A Zeiss LSM 880 in the Fralin Imaging Center was used to collect the calcium imaging data. We acknowledge Dr. Michelle L Olsen and Yuhang Pan for their assistance with the IMARIS software. We acknowledge Dr. Lenwood S. Heath for constructive comments on the manuscript and Steven Giavasis for comments on the GitHub README file. This work was supported by NIH R21MH122987 (https://www.nimh.nih.gov/index.shtml) and NIH R01GM140130 (https://www.nigms.nih.gov/) to L.N. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Calcium imaging
<ol>
	<li>Fly larvae preparation 
	NOTE: Flies and larvae are maintained at 25 &#176;C under a 12 h:12 h light:dark cycle.
	<ol>
		<li>Anesthetize the flies with CO2. Sort 20-45 males and 20-45 females into each fly vial, and give them at least 24 h to 48 h to recover from the CO2 exposure. 
		NOTE: Fly exposure to CO2 should last for the shortest amount of time possible.</li>
		<li>To synchronize the larvae age, tap over the flies into new vials containing yeast granules, and allow them 4-8 h to lay eggs. Remove the flies by flipping them into new vials.</li>
		<li>Collect the larvae at 72 h using 10 mL of 20% w/v sucrose solution.</li>
	</ol>
	</li>
	<li>Microscope and temperature control setup
	<ol>
		<li>Perform the imaging on a confocal microscope (see the Table of Materials) and a z-axis piezo stage with a stage insert using the following settings: laser, Argon; scan mode, frame; frame size, 512 x 512; speed, maximum; channels/bit depth, 1/8 Bit; zoom, 1.5; z-stack, slice =15, Keep = slice; focus devices and strategy, Definite focus; focus, Definite focus on.</li>
		<li>Attach a Peltier cooling module to a heat sink by the heat transfer compound to build a thermoelectric cooler. The Peltier is powered by a 2 A power supply.</li>
		<li>Attach a thermocouple microprobe to a data acquisition device to record the temperature.</li>
	</ol>
	</li>
	<li>Calcium imaging
	<ol>
		<li>Rinse the larvae 3x in 1x phosphate-buffered saline (PBS).</li>
		<li>Pipette 75 &#956;L of 1x PBS onto the center of a glass slide.</li>
		<li>Put one or two larvae in 1x PBS and place the thermocouple microprobe near the larvae. 
		NOTE: The distance between the larvae and the microprobe should be ~5 mm. The larvae may move if the microprobe is positioned too close to them. A great distance could result in inaccurate temperature readings.</li>
		<li>Cover the larvae and thermocouple microprobe with a glass coverslip. Seal the coverslip with nail polish.</li>
		<li>Place the slide on the microscope stage, find the focus using a 25x objective, and place the thermoelectric cooler on the slide. 
		NOTE: The Peltier is placed directly on the slide where the larvae are to deliver the temperature stimuli.</li>
		<li>In confocal software, focus on the fluorescent cells of interest. Adjust the laser power to avoid oversaturation. Set the first and last slice positions in the z-stack setting. 
		&#8203;NOTE: Use the lowest possible laser power before recording to prevent photobleaching.</li>
		<li>Start the z-stack scanning and temperature recording at the same time.</li>
		<li>Control the power supply that powers the Peltier to change the Peltier surface temperature. Turn on the power supply to decrease the temperature and turn it off to increase the temperature.</li>
		<li>Stop the z-stack scanning and temperature recording.</li>
	</ol>
	</li>
</ol>
2. Analysis of the 3D calcium imaging data
<ol>
	<li>Export the calcium imaging data to TIFF files and save them in a folder with the same name as the base name of the TIFF files inside. 
	NOTE: The filename must not contain any commas.
	<ol>
		<li>Use the following parameters to export the calcium imaging data from ZEN (black edition): file type, TIFF; compression, LZW; channels, 2; Z-position, all; time, all; phase, 1; region, full.</li>
	</ol>
	</li>
	<li>Installation
	<ol>
		<li>Download TACI-Calcium_Imaging.jar from Github (https://github.com/niflylab/TACI_CalciumImagingPlugin/releases).</li>
		<li>Install the plugin in FIJI by clicking on Plugins in the menu bar and then clicking on Install in the dropdown menu (Plugins | Install). Then, restart FIJI. 
		NOTE: Do NOT use Plugins | Install Plugin.</li>
		<li>Run the plugin by clicking on Plugins and then choosing TACI-Calcium Imaging (Plugins | TACI-Calcium Imaging).</li>
	</ol>
	</li>
	<li>Use the RENAME function to convert the TIFF filenames to the required structure. 
	NOTE: The tool uses filename_h#t#z#c#.tif as the default structure (h# and c# are optional; #: a positive integer). If the image filenames are not in the default structure, the RENAME function must be executed.
	<ol>
		<li>Choose the folder in which the TIFF images need renaming by clicking Browse Folders.</li>
		<li>Fill in the parameter information. Five parameters are listed, including Filename, Phase, Max T-Position, Max Z-Position, and Channel. Each parameter has three values: Preceding Text, Parameter Value, and Order. 
		NOTE: The information is case-sensitive. If the image filenames contain a phrase before a parameter, fill in the phrase as the Preceding Text of the corresponding parameter. If the image filenames contain a phrase after all parameters, fill in the phrase as the Post Text.
		<ol>
			<li>Be sure to enter Filename, Max T-Position, and Max Z-Position and that the parameter values for Max T-Position and Max Z-Position include all digits.</li>
			<li>Wait for the Filename to be automatically filled using the folder name.</li>
			<li>If the TIFF filenames do not include the phase and channel, leave the corresponding parameter value blank, and choose Na in Order.</li>
		</ol>
		</li>
		<li>Click Rename to create a folder with the same name and _r. Observe that in the folder, the TIFF filenames have been restructured to be compatible with the ORGANIZE function. 
		NOTE: _r has been added to the filenames of the TIFF images.</li>
	</ol>
	</li>
	<li>Use the ORGANIZE function to save the TIFF images from the same z-position in one folder. 
	NOTE: The folder name must have the same name as the base name of the TIFF files inside and must NOT have any commas.
	<ol>
		<li>Choose the folder in which the TIFF images need organizing by clicking Browse Folders.</li>
		<li>If the parameter CSV file (param.csv) exists, wait for the parameter values to be filled in automatically. 
		NOTE: The param.csv file has a required format. The parameters, including filename, phase, position_t, position_z, channel, and is_gray, must be filled in in row 1 from left to right starting from column A. Corresponding values for each parameter must be filled in in row 2.</li>
		<li>If the parameter CSV file (param.csv) does not exist, manually fill in the parameter values. Ensure that the parameter values of Phase and Channel include letters, while the parameter values of T Position and Z Position should be the largest numbers of the t- and z-positions. If the image filenames do not include the phase or channel, enter Na.</li>
		<li>Create grayscale TIFF images when needed. Leave the box of Are images gray? unchecked to grayscale the images.</li>
		<li>Click Organize to create a folder with the same name and _gray_stacks and to generate folders with the same name and _# (#: z-positions) in the folder. Observe that the TIFF files are sorted into corresponding folders by z-positions and that a file named param.csv is generated, in which the parameters and their values can be found.</li>
	</ol>
	</li>
	<li>Use TrackMate in FIJI to extract the fluorescence intensities of the cells of interest from each z-position. 
	NOTE: This step can also be accomplished by other imaging software.
	<ol>
		<li>Open the TIFF images in a z-position folder by FIJI.</li>
		<li>Run TrackMate by clicking on Plugins | Tracking | TrackMate, and adjust the following parameters if necessary.
		<ol>
			<li>Use DoG or LoG detectors.</li>
			<li>Change the blob diameter, threshold, and median filter. Adjust the blob diameter to be similar to the diameter of the cells. If the cells are oval, adjust the blob diameter to be similar to the minor axis. 
			NOTE: Increasing the threshold helps avoid background noise being picked up as signals without affecting the signal intensities (Supplementary Figure 1). However, an increase in the threshold may miss true signals (Supplementary Figure 1). If the signals are strong, use the median filter to decrease the Salt and Pepper noise.</li>
			<li>Set the filters to remove some, if not all, of the irrelevant signals. Filters X and Y are spatial filters. Use filters X and Y to remove the irrelevant signals that are distant from the real signals. 
			NOTE: When filters are set on one image, it is crucial to check all the other images to ensure that the real signals are not removed.</li>
			<li>Set the linking max distance, gap-closing max distance, and gap-closing max frame gap. Set the linking max distance and gap-closing max distance to be 3x-5x the blob diameter, especially when the samples move significantly over time, to help decrease the number of tracks. Set the gap-closing max frame gap to the number of images in the stack.</li>
			<li>Export the fluorescence intensities of the regions of interest (ROIs) to a CSV file. If an old TrackMate version is used, choose Export all spots statistics in the Select an action window. If the TrackMate version is 7.6.1 or higher, choose the Spots in the Display options window. Export or save as the interactive files to CSV files. 
			NOTE: Both files are interactive with the image window; highlighting an ROI displays the corresponding ROI in the image window. These files include the mean intensities (MEAN_INTENSITY or MEAN_INTENSITY_CH1) of the cells of interest at corresponding time points (POSITION_T). The same TRACK_IDs should represent the same ROIs at different time points. However, this is not always true and may need to be corrected manually, when necessary. If TrackMate does not recognize the ROIs at some time points, those time points are not displayed.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Extract the background fluorescence intensity, and create the Background_list.csv file. 
	NOTE: In this study, the background intensity for each z-position was estimated by using the average value of three to five neighboring same-size blobs that did not contain fluorescence signals and were from different time points.
	<ol>
		<li>The Background_list.csv file has a required format: each column contains the information of one neuron, starting from Neuron 0. Fill in the neuron numbers, such as Neuron 0, in row 1. Then, provide the background intensity for each z-position analyzed-if five z-positions are analyzed for Neuron 0, fill in five background intensity values below Neuron 0. 
		NOTE: The Background_list.csv file is required for the TACI EXTRACT function. If the background is negligible, the Background_list.csv with the zero-background intensity of every neuron must be provided.</li>
	</ol>
	</li>
	<li>Use the EXTRACT function to identify the maximum fluorescence intensities at each t-position and calculate &#916;F/F0&#160;as shown in equation (1). 
	<img alt="Equation 1" src="/files/ftp_upload/64953/64953eq02.jpg" style="margin: auto;" />&#160; &#160; (1)
	<ol>
		<li>Create a folder and name it using the neuron number, starting from Neuron 0. Save the CSV files containing the fluorescence information of the corresponding neuron in the folder. Each CSV file contains the information of one z-position, so the number of CSV files equals the number of z-positions analyzed.&#160;Name the CSV files as Mean_Intensity#.csv (# represents the z-position)&#160;and each CSV file includes at least two columns: POSITION_T and MEAN_INTENSITY or MEAN_INTENSITY_CH1. 
		NOTE: Create a folder for every cell of interest.</li>
		<li>Save the Background_list.csv file and folders created in step 2.7.1 in one folder. Choose this folder by clicking on Browse Files. 
		NOTE: The number of background values in the Background_list.csv file must match the number of the Mean_Intensity#.csv files for each neuron.</li>
		<li>The Background File is automatically filled in. Fill in the largest number of t-positions for the Number of T Positions.</li>
		<li>Click Extract to create a results folder, including the CSV files and plots for each neuron. The CSV files include information on the maximum fluorescence intensity and &#916;F/F0 at each t-position. The plots are line charts of &#916;F/F0 over t-positions. 
		NOTE: In this study, &#916;F/F0 was calculated using equation (1). The first value of each z-position was used as F0. If this F0 is not appropriate20, the plugin provides files including raw data for each neuron in the python_files folder.</li>
	</ol>
	</li>
	<li>Use the MERGE function to average each neuron&#39;s &#916;F/F0, calculate the SEM, and plot the average &#916;F/F0 over t-positions.
	<ol>
		<li>Choose the results folder created by the EXTRACT function by clicking on Browse Files.</li>
		<li>Fill in the Number of T Positions with the largest number of t-positions.</li>
		<li>Click Merge to create a merged_data folder, including a merged_data.csv file and an Average_dF_F0.png plot. The CSV file includes the information on the average and SEM &#916;F/F0 at each t-position. The plot is a line chart of the average &#916;F/F0 over t-positions.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Grienberger, C., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+in+neurons.">Imaging calcium in neurons.</a> Neuron. 73 (5), 862-885 (2012).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nature Biotechnology. 19 (2), 137-141 (2001).</li><li>Zhang, 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=jGCaMP8+fast+genetically+encoded+calcium+indicators.">jGCaMP8 fast genetically encoded calcium indicators.</a> Janelia Research Campus. , (2020).</li><li>Robbins, M., Christensen, C. N., Kaminski, C. F., Zlatic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+analysis+-+How+far+have+we+come.">Calcium imaging analysis - How far have we come.</a> F1000Research. 10, 258 (2021).</li><li>Oh, J., Lee, C., Kaang, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+analysis+of+genetically+encoded+calcium+indicators+linking+neural+circuits+and+behaviors.">Imaging and analysis of genetically encoded calcium indicators linking neural circuits and behaviors.</a> The Korean Journal of Physiology &amp; Pharmacology. 23 (4), 237-249 (2019).</li><li>Stringer, C., Pachitariu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+processing+of+neural+recordings+from+calcium+imaging+data.">Computational processing of neural recordings from calcium imaging data.</a> Current Opinion in Neurobiology. 55, 22-31 (2019).</li><li>Pnevmatikakis, E. A., Giovannucci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NoRMCorre:+An+online+algorithm+for+piecewise+rigid+motion+correction+of+calcium+imaging+data.">NoRMCorre: An online algorithm for piecewise rigid motion correction of calcium imaging data.</a> Journal of Neuroscience Methods. 291, 83-94 (2017).</li><li>Nguyen, J. P., Linder, A. N., Plummer, G. S., Shaevitz, J. W., Leifer, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatically+tracking+neurons+in+a+moving+and+deforming+brain.">Automatically tracking neurons in a moving and deforming brain.</a> PLoS Computational Biology. 13 (5), 1005517 (2017).</li><li>Lagache, T., Hanson, A., P&#233;rez-Ortega, J. E., Fairhall, A., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMC2:+A+versatile+algorithm+for+robust+tracking+of+calcium+dynamics+from+individual+neurons+in+behaving+animals.">EMC2: A versatile algorithm for robust tracking of calcium dynamics from individual neurons in behaving animals.</a> bioRxiv. , (2021).</li><li>Giovannucci, A., 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=CaImAn+an+open+source+tool+for+scalable+calcium+imaging+data+analysis.">CaImAn an open source tool for scalable calcium imaging data analysis.</a> Elife. 8, 38173 (2019).</li><li>Delestro, 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=In+vivo+large-scale+analysis+of+Drosophila+neuronal+calcium+traces+by+automated+tracking+of+single+somata.">In vivo large-scale analysis of Drosophila neuronal calcium traces by automated tracking of single somata.</a> Scientific Reports. 10, 7153 (2020).</li><li>Cantu, D. A., 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=EZcalcium:+Open-source+toolbox+for+analysis+of+calcium+imaging+data.">EZcalcium: Open-source toolbox for analysis of calcium imaging data.</a> Frontiers in Neural Circuits. 14, 25 (2020).</li><li>Eglen, S. J., 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=Toward+standard+practices+for+sharing+computer+code+and+programs+in+neuroscience.">Toward standard practices for sharing computer code and programs in neuroscience.</a> Nature Neuroscience. 20 (6), 770-773 (2017).</li><li>Pachitariu, 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=Suite2p:+Beyond+10,000+neurons+with+standard+two-photon+microscopy.">Suite2p: Beyond 10,000 neurons with standard two-photon microscopy.</a> bioRxiv. , (2017).</li><li>Corder, G., 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=An+amygdalar+neural+ensemble+that+encodes+the+unpleasantness+of+pain.">An amygdalar neural ensemble that encodes the unpleasantness of pain.</a> Science. 363 (6424), 276-281 (2019).</li><li>Lagache, T., Hanson, A., P&#233;rez-Ortega, J. E., Fairhall, A., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+calcium+dynamics+from+individual+neurons+in+behaving+animals.">Tracking calcium dynamics from individual neurons in behaving animals.</a> PLoS Computational Biology. 17 (10), 1009432 (2021).</li><li>Kolar, K., Dondorp, D., Zwiggelaar, J. C., H&#248;yer, J., Chatzigeorgiou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesmerize+is+a+dynamically+adaptable+user-friendly+analysis+platform+for+2D+and+3D+calcium+imaging+data.">Mesmerize is a dynamically adaptable user-friendly analysis platform for 2D and 3D calcium imaging data.</a> Nature Communications. 12, 6569 (2021).</li><li>Moein, 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=CaSiAn:+A+Calcium+Signaling+Analyzer+tool.">CaSiAn: A Calcium Signaling Analyzer tool.</a> Bioinformatics. 34 (17), 3052-3054 (2018).</li><li>Zhou, P., 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=Efficient+and+accurate+extraction+of+in+vivo+calcium+signals+from+microendoscopic+video+data.">Efficient and accurate extraction of in vivo calcium signals from microendoscopic video data.</a> Elife. 7, 28728 (2018).</li><li>Neugornet, A., O'Donovan, B., Ortinski, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+effects+of+event+detection+methods+on+the+analysis+and+interpretation+of+Ca(2+)+imaging+data.">Comparative effects of event detection methods on the analysis and interpretation of Ca(2+) imaging data.</a> Frontiers in Neuroscience. 15, 620869 (2021).</li><li>Tinevez, J. 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=TrackMate:+An+open+and+extensible+platform+for+single-particle+tracking.">TrackMate: An open and extensible platform for single-particle tracking.</a> Methods. 115, 80-90 (2017).</li><li>Schindelin, J., 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Fazeli, 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=Automated+cell+tracking+using+StarDist+and+TrackMate.">Automated cell tracking using StarDist and TrackMate.</a> F1000Research. 9, 1279 (2020).</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>Ni, L., 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=The+ionotropic+receptors+IR21a+and+IR25a+mediate+cool+sensing+in+Drosophila.">The ionotropic receptors IR21a and IR25a mediate cool sensing in Drosophila.</a> Elife. 5, 13254 (2016).</li><li>Omelchenko, A. A., 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=Cool+and+warm+ionotropic+receptors+control+multiple+thermotaxes+in+Drosophila+larvae.">Cool and warm ionotropic receptors control multiple thermotaxes in Drosophila larvae.</a> Frontiers in Molecular Neuroscience. , (2022).</li><li>Sanchez-Alcaniz, J. A., 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=An+expression+atlas+of+variant+ionotropic+glutamate+receptors+identifies+a+molecular+basis+of+carbonation+sensing.">An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing.</a> Nature Communications. 9 (1), 4252 (2018).</li><li>Hernandez-Nunez, L., 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=Synchronous+and+opponent+thermosensors+use+flexible+cross-inhibition+to+orchestrate+thermal+homeostasis.">Synchronous and opponent thermosensors use flexible cross-inhibition to orchestrate thermal homeostasis.</a> Science Advances. 7 (35), (2021).</li><li>Klein, 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=Sensory+determinants+of+behavioral+dynamics+in+Drosophila+thermotaxis.">Sensory determinants of behavioral dynamics in Drosophila thermotaxis.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (2), 220-229 (2015).</li></ol>

The authors have no conflicts of interest to disclose.

This study developed a new ImageJ plugin, TACI, and described a workflow analyzing 3D calcium imaging. Many currently available tools focus on correcting the x/y motion, although motion on the z-axis also needs to be explicitly diagnosed or corrected6. During image acquisition in a live organism, movement on the z-axis is unavoidable even when the organism is immobilized, and some stimuli, such as temperature change, often cause significant z-drift. Increasing the height of the z-stacks will allow...

The level of intracellular calcium is a precise marker of neuronal excitability. Calcium imaging measures the changes in intracellular calcium to understand neuronal activity1. Studies in neuroscience have increasingly used this method due to the development of techniques for measuring intracellular calcium concentration, including genetically encoded calcium indicators (GECIs), such as GCaMP2,3, which can be noninvasively expressed in specific sets of neurons through genetic approaches. The lower costs of lasers and microscope components have also increased the use of calcium imaging4. Importantly, calcium imaging allows for recording and studying single neurons as well as large neuron populations simultaneously in freely moving animals5.
Nevertheless, the analysis of calcium imaging data is challenging because (1) it involves tracking the changes in fluorescence of individual cells over time, (2) the fluorescence signal intermittently disappears or reappears with neuronal responses, and (3) the neurons may move in all directions, specifically in and out of a focal plane or appearing on multiple planes4,6. Manual analysis is time-consuming and becomes impractical as the length of recordings and the number of neurons increases. Various software programs have been developed to accelerate the process of analyzing calcium imaging. Previously, software was designed in a limited experimental context, making it difficult for other laboratories to adopt it. Recent efforts to meet modern standards for software sharing have led to the development of several tools that can consistently analyze calcium imaging data across different groups7,8,9,10,11,12,13,14,15,16,17,18,19. However, most of these tools require programming knowledge and/or depend on commercial software. A lack of programming knowledge and software paywalls deter researchers from adopting these methods. Moreover, many of these tools focus on correcting the x/y motion, although motion on the z-axis also needs to be explicitly diagnosed and corrected6. There is a need for a computational tool to analyze 3D calcium imaging that focuses on neurons exhibiting z-drift and appearing on multiple z-planes. Ideally, this tool should use open-source software and not require programming knowledge to allow other laboratories to readily adopt it.
Here, we developed a new ImageJ plugin, TACI, to analyze 3D calcium imaging data. First, the software renames, if needed, and organizes the 3D calcium imaging data by z-positions. The cells of interest are tracked in each z-position, and their fluorescence intensities are extracted by TrackMate or other computational tools. TACI is then applied to examine the motion on the z-axis. It identifies the maximum value of a z-stack and uses it to represent a cell&#39;s intensity at the corresponding time point. This workflow is suited to analyzing 3D calcium imaging with motion in all directions and/or with neurons overlapping in the lateral (x/y) direction but appearing in different z-positions. To validate this workflow, 3D calcium imaging datasets from fly larval thermosensitive neurons and mushroom neurons in the brain were used. Of note, TACI is an open-source ImageJ plugin and does not require any programming knowledge.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blunt Fill Needel</td>
			<td>BD</td>
			<td>303129</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fisher Scientific&#160;</td>
			<td>10035-04-8</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Carbon dioxide</td>
			<td>Airgas</td>
			<td>UN1013</td>
			<td>Size 200 High Pressure Steel Cylinder</td>
		</tr>
		<tr>
			<td>CO2 bubbler kit</td>
			<td>Genesee</td>
			<td>59-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope LSM880</td>
