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>

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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.

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			<td>Fly line: Ir21a-Gal80</td>
			<td>Dr. Lina Ni lab</td>
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			<td>Fly line: Ir93a-Gal4</td>
			<td>Dr. Paul Garrity lab</td>
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			<td>Fly line: UAS-GCaMP6</td>
			<td>Bloomington Drosophila Stock Center</td>
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			<td>Objective&#160;</td>
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			<td>49-102</td>
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			<td>Circuit Specialists</td>
			<td>CSI1802X</td>
			<td>10 volt DC 2.0 amp linear bench power supply</td>
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			<td>Princeton Artist Brush Nepture</td>
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			<td>432339-9030-000</td>
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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

Research

JoVE Journal

Neuroscience

3.7K 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). ...

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 ...

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 ...

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 ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...