This work is supported by the following grants: the Japan Science and Technology Agency (JST)/ Precursory Research for Embryonic Science and Technology (PRESTO) (JPMJPR15F8, Japan); the Japan Society for the Promotion of Science (JSPS)/Grants in Aid for Scientific Research (KAKENHI) (JP18H05414, JP17H05710, JP16K07316), Takeda Foundation. The authors thank Haruhiko Bito (University of Tokyo) for providing RCaMP2 and Arthur J.Y. Huang and Thomas McHugh (RIKEN CBS) for providing AAV vectors and for instructions regarding AAV preparation. The authors would&#160;also like to thank&#160;editors at the Journal of Visualized Experiments for their help with video filming and editing.&#160;

All the experiments described here were approved by the RIKEN safety committee and animal experiment committee, according to the guideline issued by the Japanese Ministry of Education, Culture, Sports, Science, and Technology.&#160;
1. Preparation of Cells&#160;
<ol>
	<li>Preparation of poly(ethyleneimine)-coated coverslips 
	NOTE: Poly(ethyleneimine) (PEI) coating is recommended for the glass apparatus, as it allows neurons and astrocytes to attach tightly to the coverslips without preventing their development. However, other coating methods (e.g., poly-ornithine, poly L-lysine, laminin coating) are also available, if necessary, for glass-bottom dishes.&#160;
	<ol>
		<li>Place an 18 mm-diameter glass coverslip in each well of a 12-well plate. Prepare 0.04% PEI solution (12.5 mL/12-well plate) using sterilized water.</li>
		<li>Add 1 mL of 0.04% PEI solution to each well. Ensure that there are no bubbles underneath the coverslips.</li>
		<li>Incubate the plates in a CO2 incubator overnight at 37 &#176;C.</li>
		<li>The next day, wash the coated coverslips 3x with 1 mL of sterilized water. Remove the PEI solution with an aspirator, add 1 mL of sterilized water to each well, and shake the 12-well plate so that the PEI solution between the coverslip and the plate can be washed out thoroughly. As the remaining PEI is toxic for cells, ensure that the water after the final wash is aspirated completely.</li>
		<li>Dry and sterilize the coverslips inside the hood with ultraviolet (UV) light for at least 15 min. The PEI-coated dish can be stored at 4 &#176;C for up to 2 months. Illuminate the dishes with UV light for 15 min just before use.</li>
		<li>Add 5 mL of sterile distilled water in the space between the wells to prevent evaporation of the culture medium.</li>
	</ol>
	</li>
	<li>Plating cell lines 
	NOTE: This protocol provides just one example for transfection into cells from mammalian cell lines, such as HeLa cells and COS-7 cells. Users can apply other transfection protocols that are optimized for their experiments. In this section, we will describe the HeLa cell culture protocol, which is also applicable to COS-7 cells.
	<ol>
		<li>On the day before the transfection, culture the cells in a 10 cm culture dish until they attain 70%&#8211;90% confluence.</li>
		<li>Prewarm the culture medium (see Table of Materials) to 37 &#176;C.</li>
		<li>Wash the cells 2x with phosphate-buffered saline without Ca2+ and Mg2+ (PBS [-]).</li>
		<li>Aspirate the PBS(-), add 1 mL of 0.5% trypsin-ethylenediaminetetraacetic acid (EDTA) solution, and incubate the cells at 37 &#176;C for 90 s until they attain a round shape.</li>
		<li>Add 9 mL of prewarmed culture medium to stop the trypsinization. Dilute the cells with culture medium at a ratio of 1:6.</li>
		<li>Seed 1 mL of the diluted cells on PEI-coated coverslips in the 12-well plates.</li>
	</ol>
	</li>
	<li>Preparation of hippocampal neuron-astrocyte mixed culture from rats or mice 
	NOTE: Sections 1.3 and 1.4 must first be reviewed and approved by an Institutional Animal Care and Use Committee and must follow officially approved procedures for the care and use of laboratory animals. The flowchart of the neuronal culture protocol is shown in Figure 2.
	<ol>
		<li>Prepare all reagents under the laminar flow hood. Place Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) into two 100 mm culture dishes (approximately 20 mL/dish) that are in an icebox.&#160;</li>
		<li>Prepare 50 mL of the dissection medium composed of DMEM and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (see Table of Materials) and dispense the medium into three 60 mm culture dishes (approximately 7 mL/dish) and eight 35 mm culture dishes (approximately 2 mL/dish). Place the dishes in another icebox.&#160;</li>
		<li>Prepare 50 mL of the incubation saline, composed of Hanks&#8217; balanced salt solution supplemented with 20 mM HEPES (see Table of Materials), and place 8 mL of the saline in a 15 mL conical tube on ice.</li>
		<li>Prepare the plating medium with minimum essential medium (MEM) supplemented with B-27, glutamine, and penicillin-streptomycin (see Table of Materials). Maintain this medium at room temperature (20&#8211;28 &#176;C).</li>
		<li>Sterilize the surgical instruments with 70% ethanol.&#160;</li>
		<li>Place a paper towel in a glass jar with a lid and 1 mL of isoflurane. Let the isoflurane evaporate for 1 min.</li>
		<li>Place a pregnant rat or a mouse in the jar prepared according to step 1.3.6 and keep the animal in the jar until it is deeply anesthetized (approximately 30 s to 1 min).</li>
		<li>Take the anesthetized animal out of the jar and disinfect the animal and the dissection equipment by spraying them with 70% ethanol. Cut the ventral midline with standard dissecting scissors and tweezers and extract the uterus from the pregnant rat or mouse.</li>
		<li>Extract E18&#8211;19 embryos from the uterus of an anesthetized female rat or mouse, using delicate dissecting scissors, and place the extracted embryos with a ring forceps into ice-cold DMEM in a 10 cm dish for cold anesthesia.</li>
		<li>Decapitate the embryo with fine dissection scissors and place the head in ice-cold DMEM in a 10 cm dish.</li>
		<li>Extract the brain from each embryo with a 13 cm curved Semken forceps and forceps with fine tips. Keep the brain in the ice-cold dissection medium in a 60 mm dish.</li>
		<li>Remove the hippocampi, using two forceps with fine tips, in the ice-cold dissection medium in 35 mm dishes and maintain the isolated tissue in the incubation saline placed in a 15 mL conical tube on ice.</li>
		<li>Wash the hippocampi with incubation saline and incubate with trypsin (1.25 mg/mL) and DNase I (0.25 mg/mL) in the incubation saline for 5 min at 37 &#176;C. The recommended incubation volume is 2.7 mL of the incubation saline, 150 &#181;L of 20x stock trypsin, and 150 &#181;L of 20x stock DNase (see Table of Materials).</li>
		<li>Wash the hippocampi 3x with ice-cold incubation saline.</li>
		<li>Aspirate the incubation saline and add 1 mL of the plating medium containing DNase I (10 &#181;L of stock solution; see Table of Materials). Suspend the tissue by pipetting no more than 20x and measure the density of viable cells, using a cell counter and Trypan blue assay.</li>
		<li>Dilute the hippocampal cells to a density of 1.4 x 105 viable cells/mL for rats and 2.5 x 105 viable cells/mL for mice. Seed 1 mL of the diluted cell suspensions onto the PEI-coated coverslips in 12-well culture plates.</li>
		<li>Maintain the cells at 37 &#176;C in a CO2 incubator for 2&#8211;3 days.</li>
		<li>Remove the plating medium. Do not let the cells dry out. Gently and quickly add the prewarmed maintenance medium (see Table of Materials).&#160;</li>
	</ol>
	</li>
	<li>Preparation of rat cortical neuron-astrocyte mixed culture and frozen cells, and the revival of frozen cultures 
	NOTE: A cryopreservation method for cortical cells was described previously11. Here, a modified protocol in which cortical cells can be stored at -80 &#176;C for at least 3 months is provided. The flowchart for this protocol is shown in Figure 2.
	<ol>
		<li>Prepare DMEM, dissection medium, incubation saline, and plating medium as indicated in steps 1.3.1&#8211;1.3.4, and the Table of Materials.&#160;</li>
		<li>Prepare the wash medium constituted of DMEM, heat-inactivated fetal bovine serum, and penicillin-streptomycin (see Table of Materials), if necessary.</li>
		<li>Extract E18&#8211;19 embryos from the uterus of an anesthetized female rat or mouse, using delicate dissection scissors, and use ring forceps to place each extracted embryo into ice-cold DMEM for cold anesthesia.</li>
		<li>Remove the brains from the embryos and keep them in ice-cold dissection medium. Remove the cortexes and maintain them in the incubation saline placed in a 15 mL conical tube on ice.</li>
		<li>Wash the cortexes with incubation saline and incubate the cortexes with trypsin (1.25 mg/mL) and DNase I (0.25 mg/mL) in incubation saline for 5 min at 37 &#176;C. The recommended incubation volume for 12 cortexes is 5.4 mL of incubation saline, 300 &#181;L of 20x stock trypsin, and 300 &#181;L of 20x stock DNase I.</li>
		<li>Wash the cortexes 3x with ice-cold incubation saline.</li>
		<li>Remove the supernatant and add 2 mL of plating medium supplemented with 150 &#181;L of DNase I stock. Dissociate the cells by pipetting less than 20 strokes, and filter the cells using a cell strainer with a pore size of 70 &#181;m.</li>
		<li>Wash the cell strainer with 20 mL of the plating medium for plating. For the preparation of frozen cell stock, wash the cells with 20 mL of the wash medium (see Table of Materials)</li>
		<li>Measure the density of the viable cells, using a cell counter and the Trypan blue method.</li>
		<li>Dilute the cortical cells to a density of 1.4 x 105 viable cells/mL with the plating medium and add 1 mL of the diluted cell suspension to PEI-coated coverslips in the 12-well culture plates.</li>
		<li>Maintain the cells at 37 &#176;C in a CO2 incubator for 2&#8211;3 days and change the culture medium to the maintenance medium.</li>
		<li>After step 1.4.9, prepare the frozen cortical cell stock by centrifuging the cells at 187 x g for 3 min, using a swing rotor.&#160;</li>
		<li>Aspirate the supernatant and add the cryopreservation medium (see Table of Materials) kept at 4 &#176;C, to obtain a cell density of 1 x 107 cells/mL. Aliquot 1 mL of the cell suspension into cryogenic tubes.</li>
		<li>Place the tubes in a cell freezing container with a freezing rate of -1 &#176;C/min, until a temperature of -80 &#176;C is reached, and transfer the freezing container to a -80 &#176;C freezer. The cells can be stored for at least 3 months at -80 &#176;C.</li>
		<li>To revive frozen cells, prewarm the wash medium (approximately 13 mL for each cryogenic tube) and maintenance medium for frozen cortical cells (see Table of Materials).</li>
		<li>Thaw the frozen cells rapidly at 37 &#176;C in a water bath.</li>
		<li>Dilute the thawed cells gently with prewarmed wash medium. Centrifuge the cells at 187 x g for 3 min, using a swing rotor.</li>
		<li>Suspend the pellet in 1 mL of wash medium and measure the viable cell density.</li>
		<li>Dilute the cells with the maintenance medium for frozen cortical cells to yield a cell density of 3.0 x 105 viable cells/mL, and seed 1 mL of the cell suspension in the PEI-coated 12-well plates.</li>
	</ol>
	</li>
</ol>
2. Expression of Membrane-targeted GECIs
<ol>
	<li>Transfection of cells&#160;
	<ol>
		<li>Add 250 ng of the GECI plasmid (i.e., Lck-GCaMP6f, Lck-RCaMP2, or OER-GCaMP6f with CMV promoter)7,8,9 to 100 &#181;L of the reduced serum medium (see Table of Materials) per well. For the cotransfection of Lck-RCaMP2 and OER-GCaMP6f, use 250 ng of each plasmid in 100 &#181;L of reduced serum medium in each well.</li>
		<li>Add 0.5 &#181;L of transfection reagent (see Table of Materials) per well into the plasmid-reduced serum medium mixture. For the cotransfection of Lck-RCaMP2 and OER-GCaMP6f, add 0.5 &#181;L of transfection reagent per well.</li>
		<li>Incubate the mixture for 30 min at room temperature (20&#8211;28 &#176;C).</li>
		<li>Add 100 &#181;L of the mixture to each coverslip in a drop-wise manner.</li>
		<li>Incubate the cells for 48&#8211;72 h in a CO2 incubator at 37 &#176;C to allow the expression of the GECIs.</li>
	</ol>
	</li>
	<li>Transfection and adeno-associated virus infection of hippocampal or cortical neurons 
	NOTE: Transfection for 3&#8211;5 days in vitro (DIV) results in a higher transfection rate for neurons. Transfection at 6&#8211;8 DIV is preferred for the optimal expression of GECIs in astrocytes. For the expression of GECIs in dissociated culture neurons after 9 DIV, the infection of the adeno-associated virus (AAV) vectors provides a better expression efficacy. AAV vectors for the expression of Lck-GCaMP6f, Lck-RCaMP2, and OER-GCaMP6f under the EF1a promoters were prepared as described previously, using HEK293 cells12 (see Table of Materials).&#160;
	<ol>
		<li>For transfection 3&#8211;8 days after plating, label two tubes, one for plasmid DNA and the other for transfection reagent.</li>
		<li>Add 50 &#181;L of reduced serum medium (see Table of Materials) per well to each tube.</li>
		<li>Add 0.5 &#181;g of plasmid DNA per coverslip and 1 &#181;L of supplement accompanied by transfection reagent for neurons (see Table of Materials) per well to the plasmid DNA tube. For the cotransfection of Lck-RCaMP2 and OER-GCaMP6f, 0.5 &#181;g of each plasmid and 1 &#181;L of supplement are mixed in 50 &#181;L of reduced serum medium per well.&#160;</li>
		<li>Add 1 &#181;L of transfection reagent (see Table of Materials) per well in the transfection reagent tube. The same amount of transfection reagent is used for cotransfection.</li>
		<li>Vortex both tubes for 1&#8211;2 s.</li>
		<li>Add the transfection reagent mixture (from step 2.2.4) to the DNA mixture (from step 2.2.3). Mix by pipetting gently and incubate the mixture (100 &#181;L per coverslip) for 5 min at room temperature.</li>
		<li>Load this mixture onto the cells in a drop-wise manner.&#160;</li>
		<li>Incubate the cells in a CO2 incubator for 2&#8211;3 days until the marker proteins are expressed.</li>
		<li>For AAV infection, add 3 &#181;L of AAV per well to the mixed neuron-astrocyte culture. Mix gently by rocking the dish. For the double infection of Lck-RCaMP2 and OER-GCaMP6f, 3 &#181;L of each AAV is introduced per well. In case the numbers of cells expressing GECIs are insufficient, the optimal AAV amount for infection should be determined.</li>
		<li>Maintain the culture for 1&#8211;2 weeks until the GECIs are expressed.</li>
	</ol>
	</li>
</ol>
3. Ca2+ Imaging
<ol>
	<li>Simultaneous imaging of cells expressing Lck-RCaMP2 and OER-GCaMP6f 
	NOTE: To simultaneously record the Lck-RCaMP2 and OER-GCaMP6f signals, image-splitting optics are required. The optics enable the separation of RCaMP2 and GCaMP6f and their projection onto the same photographic frame of the camera (Figure 3A). Simultaneous imaging also requires (1) light sources that can simultaneously emit excitation light in the blue (450&#8211;490 nm) and green (500&#8211;560 nm) spectra, (2) double-band filter and dichroic mirror sets in the microscope, and (3) emission filters for RCaMP2 and GCaMP6f. For details, refer to the Table of Materials.&#160;
	<ol>
		<li>Turn on the imaging devices and computers at least 30 min before the recording. Prewarm the microscope heating chamber to 37 &#176;C. Set up the image-splitting device, filters, and light source. Align the image-splitting optics so that the same field of view appears on the camera. Choose the appropriate objective lens (see Table of Materials).</li>
		<li>Mount the coverslip containing the cells transfected with Lck-RCaMP2 and OER-GCaMP6f in the recording chamber, add the appropriate imaging medium or buffer (400 &#181;L for 18 mm coverslips) in the chamber, and place it on the microscope stage. Place a lid on the recording chamber to avoid the evaporation of the medium.</li>
		<li>Locate the cells expressing both Lck-RCaMP2 and OER-GCaMP6f by fluorescent imaging. Minimize the excitation light intensity to prevent photobleaching and phototoxicity.</li>
		<li>Remove the lid and start a time-lapse recording at 10 Hz. During this recording, add the agonist to the chamber to evoke Ca2+ responses (e.g., histamine for HeLa cells, ATP for COS-7 cells).&#160;</li>
		<li>Save the time-lapse data in the hard disk drive (HDD).</li>
		<li>Analyze the data using image analysis software.</li>
	</ol>
	</li>
	<li>Recording spontaneous activities of astrocytes expressing Lck-RCaMP2 and OER-GCaMP6f 
	NOTE: Without image-splitting optics, the Ca2+ signals at the plasma membrane and those around the ER can be monitored in the same cell. Here, the sequential recording of Lck-RCaMP2 and OER-GCaMP6f in the same astrocytes is described. An oil-immersion objective with a numerical aperture larger than 1.3 is highly recommended for spontaneous Ca2+ activity.&#160;
	<ol>
		<li>Turn on the microscope, camera, light source, and the microscope heating chamber at least 30 min before recording.</li>
		<li>Mount the coverslip containing the cells transfected with Lck-RCaMP2 and OER-GCaMP6f in the recording chamber and add 400 &#181;L of the imaging medium. Place a lid on top of the chamber.</li>
		<li>Choose the filter set for GCaMP6f and the light source (blue excitation light, e.g. 470&#8211; 490 nm; see Table of Materials). Locate the astrocytes expressing OER-GCaMP6f.</li>
		<li>Choose a filter set and the light source for RCaMP2 (green excitation light, e.g., 510&#8211;560 nm; see Table of Materials) and confirm whether Lck-RCaMP2 is expressed in the same astrocytes. Avoid long exposure to the light source to prevent photobleaching.&#160;</li>
		<li>Record time-lapse images of Lck-RCaMP2 at 2 Hz for 2 min. Save the imaging data on the HDD.</li>
		<li>Change the filter set to that for GCaMP6f. Record time-lapse images of OER-GCaMP6f in the same field of view, at 2 Hz for 2 min. Save the data on the HDD.</li>
		<li>Analyze the data using the image analysis software.</li>
	</ol>
	</li>
	<li>Recording spontaneous neuronal activity and induced Ca2+ elevation in neurons 
	NOTE: The microscope setup for neuronal imaging is the same as that described in section 3.2. Here, the imaging of spontaneous Ca2+ elevation due to Lck-GCaMP6f and Ca2+ elevation induced by mGluR activation due to OER-GCaMP6f is described.&#160;
	<ol>
		<li>To record spontaneous neuronal activity, mount the coverslip containing the cells expressing Lck-GCaMP6f in the recording chamber and add 400 &#181;L of imaging medium. Place a lid on top of the chamber.</li>
		<li>Set the filter and the light source (blue excitation, e.g. 470&#8211;790 nm; see Table of Materials) to those for GCaMP6f. Find the neurons expressing Lck-GCaMP6f and showing spontaneous activity.</li>
		<li>Acquire images at 2 Hz or faster. Save the data on the HDD.</li>
		<li>Analyze the data using the image analysis software.</li>
		<li>To record induced Ca2+ elevation, mount the coverslips containing the cells expressing OER-GCaMP6f with 400 &#181;L imaging medium. Place a lid on top of the chamber.&#160;</li>
		<li>Using the filter set for GCaMP6f, find the neurons expressing OER-GCaMP6f.</li>
		<li>Remove the lid. Start the time-lapse recording at 2 Hz or faster. During the recording, add the agonists for a Gq-protein-coupled receptor (e.g., mGluR5 agonist [RS]-3,5-dihydroxyphenylglycine [DHPG]) to evoke a Ca2+ release from the ER.</li>
		<li>Save the time-lapse images on the HDD and analyze the data.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling.">Calcium signaling.</a> Cell. 131 (6), 1047-1058 (2007).</li><li>Bagur, R., Hajnoczky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca(2+)+Sensing:+Its+Role+in+Calcium+Homeostasis+and+Signaling.">Intracellular Ca(2+) Sensing: Its Role in Calcium Homeostasis and Signaling.</a> Molecular Cell. 66 (6), 780-788 (2017).</li><li>Bannai, H., 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=Bidirectional+Control+of+Synaptic+GABAAR+Clustering+by+Glutamate+and+Calcium.">Bidirectional Control of Synaptic GABAAR Clustering by Glutamate and Calcium.</a> Cell Reports. 13 (12), 2768-2780 (2015).</li><li>Tojima, T., Hines, J. H., Henley, J. R., Kamiguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+messengers+and+membrane+trafficking+direct+and+organize+growth+cone+steering.">Second messengers and membrane trafficking direct and organize growth cone steering.</a> Nature Reviews Neuroscience. 12 (4), 191-203 (2011).</li><li>Shigetomi, E., Kracun, S., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+targeted+optical+sensor+to+monitor+calcium+signals+in+astrocyte+processes.">A genetically targeted optical sensor to monitor calcium signals in astrocyte processes.</a> Nature Neuroscience. 13 (6), 759-766 (2010).</li><li>Chen, T. W., 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=Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity.">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li><li>Niwa, F., 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=Dissection+of+local+Ca(2+)+signals+inside+cytosol+by+ER-targeted+Ca(2+)+indicator.">Dissection of local Ca(2+) signals inside cytosol by ER-targeted Ca(2+) indicator.</a> Biochemical and Biophysical Research Communications. 479 (1), 67-73 (2016).</li><li>Vervliet, T., 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=Basal+ryanodine+receptor+activity+suppresses+autophagic+flux.">Basal ryanodine receptor activity suppresses autophagic flux.</a> Biochemical Pharmacology. 132, 133-142 (2017).</li><li>Sakuragi, S., Niwa, F., Oda, Y., Mikoshiba, K., Bannai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglial+Ca2++signaling+is+generated+by+the+coordination+of+IP3R+and+store-operated+Ca2++channels.">Astroglial Ca2+ signaling is generated by the coordination of IP3R and store-operated Ca2+ channels.</a> Biochemical and Biophysical Research Communications. 486 (4), 879-885 (2017).</li><li>Inoue, M., 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=Rational+design+of+a+high-affinity,+fast,+red+calcium+indicator+R-CaMP2.">Rational design of a high-affinity, fast, red calcium indicator R-CaMP2.</a> Nature Methods. 12 (1), 64-70 (2015).</li><li>Quasthoff, K., 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=Freshly+frozen+E18+rat+cortical+cells+can+generate+functional+neural+networks+after+standard+cryopreservation+and+thawing+procedures.">Freshly frozen E18 rat cortical cells can generate functional neural networks after standard cryopreservation and thawing procedures.</a> Cytotechnology. 67 (3), 419-426 (2015).</li><li>Boehringer, R., 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=Chronic+Loss+of+CA2+Transmission+Leads+to+Hippocampal+Hyperexcitability.">Chronic Loss of CA2 Transmission Leads to Hippocampal Hyperexcitability.</a> Neuron. 94 (3), 642-655 (2017).</li><li>Arizono, M., 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=Receptor-selective+diffusion+barrier+enhances+sensitivity+of+astrocytic+processes+to+metabotropic+glutamate+receptor+stimulation.">Receptor-selective diffusion barrier enhances sensitivity of astrocytic processes to metabotropic glutamate receptor stimulation.</a> Science Signaling. 5 (218), ra27 (2012).</li><li>Kanemaru, K., 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=In+vivo+visualization+of+subtle,+transient,+and+local+activity+of+astrocytes+using+an+ultrasensitive+Ca(2+)+indicator.">In vivo visualization of subtle, transient, and local activity of astrocytes using an ultrasensitive Ca(2+) indicator.</a> Cell Reports. 8 (1), 311-318 (2014).</li><li>Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TI+Workbench,+an+integrated+software+package+for+electrophysiology+and+imaging.">TI Workbench, an integrated software package for electrophysiology and imaging.</a> Microscopy (Oxford, UK). 67 (3), 129-143 (2018).</li><li>Miyamoto, A., Bannai, H., Michikawa, T., Mikoshiba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+microscopic+systems+for+long-term+imaging+of+intracellular+calcium+using+a+ratiometric+genetically-encoded+calcium+indicator.">Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator.</a> Biochemical and Biophysical Research Communications. 434 (2), 252-257 (2013).</li><li>Srinivasan, R., 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=New+Transgenic+Mouse+Lines+for+Selectively+Targeting+Astrocytes+and+Studying+Calcium+Signals+in+Astrocyte+Processes+In+Situ+and+In+Vivo.">New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.</a> Neuron. 92 (6), 1181-1195 (2016).</li></ol>

The authors have nothing to disclose.

Diverse biological outputs are initiated by Ca2+ signals. Ca2+ is a versatile intracellular signaling messenger. Decoding Ca2+ signals to evoke specific outputs has been a fundamental biological question, and Ca2+ imaging techniques to describe the diversity of Ca2+ signals are required. The presently detailed protocol enables the detection of distinct Ca2+ signals at the plasma membrane and ER (Figure 3 and Figure 4) and local Ca2+ microdomains inside a cell (Figure 4 and Figure 5). This contributes to describing the diversity of intracellular Ca2+ signals. The temporal resolution of Ca2+ signals was also improved by targeting GECIs in the plasma membrane and ER because it can avoid the effect of a three-dimensional diffusion of the Ca2+ and GECIs themselves, and it has the potential to detect the very moment of Ca2+ influx or Ca2+ release, which occurs on the membrane.&#160;
The protocol has some limitations. Users should keep in mind that the detected signals are the summation of &#8220;the moment of Ca2+ influx or release&#8221; and &#8220;Ca2+ diffused out from the original Ca2+ source&#8221;, especially for large Ca2+ signals. For example, although His stimulation in HeLa cells evokes Ca2+ release from the ER, its resultant Ca2+ signals are detected not only by ER-targeted OER-GCaMP6f but also by plasma-membrane-targeted Lck-RCaMP2 (Figure 3). Another limitation is that the spatiotemporal pattern of Ca2+ signals may not be the only determinant of the output of Ca2+ signals. The distribution of downstream effector proteins (such as Ca2+-dependent kinases and phosphatases) may also be a determining factor2. To completely decode the intracellular Ca2+ signals, analysis of downstream enzyme behavior, which is not covered in this protocol, is absolutely necessary.
One of the most critical aspects for successful Ca2+ imaging is the imaging setup and image acquisition conditions, as well as for other live-imaging studies. We previously showed that Ca2+ responses in the cell are highly dependent on the duration and intensity of excitation and on image acquisition conditions, including exposure time and acquisition frequency16. Excitation illumination power is the most critical factor, as it can cause light toxicity and photobleaching of GECIs. The recording conditions of exposure time, recording frequency, excitation light intensity, and duration of recording should be optimized according to the purpose of the experiment. We recommend reducing the exposure time and the excitation light intensity as much as possible to avoid photobleaching and phototoxicity to the cell. The recording frequency and the duration of recording should be sufficient to cover the Ca2+ elevation events of interest but should be kept as low as possible to avoid photobleaching and phototoxicity also. We recommend determining the recording frequency and the duration first and optimizing the light intensity and the exposure time so that the photobleaching of the GECIs is minimized. Another important factor is the expression level of the GECIs. GECIs have a Ca2+-buffering effect as they are Ca2+-binding proteins. Therefore, the overexpression of GECIs results in the buffering of Ca2+, which is physiologically necessary for the cells. The amount of GECI expression should be minimized to avoid imaging cells expressing high amounts of GECIs. 
&#160; 
In conclusion, the dissection of Ca2+ signals at a subcellular resolution is one of the most important steps for decoding intracellular Ca2+ signals that determine the output biological phenomenon. This protocol provides a new method for the dissection of Ca2+ signals to describe the diversity among these signals. Presently, this technique is limited for in vitro experiments. However, Lck-GCaMP6f is already being used for in vivo Ca2+ imaging in mice17, and OER-GCaMP6f was confirmed to monitor Ca2+ signals in vivo in the VD motor neurons in C. elegans7. Therefore, targeting GECIs in the subcellular compartment has the potential to be expanded to in vivo imaging in the future, thus enabling Ca2+ dissection in vivo.

Ca2+ signals represent the elevation of the intracellular Ca2+ concentration. Ca2+ is the universal second messenger for eukaryotic cells. Using Ca2+, cells function via diverse intracellular signaling pathways and induce various biological outputs. For example, in neurons, synaptic vesicle release at the presynaptic terminal, gene expression in the nucleus, and induction of synaptic plasticity at the postsynapse are regulated by distinct Ca2+ signals that precisely activate the appropriate downstream enzymes at the right sites and with precise timing1.&#160;
Specific spatiotemporal patterns of Ca2+ signals activate the specific downstream enzymes. Ca2+ signals are generated by the coordination between two different Ca2+ sources: Ca2+ influx from the extracellular space and Ca2+ release from the endoplasmic reticulum (ER), which serves as an intracellular Ca2+ store. The meaningful spatiotemporal Ca2+ signaling pattern to induce a specific cell function is also supported by nanodomains of 10&#8211;100 &#181;M Ca2+ generated in the vicinity of Ca2+ channels on the plasma membrane or ER membrane2. Importantly, the source of Ca2+ signals is one of the most critical factors determining the downstream biological output. In neurons, Ca2+ influx and Ca2+ release have opposite effects on the clustering of gamma-aminobutyric acid (GABA)A receptors (GABAAR) at the GABAergic synapses, which is responsible for the inhibition of neuronal excitability3. Ca2+ influx accompanied by massive neuronal excitation induces the dispersion of synaptic GABAAR clusters, whereas persistent Ca2+ release from the ER promotes the clustering of synaptic GABAARs. Other groups have also reported that the tuning direction of growth cones is critically dependent on the source of the Ca2+ signal: Ca2+ influx induces repulsion, while Ca2+ release guides the attraction of the neuronal growth cone4. Therefore, to fully understand the Ca2+ signaling pathways underlying specific cellular outputs, it is important to identify the source of Ca2+ signals by describing Ca2+ signals at the subcellular resolution.
In this protocol, we describe a Ca2+ imaging method to report Ca2+ signals at the subcellular resolution, which allows the estimation of the Ca2+ signal sources (Figure 1). Ca2+ microdomains just beneath the plasma membrane are successfully monitored by genetically encoded Ca2+ indicators (GECIs) targeted to the plasma membrane via the attachment of the plasma membrane-localization signal Lck within Src kinase to the N-termini of GECIs5. To detect the Ca2+ signal pattern in the vicinity of the ER at a better spatial and temporal resolution, we recently developed OER-GCaMP6f, in which GCaMP6f6 targets the ER outer membrane, using the ER transmembrane protein. OER-GCaMP6f can sensitively report Ca2+ release from the ER at a better spatiotemporal resolution than conventional nontargeted GCaMP6f in COS-7 cells7 and HEK293 cells8, by avoiding the diffusion of Ca2+ and GECIs. We also confirmed that the spontaneous Ca2+ elevation in cultured hippocampal astrocytes reported by OER-GCaMP6f showed a different spatiotemporal pattern compared to that monitored by plasma membrane-targeted GCaMP6f (Lck-GCaMP6f)7,9, indicating that Ca2+ imaging with OER-GCaMP6f in combination with Lck-GCaMP6f contributes to the dissection of Ca2+ signals at the subcellular resolution to identify their sources.
Presently, we detail the protocol for the Ca2+ signal dissection in HeLa cells and neuron-astrocyte mixed cultures plated on glass coverslips. The Ca2+ imaging technique with GECIs indicated here, Lck-GCaMP6f, plasma-membrane-targeted RCaMP210 (Lck-RCaMP2), and OER-GCaMP6f (Figure 1) are applicable to all cells in which these GECIs can be expressed.

<table border="0" cellpadding="0" cellspacing="0" style="width:693px;" width="692">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">(RS)-3,5-Dihydroxyphenylglycine (DHPG)</td>
			<td style="width:179px;">Tocris</td>
			<td style="width:97px;">#0342</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:207px;">0.5% DNase I stock solution</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:97px;">#11284932001</td>
			<td>Prepare 0.5% DNase I (w/v) in Hanks&#39; Balanced Salt Solution&#160; supplemented with 120 mM MgSO4. Prepare 160 &#181;L aliquots and store at -30&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">0.5% Trypsin-EDTA solution</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:97px;">#25300054</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100 mM L-glutamine (&#215;100 stock)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:97px;">#25030081</td>
			<td style="width:211px;">Preparing small aliquots of 250--750 &#181;L and store at -30&#176;C.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">100 mM Sodium pyruvate (&#215;100 stock)</td>
			<td>Thermo Fisher Scientific</td>
			<td>#11360070</td>
			<td style="width:211px;">Aliquots (10 mL) can be stored at -20&#176;C. After thawing, the solution can be maintained at 4&#176;C for 2 months.</td>
		</tr>
		<tr height="133">
			<td height="133" style="height:133px;width:207px;">12-Well multiwell culture plates with low-evaporation lid</td>
			<td>Falcon</td>
			<td style="width:97px;">#353043</td>
			<td style="width:211px;">Low-evaporation lid is critical for culturing neuron-glia mixed culture. For cell line cells, alternative culture dishes can be used.</td>
		</tr>
		<tr height="74">
			<td height="74" style="height:75px;width:207px;">18-mm diameter circular coverslips</td>
			<td style="width:179px;">Karl Hecht &#34;Assistent&#34;</td>
			<td style="width:97px;">#41001118</td>
			<td style="width:211px;">Thickness 1, 18-mm diameter circular coverslips; alternative coverslips can be used.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:207px;">1M HEPES</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:97px;">#15630080</td>
			<td style="width:211px;">pH 7.2 - 7.6</td>
		</tr>
		<tr height="256">
			<td height="256" style="height:256px;width:207px;">2.5% Trypsin stock solution (&#215;20 stock)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:97px;">#T4674</td>
			<td style="width:211px;">Prepare 150 &#181;L aliquot and store at -30&#176;C.</td>
		</tr>
		<tr height="144">
			<td height="144" style="height:144px;width:207px;">50% Poly(ethyleneimine) (PEI) solution&#160;</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td>#P3143</td>
			<td style="width:211px;">Prepare 2% (V/V) PEI stock solution (&#215;50) with distilled water sterilized by filtration. Store stock solution at -30&#176;C after preparing small aliquots of 250-750 &#181;L. Prepare 0.04% PEI solution with distilled water on the day of coverslip coating.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">70% Ethanol</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Kept in a spray bottle to be used for surface disinfection.</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:207px;">Adeno-associated virus (AAV) for Lck-GCaMP6f, Lck-RCaMP2, and OER-RCaMP2 expression under the direction of the EF1a promoter</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">AAV can be prepared using AAV Helper Free System (Agilent Technologies) and HEK293 cells, or alternative methods. pAAV.EF1a.Lck-GCaMP6f, pAAV.EF1a.Lck-RCaMP2, and&#160; pAAV.EF1a.OER-GCaMP6f are available upon request.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;">B-27 supplement (&#215;50 stock)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:97px;">#17504044</td>
			<td style="width:211px;">This can be replaced by B-27 plus supplement (Thermo Fisher Scientific; #A3582801) or MACS NeuroBrew-21 (Miltenyi Biotec, Bergisch Gladbach, Germany; #130-093-566).</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:107px;width:207px;">B57BL/6</td>
			<td style="width:179px;">Japan SLC, Inc.</td>
			<td style="width:97px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:207px;">Camera for microscopic image recording</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">The following cameras were available for use: cooled-CCD camera (e.g., Hamamatsu Photonics, OECA-ER), EM-CCD camera (e.g., Hamamatsu Photonics,&#160; ImagEM; Andor, iXon) or&#160; CMOS camera (e.g., Hamamatsu Photonics ORCA-Flash4.0)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Cell freezing container</td>
			<td style="width:179px;">Sarstedt K.K.</td>
			<td style="width:97px;">#95.64.253</td>
			<td style="width:211px;">Alternative cell freezing container can be used.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Cell strainer</td>
			<td style="width:179px;">Falcon</td>
			<td style="width:97px;">#352350</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:207px;">CO2 incubator</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Maintain at 37&#176;C, 5% CO2.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Cryogenic tube</td>
			<td style="width:179px;">Corning</td>
			<td style="width:97px;">#430661</td>
			<td style="width:211px;">Alternative cryogenic tubes can be used.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:207px;">Cryopreservation medium</td>
			<td style="width:179px;">Zenoaq</td>
			<td style="width:97px;"></td>
			<td style="width:211px;">&#34;CELLBANKER1&#34;</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:207px;">Culture medium (for HeLa cells)</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, and penicillin-streptomycin solution (final concentration: Penicillin 100 units/mL and Streptomycin 100 &#181;g/mL)</td>
		</tr>
		<tr height="94">
			<td height="94" style="height:95px;width:207px;">Dissection medium&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">One milliliter of 1 M HEPES (final concentration 20 mM) to 49 mL DMEM&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:207px;">DMEM</td>
			<td style="width:179px;">Nacalai</td>
			<td style="width:97px;">#08456-65</td>
			<td style="width:211px;">Alternative DMEM can be used.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">DMEM</td>
			<td style="width:179px;">Nacalai</td>
			<td style="width:97px;">#08456-65</td>
			<td style="width:211px;">Low glucose</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">DNA transfection reagent</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:97px;">#6366244001</td>
			<td style="width:211px;">&#34;X-tremegene HP DNA transfection reagent&#34; 
			Alternative transfection reagents can be used.</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:207px;">Glass jar with a lid</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">500 mL jar (for mouse) or 1500 mL jar (for rat) to anesthetize the animal&#160;</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:207px;">HBSS</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:97px;">#14170161</td>
			<td style="width:211px;">HBSS free of calcium and magnesium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Heat inactivated bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>#10100147</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:207px;">HeLa cells</td>
			<td style="width:179px;">RIKEN BioResource Center</td>
			<td style="width:97px;">#RCB0007</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Histamine</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:97px;">#H7125</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Image analysis software</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Such as Metamorph (Molecular Devices), Image J (NIH), and TI Workbench14 (custom made)&#160;</td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:207px;">Image splitting optics</td>
			<td style="width:179px;">Hamamatsu Photonics</td>
			<td style="width:97px;">#A12801-01</td>
			<td style="width:211px;">W-view GEMINI</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Image splitting optics dichroic mirror</td>
			<td style="width:179px;">Semrock</td>
			<td style="width:97px;">#FF560-FDi01-25&#215;36</td>
			<td style="width:211px;">For separation of green fluorescent protein/red fluorescent protein&#160; (GFP/RFP) signal</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Image splitting optics emission filters</td>
			<td style="width:179px;">Semrock</td>
			<td style="width:97px;">#FF01-512/25-25, #FF01-630/92-25</td>
			<td style="width:211px;">For emission of GFP/RFP signal, respectively</td>
		</tr>
		<tr height="164">
			<td height="164" style="height:164px;width:207px;">Imaging medium and buffer</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Use optimal medium or buffer for the experiment. When medium is used, medium without phenol red is desirable to reduce background fluorescence. Add 20 mM HEPES to maintain pH outside of CO2 incubator.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Incubation saline&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Add 1 mL of 1 M HEPES (20 mM) to 49 mL HBSS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Inverted fluorescence microscope</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Such as IX73 (Olympus) or&#160; Eclipse TI (Nikon Instech)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Isoflurane</td>
			<td style="width:179px;">Pfizer</td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Used for anesthesia</td>
		</tr>
		<tr height="130">
			<td height="130" style="height:131px;width:207px;">Maintenance medium (for 4 &#215; 12 well dishes)</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">48.5 mL Neurobasal-A medium supplemented with 1 mL B-27, 500 &#181;L of L-glutamine stock and 25 &#181;L Penicillin-Streptomycin solution.</td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;width:207px;">Maintenance medium for frozen cortical cells (for 1 &#215; 12 well dishes)&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">12.2 mL Neurobasal plus medium supplemented with 250 &#181;L B-27, 125 &#181;L of L-glutamine stock and 6.2 &#181;L Penicillin-Streptomycin solution.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">MEM (Minimum Essential Medium)</td>
			<td>Thermo Fisher Scientific</td>
			<td>#11090-081</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:207px;">Microscope filter set for GCaMP6f imaging</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Appropriate filter for GFP (excitation, 480 &#177; 10 nm; emission, 530 &#177; 20 nm)</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:207px;">Microscope filter set for RCaMP2 imaging</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Appropriate filter for RFP (excitation, 535 &#177; 50 nm; emission, 590 nm long pass)</td>
		</tr>
		<tr height="216">
			<td height="216" style="height:216px;width:207px;">Microscope filter sets for double imaging of RCaMP2 and GCaMP6f</td>
			<td style="width:179px;">Semrock</td>
			<td style="width:97px;">#FF01-468/553-25, #FF493/574-FDi01-25&#215;36, #FF01-512/630-25</td>
			<td style="width:211px;">Dual excitation filter, Dual dichroic mirror, and emission filter for GFP/RFP imaging.</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:207px;">Microscope heating system</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">A heating system to maintain cells at 37&#176;C during the imaging. To avoid drift caused by thermal expansion, heating systems covering the entire microscope itself (e.g., Tokai Hit, Thermobox) are recommended.</td>
		</tr>
		<tr height="200">
			<td height="200" style="height:200px;width:207px;">Microscope light source for excitation</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Mercury lamp (100 W), xenon lamp (75 W), Light-emitting diode (LED) illumination system (e.g., CoolLED Ltd., precisExcite; Thorlabs Inc., 4-Wavelength LED Source; Lumencor, SPECTRA X light engine). In case of mercury lump and xenon lamp, use ND filter to reduce the excitation intensity.</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:207px;">Microscope objective lens</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Plan-Apochromat oil immersion objective with numerical aperture higher than 1.3 is highly recommended for the recording of spontaneous Ca2+ activity in neurons and astrocytes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Neurobasal plus medium</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:97px;">#A3582901</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="174">
			<td height="174" style="height:175px;width:207px;">Neurobasal-A Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:97px;">#10888022</td>
			<td style="width:211px;">Neurobasal plus medium (Thermo Fisher, A3582901) can be used instead of Neurobasal-A medium.</td>
		</tr>
		<tr height="158">
			<td height="158" style="height:159px;width:207px;">PBS(-): Phosphate-buffered saline free of Ca2+ and Mg2+&#160;</td>
			<td style="width:179px;">Fujifilm Wako Pure Chemical Cooperation</td>
			<td style="width:97px;">#164-23551</td>
			<td style="width:211px;">The absence of Ca2+ and Mg2+ is critical not to inhibit the trypsin activity. An alternative to PBS(-) can be used.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">PC and image acquisition software</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Such as Metamorph (Molecular Devices); Micromanager; TI Workbench14.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Penicillin-Streptomycin solution</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:97px;">#15140122</td>
			<td style="width:211px;">Penicillin 10,000 units/mL and Streptomycin 10,000 &#181;g/mL&#160;</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:207px;">Plasmid for Lck-GCaMP6f, Lck-RCaMP2, and OER-RCaMP2 expression under cytomegalovirus promoter 7-9</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Available upon request</td>
		</tr>
		<tr height="280">
			<td height="280" style="height:280px;width:207px;">Plating medium (for 4 &#215; 12 well dishes)</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">48 mL MEM supplemented with 1 mL B-27 supplement, 500 &#181;L L-glutamine stock (final concentration: 2 mM), 500 &#181;L of sodium pyruvate stock (1 mM) and 25 &#181;L Penicillin-Streptomycin solution (penicillin 5 u/mL, streptomycin 5 &#181;g/mL). This concentration of Penicillin-Streptomycin, which is 1/20 of&#160; the concentration recommended by the manufacturer, is critical for neuronal survival.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Recording chamber</td>
			<td style="width:179px;">Elveflow</td>
			<td style="width:97px;">Ludin Chamber</td>
			<td style="width:211px;">This recording chamber is for 18 mm diameter round coverslips.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Reduced serum media</td>
			<td>Thermo Fisher</td>
			<td style="width:97px;">#11058021</td>
			<td style="width:211px;">Opti-MEM</td>
		</tr>
		<tr height="272">
			<td height="272" style="height:272px;width:207px;">Stereomicroscope</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Used to dissect hippocampi. Olympus SZ60 or equivalent stereomicroscopes are available.</td>
		</tr>
		<tr height="220">
			<td height="220" style="height:220px;width:207px;">Surgical instruments</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Standard dissecting scissors to cut the abdomen of a mouse or a rat, tweezers to pinch the uterus, delicate dissecting scissors to cut the uterus and the head of embryo, ring forceps to pinch the embryos, 13-cm curved Semken forceps (Fine Science Tools #11009-13) to extract brains,&#160; 3 forceps with fine tips&#160; (Dumont Inox #5)</td>
		</tr>
		<tr height="197">
			<td height="197" style="height:197px;width:207px;">Transfection reagent for neuron</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:97px;">#L3000008</td>
			<td style="width:211px;">&#34;Lipofectamine 3000&#34; reagent. 
			It is composed of the the &#34;supplement (P3000)&#34; that should be mixed with plasmid DNA in the step 2.2.3, and the &#34;transfection reagent (lipofectamine 3000)&#34; used in the step 2.2.4.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Trypan blue (0.4%)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:97px;">#15250061</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Wash medium for frozen cortical cells&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:97px;"></td>
			<td style="width:211px;">25 mL DMEM, supplemented with 250 &#181;L heat-inactivated fetal bovine serum + 12.5 &#181;L Penicillin Streptomycin.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Wistar rats</td>
			<td style="width:179px;">Japan SLC, Inc</td>
			<td style="width:97px;"></td>
			<td style="width:211px;">Pregnant rats (E18)</td>
		</tr>
	</tbody>
</table>

dissection of local ca2+ signals in cultured cells by membrane-targeted ca2+ indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Lck-RCaMP2 and OER-GCaMP6f were expressed in HeLa cells, and both signals were recorded simultaneously using image-splitting optics, 24 h after transfection (Figure 3A and Video 1). The images were acquired at 10 Hz. Histamine (His, 1 &#181;M), which induces Ca2+ release from the ER, was added during the recording. Upon the application of His, the signal intensity of Lck-RCaMP2 and OER-GCaMP6f increased, as shown by the pseudocolor display of &#916;F/F0, which represents the change from the initial fluorescence intensity (Figure 3B). The time courses of Ca2+ elevation (&#916;F/F0) reported by Lck-RCaMP2 and OER-GCaMP6f were compared in the same region of interest (ROI) (Figure 3C). The &#916;F/F0 values were normalized to their peak values to enable the time course comparison between the two different GECIs, which have different expression levels and distributions. Both sensors reported an oscillation-like Ca2+ elevation. Lck-RCaMP2 and OER-GCaMP6f showed the same time course for Ca2+ elevation in two cells among the five cell types examined (Figure 3C, ROI 1 and 3). However, Ca2+ elevations shown by Lck-RCaMP2 remained at a higher level compared to that shown by OER-GCaMP6f (Figure 3C, ROI 2, 4, and 5). The results indicate that the Ca2+ elevation is prolonged in the vicinity of the plasma membrane, while it is terminated earlier around the ER, which is the source of this Ca2+ signal induced by His stimulation.&#160;
Spontaneous Ca2+ signals from astrocytes in the neuron-astrocyte mixed culture from rat hippocampi (Figure 4A) and cortexes (Figure 4B) were shown by Lck-RCaMP2 and OER-GCaMP6f (Figure 4, Video 2, Video 3, Video 4, and Video 5). Cortical cultures were revived from the frozen stock that was prepared as described in this protocol. Lck-RCaMP2 and OER-GCaMP6f signals were sequentially recorded at 2 Hz from the same cells. Three ROIs were selected in the area that showed Ca2+ elevation by each GECI, and the time course of &#916;F/Fbase (i.e., the fluorescence intensity) changed from the average fluorescence intensity during the entire recording period (Fbase). When the baseline fluorescence is stable and Ca2+ elevations are less frequent, Fbase becomes a useful baseline to detect Ca2+ elevation events. Spontaneous Ca2+ elevations were visible only at the astrocytic process, not at the cell body. This result is consistent with the previous reports on astrocytic spontaneous Ca2+ signals by other GECIs visualized in vitro13 and in vivo14. In both hippocampal and cortical astrocytes, Ca2+ elevations shown by Lck-RCaMP2 (top) were more frequent than those shown by OER-GCaMP6f. This result is consistent with our previous demonstration that Ca2+ elevations in astrocytes due to Lck-GCaMP6f were more frequently detected than those due to OER-GCaMP6f7 and suggests that this notion is also applicable at the single-cell level.
Spontaneous Ca2+ elevations by Lck-GCaMP6f in immature rat hippocampal neurons (10 DIV) were seen at 2 Hz (Figure 5A and Video 6). The time courses of &#916;F/F0 in five different ROIs suggest that these Ca2+ elevations are locally confined to the subcellular domains. Figure 5B (Video 7) shows the Ca2+ responses in mature mouse hippocampal neurons (30 DIV) infected with OER-GCaMP6f-expression AAV vectors. Neurons were stimulated with 100 &#181;M DHPG, which is the agonist for metabotropic glutamate receptors, inducing Ca2+ release. DHPG-induced Ca2+ release due to OER-GCaMP6f was detected.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59246/59246fig1.jpg" /> 
Figure 1: Diagram showing membrane-targeted GECIs. Schematic diagram of plasma membrane-targeted GECIs (Lck-GCaMP6f and Lck-RCaMP2) and outer ER membrane-targeted GCaMP6f (OER-GCaMP6f).&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59246/59246fig2.jpg" /> 
Figure 2: Flowchart for hippocampal and cortical cell preparation, plasmid transfection, and AAV infection. The microscopic images are representative, freshly plated DIV-6 cortical cells (left) and revived cells from the frozen stock (right). Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59246/59246fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59246/59246fig3.jpg" /> 
Figure 3: Example of the simultaneous imaging of Lck-RCaMP2 and OER-GCaMP6f in HeLa cells. (A) Schematic representation of signal separation with image-splitting optics. The same field of view for Lck-RCaMP2 and OER-GCaMP6f is simultaneously projected on the camera. A representative recording acquired at 10 Hz by a CMOS camera is provided in Video 1, and one frame of this recording is shown in panel A (right). (B) Pseudo-color images of &#916;F/F0 for Lck-GCaMP2 (top) and OER-GCaMP6f (bottom). Histamine (His, 1 &#181;M) was added at 0 s. (C) Representative normalized &#916;F/F0 time course of Lck-GCaMP2 (magenta) and OER-GCaMP6f (green). Data were normalized to the maximum &#916;F/F0 value for each plot. The gray bars indicate the timing of His application. Data were analyzed with a custom-made software TI Workbench15. Scale bar&#160;= 50 &#181;m (microscopic image).&#160; <a href="https://www.jove.com/files/ftp_upload/59246/59246fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59246/59246fig4.jpg" /> 
Figure 4: Spontaneous Ca2+ elevation in astrocytes monitored for Lck-RCaMP2 and OER-GCaMP6f expression. (A) Representative hippocampal and (B) cortical astrocytes transfected with Lck-RCaMP2 (top) and OER-GCaMP6f (bottom). Cortical cells were revived from frozen stock cultures. Lck-RCaMP2 and OER-GCaMP6f images were sequentially acquired in the same cell, at 2 Hz, with an EM-CCD camera. The plots on the left show the time courses of &#916;F/Fbase measured in the ROIs indicated in the microscopic image. Data were analyzed with TI Workbench. Actual movies are provided in Video 2, Video 3, Video 4, and Video 5. The scale bar = 20 &#181;m (microscopic image). The baseline drift suggests the changes in the global Ca2+ level in the cell.&#160; <a href="https://www.jove.com/files/ftp_upload/59246/59246fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59246/59246fig5.jpg" /> 
Figure 5: Examples of Ca2+ imaging in neurons with Lck-GCaMP6f and OER-GCaMP6f. (A) Representative rat hippocampal neurons expressing Lck-GCaMP6f at DIV 10 (left) and plots showing the time courses of &#916;F/F0 measured in the ROIs (yellow circles) have been indicated in the image (right). The numbers in the time course correspond to the ROI numbers in the image. Note that the temporal pattern of Ca2+ elevation is different among the various regions of interest. The baseline drift suggests the increase in the global Ca2+ level in this neuron. (B) An example of mature mouse hippocampal neurons (DIV 30) infected with OER-GCaMP6f expression AAV vectors (left). The time course plot of &#916;F/F0 measured shows the Ca2+ response to 100 &#181;M (RS)-3,5-dihydroxyphenylglycine (DHPG) applied at the timing shown by the gray bar. Yellow circles show the position of ROIs where the time course was obtained. The images were acquired at 2 Hz with a cooled-CCD camera (panel A) or an EM-CCD camera (panel B) and analyzed with TI Workbench. Scale bar = 20 &#181;m (microscopic image). <a href="https://www.jove.com/files/ftp_upload/59246/59246fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/59246/59246vid1.jpg" /> 
Video 1: Example of simultaneous imaging of Lck-RCaMP2 and OER-GCaMP6f in HeLa cells. Representative recording acquired at 10 Hz and presented in Figure 3. Scale bar = 50 &#181;m.&#160; <a href="https://www.jove.com/files/ftp_upload/59246/Video1.mp4" target="_blank">Please click here to download this video.</a>
<img alt="Video 2" src="/files/ftp_upload/59246/59246vid2.jpg" /> 
Video 2: Spontaneous Ca2+ transient observed in Lck-RCaMP2 in hippocampal astrocyte. Representative recording acquired at 2 Hz (Figure 4A), recorded in the same field of view as Video 3. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59246/Video2.mp4" target="_blank">Please click here to download this video.</a>
<img alt="Video 3" src="/files/ftp_upload/59246/59246vid3.jpg" /> 
Video 3: Spontaneous Ca2+ transient observed in OER-GCaMP6f in a hippocampal astrocyte. Representative recording acquired at 2 Hz (Figure 4A), in the same field of view as Video 2. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59246/Video3.mp4" target="_blank">Please click here to download this video.</a>
<img alt="Video 4" src="/files/ftp_upload/59246/59246vid4.jpg" /> 
Video 4: Spontaneous Ca2+ transient observed in Lck-RCaMP2 in a cortical astrocyte Representative recording acquired at 2 Hz (Figure 4B), recorded in the same field of view as Video 5. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59246/Video4.mp4" target="_blank">Please click here to download this video.</a>
<img alt="Video 5" src="/files/ftp_upload/59246/59246vid5.jpg" /> 
Video 5: Spontaneous Ca2+ transient observed in OER-GCaMP6f in a cortical astrocyte. Representative recording acquired at 2 Hz (Figure 4B), in the same field of view as Video 4. The scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59246/Video5.mp4" target="_blank">Please click here to download this video.</a>
<img alt="Video 6" src="/files/ftp_upload/59246/59246vid6.jpg" /> 
Video 6: Example of Ca2+ imaging in a rat hippocampal neuron (DIV 10) by Lck-GCaMP6f. Example of neuronal Ca2+ signals recorded at 2 Hz (Figure 5A). Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59246/Video6.mov" target="_blank">Please click here to download this video.</a>
<img alt="Video 7" src="/files/ftp_upload/59246/59246vid7.jpg" /> 
Video 7: Ca2+ release in a mouse hippocampal neuron (DIV 30) expressing OER-GCaMP6f. Example of neuronal Ca2+ signals recorded in a mouse hippocampal neuron infected with OER-GCaMP6f expression AAV vectors (Figure 5B). The neuron was stimulated with 100 &#181;M dihydroxyphenylglycine (DHPG) applied at 30 s to evoke Ca2+ release from the ER. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59246/Video7.mov" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators at JoVE.com

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Dissezione del locale Ca2 + segnali in cellule coltivate di targeting per membrana Ca2 + indicatori

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

dissection-local-ca2-signals-cultured-cells-membrane-targeted-ca2

Research

JoVE Journal

Neuroscience

8.8K Views.  PRESTO. La dissezione dei segnali di calcio a risoluzione subcellulare, è uno dei passaggi più importanti per decodificare i segnali di calcio intracellulare che determinano il fenomeno biologico di uscita. Questo protocollo descrive un nuovo metodo di imaging del calcio che consente il monitoraggio del momento stesso dell'afflusso di calcio e del rilascio di calcio. Questo protocollo è applicabile a tutti i tipi di cellule che consentono l'espressione di indicatori di calcio geneticamente codific...

Video: Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Ricerca

Neuroscienze

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp Misure di capacità e Ca 2 + Imaging terminazioni nervose della retina singola in Fette

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Imaging intracellulare Ca<sup&gt; 2+</sup&gt; Segnali di striatale astrociti da topi adulti utilizzando indicatori di calcio geneticamente codificate

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imaging del sistema nervoso enterico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imaging Ca<sup&gt; 2+</sup&gt; Dinamica a cono fotorecettore Axon Terminali della retina di topo

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Generation of Local CA1 &amp;#947; Oscillations by Tetanic Stimulation

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

Generazione di locali oscillazioni CA1 γ di Tetanic Stimolazione

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluorescenza e bioluminescenza di subcellulare Ca<sup&gt; 2+</sup&gt; In Aged neuroni dell&#39;ippocampo

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.