			<td>Zeiss</td>
			<td>4109002107876000</td>
			<td>An inverted Axio Observer Z1, equipped with 5 lasers, 2 standard PMT detectors, 32-channel GaAsP dectectors, an Airyscan detector, and Definite Focus.2.</td>
		</tr>
		<tr>
			<td>DAQami software</td>
			<td>Measurement Computing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Genesee</td>
			<td>62-113</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Drosophila Agar</td>
			<td>Genesee</td>
			<td>66-111</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs, Inc.</td>
			<td>64-17-5</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Fly line: Ir21a-Gal4</td>
			<td>Dr. Paul Garrity lab</td>
			<td></td>
			<td>A kind gift</td>
		</tr>
		<tr>
			<td>Fly line: Ir21a-Gal80</td>
			<td>Dr. Lina Ni lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly line: Ir68a-Gal4</td>
			<td>Dr. Aravinthan DT Samuel lab</td>
			<td></td>
			<td>A kind gift</td>
		</tr>
		<tr>
			<td>Fly line: Ir93a-Gal4</td>
			<td>Dr. Paul Garrity lab</td>
			<td></td>
			<td>A kind gift</td>
		</tr>
		<tr>
			<td>Fly line: UAS-GCaMP6</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>42750</td>
			<td></td>
		</tr>
		<tr>
			<td>Flypad</td>
			<td>Genesee</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>General purpose forged brass regulator</td>
			<td>Gentec</td>
			<td>G152</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco PBS pH 7.4 (1x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Green Drosophila tubing</td>
			<td>Genesee</td>
			<td>59-124</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat transfer compound</td>
			<td>MG Chemicals</td>
			<td>860-60G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heatsink</td>
			<td>Digi-Key Electronics</td>
			<td>ATS2193-ND</td>
			<td>Resize to 12.9 x 5.5 cm</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>AmScope</td>
			<td>LED-6W</td>
			<td></td>
		</tr>
		<tr>
			<td>Inactive Dry Yeast</td>
			<td>Genesee</td>
			<td>62-108</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Pervical</td>
			<td>DR-41VL</td>
			<td>Light: dark cycle: 12h:12h; temperature: 25 &#176;C; humidity: 40-50% RH.</td>
		</tr>
		<tr>
			<td>Methyl-4-hydroxybenzoate</td>
			<td>Thermo Scientific</td>
			<td>126965000</td>
			<td>Fly food ingrediete</td>
		</tr>
		<tr>
			<td>Micro cover glass</td>
			<td>VWR</td>
			<td>&#160;48382-126</td>
			<td>22 x 40 mm</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-544-2</td>
			<td>25 x 75 x 1.0 mm</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Kleancolor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Drosophila vials</td>
			<td>Genesee</td>
			<td>32-113RL</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective&#160;</td>
			<td>Zeiss</td>
			<td>420852-9871-000</td>
			<td>LD LCI Plan-Apochromat 25x/0.8 Imm Corr DIC M27</td>
		</tr>
		<tr>
			<td>Peltier cooling module</td>
			<td>TE Technology</td>
			<td>TE-127-1.0-0.8</td>
			<td>30 x 30 mm</td>
		</tr>
		<tr>
			<td>Plugs</td>
			<td>Genesee</td>
			<td>49-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>Circuit Specialists</td>
			<td>CSI1802X</td>
			<td>10 volt DC 2.0 amp linear bench power supply</td>
		</tr>
		<tr>
			<td>Princeton Artist Brush Nepture</td>
			<td>Princeton Artist Brush Co.</td>
			<td></td>
			<td>Series 4750, size 2</td>
		</tr>
		<tr>
			<td>Sodium potassium L-tartrate tetrahydrate</td>
			<td>Thermo Scientific</td>
			<td>033241-36</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Stage insert&#160;</td>
			<td>Wienecke and Sinske</td>
			<td>432339-9030-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td>Any stereo microscope works</td>
		</tr>
		<tr>
			<td>T-Fitting</td>
			<td>Genesee</td>
			<td>59-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouple data acquisition device</td>
			<td>Measurement Computing</td>
			<td>USB-2001-TC</td>
			<td>Single channel</td>
		</tr>
		<tr>
			<td>Thermocouple microprobe</td>
			<td>Physitemp</td>
			<td>IT-24P&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Yellow Cornmeal</td>
			<td>Genesee</td>
			<td>62-101</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Z-axis piezo stage</td>
			<td>Wienecke and Sinske</td>
			<td>432339-9000-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

taci: an imagej plugin for 3d calcium imaging analysis

Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging is a widely used technique that requires sophisticated software to obtain reliable results, but many laboratories struggle to adopt computational methods when updating protocols to meet modern standards. Difficulties arise due to a lack of programming knowledge and paywalls for software. In addition, cells of interest display movements in all directions during calcium imaging. Many approaches have been developed to correct the motion in the lateral (x/y) direction.
This paper describes a workflow using a new ImageJ plugin, TrackMate Analysis of Calcium Imaging (TACI), to examine motion on the z-axis in 3D calcium imaging. This software identifies the maximum fluorescence value from all the z-positions a neuron appears in and uses it to represent the neuron&#39;s intensity at the corresponding t-position. Therefore, this tool can separate neurons overlapping in the lateral (x/y) direction but appearing&#160;on distinct z-planes. As an ImageJ plugin, TACI is a user-friendly, open-source computational tool for 3D calcium imaging analysis. We validated this workflow using fly larval thermosensitive neurons that displayed movements in all directions during temperature fluctuation and a 3D calcium imaging dataset acquired from the fly brain.

Workflow of 3D calcium imaging analysis 
In this study, we developed a new ImageJ plugin, TACI, and described a workflow to track z-drift and analyze 3D calcium imaging that pinpoints the responses of individual cells appearing in multiple z-positions (Figure 1). This tool has four functions: RENAME, ORGANIZE, EXTRACT,&#160;and MERGE. First, if the image names are not compatible with the ORGANIZ...

Watch this Scientific Journal Video about TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis at JoVE.com

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes.

Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging ...

taci-an-imagej-plugin-for-3d-calcium-imaging-analysis

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.8K Views.  Virginia Polytechnic Institute and State University. TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes. 

Video: TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

Summary

Explore More Videos

Chapters in this video

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

Functional Calcium Imaging in Developing Cortical Networks

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Subcellular Imaging of Neuronal Calcium Handling In Vivo