin</td><td>Nacalai Tesque</td><td>01493-85</td><td>Stock solution: 100mM in 0.9% saline</td></tr><tr><td>DNase</td><td>Worthington Biochemical Corporation</td><td>LK003172</td><td>Stock solution: 1000U/ml in EBSS</td></tr><tr><td>EBSS</td><td>Worthington Biochemical Corporation</td><td>LK003188</td><td></td></tr><tr><td>Glass bottom dish</td><td>IWAKI</td><td>3910-035</td><td></td></tr><tr><td>N2 supplement (100x)</td><td>invitrogen</td><td>17502-048</td><td></td></tr><tr><td>N-acetyl-cystein</td><td>Sigma</td><td>A-9165-25G</td><td></td></tr><tr><td>Papain</td><td>Worthington Biochemical Corporation</td><td>LK003178</td><td>Stock solution: 7U/ml in EBSS</td></tr><tr><td>Penicillin/Streptmycine</td><td>Nacalai Tesque</td><td>09367-34</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>P-6281</td><td>40 mg/ml in DW</td></tr><tr><td>&lt;strong&gt;Preparations for in utero electroporation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50-ml syringe</td><td>TERUMO</td><td>181228T</td><td></td></tr><tr><td>Electrode</td><td>Neppagene</td><td>7-mm</td><td></td></tr><tr><td>Electroporator</td><td>Neppagene</td><td>CUY21 EDIT</td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Gauzes</td><td>Kawamoto co.</td><td>7161</td><td></td></tr><tr><td>Micro capillary</td><td>Made in-house</td><td></td><td></td></tr><tr><td>PBS</td><td>Nacalai Tesque</td><td>14249-24</td><td></td></tr><tr><td>Pentbarbital</td><td>Kyoritsuseiyaku</td><td>Somnopentyl</td><td></td></tr><tr><td>Ring-shaped forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors, Micro scissors</td><td></td><td></td><td></td></tr><tr><td>Suture needle</td><td>Akiyama MEDICAL MFG. CO</td><td>F17-40B2</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>Seractal</td><td></td></tr><tr><td>&lt;strong&gt;Preparations for Slice culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10-cm plastic petri dish</td><td>greiner</td><td>664160-013</td><td></td></tr><tr><td>35-mm plastic petri dish</td><td>greiner</td><td>627160</td><td></td></tr><tr><td>Culture insert</td><td>Millipore</td><td>PICM01250</td><td></td></tr><tr><td>DMEM/F12</td><td>invitrogen</td><td>11039-021</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>172012-500ML</td><td></td></tr><tr><td>Fine forceps</td><td>DUMONT</td><td>INOX No.5</td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050-122</td><td></td></tr><tr><td>Micro surgical knife</td><td>Alcon</td><td>19 Gauge V-Lance</td><td></td></tr><tr><td>Multi-gas incubator</td><td>Panasonic</td><td>MCO-5MUV-PJ</td><td></td></tr><tr><td>N2/B27 media</td><td>Made in-house</td><td></td><td>ref. NPC dissociatioin culture</td></tr><tr><td>PBS</td><td>Nacalai Tesque</td><td>14249-24</td><td></td></tr><tr><td>Ring-shaped forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors, Micro scissors</td><td></td><td></td><td></td></tr><tr><td>Silicon rubber cutting board</td><td>Made in-house</td><td></td><td></td></tr></tbody></table>

real-time bioluminescence imaging of notch signaling dynamics during murine neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Expressions of the genes Hes1/7 exhibit 2 h oscillation cycle in various cell lines and during somitogenesis. Furthermore, the period of oscillation is very short and both their mRNAs and proteins are extremely unstable with the half-lives of around 20 min. If using a slow response reporter, we cannot trace such rapid dynamics, and if using a stable reporter, it gradually accumulates while the gene expression oscillates. Thus, the reporter must be rapidly degraded to monitor the rapid turnover of such cyclically...

Watch this Scientific Journal Video about Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis at JoVE.com

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

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Alternative Signaling Routes in Gene Expression

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7.5K Views.  Kyoto University. Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Video: Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetics

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuroscience

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Biology

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

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.

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

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Summary

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Chapters in this video

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice