Name: applications of spatio-temporal mapping and particle analysis techniques to quantify intracellular ca2+ signaling in situ
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ at JoVE.com

Funding was provided by the NIDDK, via P01 DK41315.

All animals used and the protocols carried out in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno.
1. Generation of KitGCaMP6f Mice
<ol>
	<li>Cross Ai95 (RCL-GCaMP6f)-D (GCaMP6f mice) and c-Kit+/Cre-ERT2 (Kit-Cre mice) to generate ICC specific GCaMP6f expressing animals (Kit-Cre-GCaMP6f mice). ...

Ca2+ imaging of specific types of cells within intact tissues or within networks of cells often reveals complex patterns of Ca2+ transients. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events and kinetics of these events as possible. STM and PTCL analysis provide an opportunity to maximize the amount of quantitative data yielded from recordings of this type.
The narrow, spindle shaped morpholo...

Ca2+ is a ubiquitous intracellular messenger which controls a wide range of cellular processes, such as muscle contraction1,2, metabolism3, cell proliferation3,4,5, stimulation of neurotransmitter release at nerve terminals6,7, and activation of transcription factors in the nucleus.7 Intracellular Ca2+ signals often take the form of transient elevations in cytosolic Ca2+, and these can be spontaneous or arise from agonist stimulation depending on the cell type8. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity, and amplitude can provide the biological information required for intracellular signalling5,7,9. Cytoplasmic Ca2+ signals can result from the influx of Ca2+ from the extracellular space or via Ca2+ release from the endoplasmic reticulum (ER) via Ca2+ release channels such as ryanodine receptors (RyRs) and inositol-tri-phosphate receptors (IP3Rs)10. RyRs and IP3Rs may both contribute to the generation of Ca2+ signals and the concerted opening of these channels, which combined with various Ca2+ influx mechanisms can result in a myriad of Ca2+ signalling patterns that are shaped by the numbers and open probability of Ca2+ influx channels, the expression profile of Ca2+ release channels, the proximity between Ca2+ influx and release channels, and expression and distribution of Ca2+ reuptake and extrusion proteins. Ca2+ signals may take the form of uniform, long lasting, high intensity global oscillations that may last for several seconds or even minutes, propagating intracellular and intercellular Ca2+ waves that may cross intracellular distances over 100 &#181;m10,11,12,13,14,15,16, or more brief, spatially localized events such as Ca2+ sparks and Ca2+ puffs that occur on tens of millisecond timescales and spread less than 5 &#181;m17,18,19,20.
Fluorescent microscopy has been used widely to monitor Ca2+ signalling in isolated and cultured cells and in intact tissues. Traditionally, these experiments involved the use of fluorescent Ca2+ indicators, ratiometric and non-ratiometric dyes such as Fura2, Fluo3/4 or Rhod2, among others 21,22,23. These indicators were designed to be permeable to cell membranes and then become trapped in cells by the cleavage of an ester group via endogenous esterases. Binding of Ca2+ to the high affinity indicators caused changes in fluorescence when the cells and tissues were illuminated by appropriate wavelengths of light. The use of cell permeable Ca2+ indicators greatly enhanced our understanding of Ca2+ signaling in living cells and permitted spatial resolution and quantification of these signals that was not possible by assaying Ca2+ signals through other means, such as electrophysiology. However, traditional Ca2+ indicators have several limitations, such as photobleaching that occurs over extended recording periods24. While newer Ca2+ indicator dyes such as Cal520/590 have greatly improved signal to noise ratios and the ability to detect local Ca2+ signals25, issues with photobleaching can still remain a concern for some investigators26,27,28. Precipitous photobleaching also restricts the magnification, rate of image acquisition, and resolution that can be used for recordings, as increased objective power and higher rates of image acquisition require increased excitation light intensity that increases photobleaching.
These limitations of traditional Ca2+ indicator dyes are exacerbated when recording Ca2+ signals in situ, for example when recording intracellular Ca2+ signals from intact tissues. Due to the problems above, visualization of Ca2+ signals in situ using cell permeable Ca2+ indicators has been limited to low power magnification and reduced rates of image capture, constraining the ability of investigators to record and quantify temporally or spatially restricted subcellular Ca2+ signals. Thus, it has been difficult to capture, analyze, and appreciate the spatial and temporal complexity of Ca2+ signals, which can be important in the generation of desired biological responses, as outlined above. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from selected regions of interest (ROI) within a field of view (FOV). The arbitrary choice of the number, size and position of ROIs, dependent on the whim of the researcher, can severely bias the results obtained. As well as inherent bias with ROI analysis, this approach ignores much of the important signaling information contained in the FOV, as dynamic Ca2+ events within an arbitrarily chosen ROI are selected for analysis. Furthermore, analysis of ROIs fails to provide information about the spatial characteristics of the Ca2+ signals observed. For example, it may not be possible to distinguish between a rise in Ca2+ resulting from a propagating Ca2+ wave and a highly localized Ca2+ release event from tabulations of Ca2+ signals within an ROI.
The advent of genetically encoded Ca2+ indicators (GECIs) has radically changed how Ca2+ imaging can be performed in situ29,30,31,32,33. There are several advantages to using GECIs over dyes. The most important perhaps is that expression of GECI can be performed in a cell specific manner, which reduces unwanted background contamination from cells not of interest. Another advantage of GECIs over traditional Ca2+ indicators is that photobleaching is reduced (as fluorescence and consequentially photobleaching only occurs when cells are active), as compared to dye-loaded specimens, particularly at high magnification and high rates of image capture34. Thus, imaging with GECIs, such as the GCaMP series of optogenetic sensors, affords investigators the ability to record brief, localized sub-cellular Ca2+ signals in situ and investigate Ca2+ signaling in cells within their native environments that have not been possible previously. To maximize recovery of information from such high-resolution recordings, appropriate spatial and temporal analysis of the Ca2+ signals is required. It should be noted that while GECIs can offer some clear advantages, recent studies have revealed that Ca2+ imaging can be successfully performed from large populations of different neurochemical classes of neurons simultaneously using conventional Ca2+ indicators that are not genetically encoded into the animal35. This approach used post hoc immunohistochemistry to reveal multiple different classes of neurons firing at high frequency in synchronized bursts, and avoided the potential that genetic modifications to the animal may have interfered with the physiological behavior the investigator seeks to understand35,36.
The protocols outlined in this paper facilitate more complete analysis and quantification of Ca2+ signals recorded from cells in situ using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signalling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC). ICC are specialized cells in the gastrointestinal (GI) tract that exhibit dynamic intracellular Ca2+ signaling, as visualized using mice expressing GCaMPs37,38,39,40,41,42. Ca2+ transients in ICC are linked to activation of Ca2+-activated Cl- channels (encoded by Ano1) that are important in regulating the excitability of intestinal smooth muscle cells (SMCs)43,44,45. Thus, the study of Ca2+ signaling in ICC is fundamental to understanding intestinal motility. The murine small intestine offers an excellent example for this demonstration, as there are two classes of ICC that are anatomically separated and can be visualized independently: i) ICC are located in the area between the circular and longitudinal smooth muscle layers, surrounding the myenteric plexus (ICC-MY). These cells serve as pacemaker cells and generate the electrical activity known as slow waves46,47,48,49; ii) ICC are also located amongst a plexus rich in the terminals of motor neurons (deep muscular plexus, thus ICC-DMP). These cells serve as mediators of responses to enteric motor neurotransmission37,39,40,50. ICC-MY and ICC-DMP are morphologically distinct, and their Ca2+ signaling behaviors differ radically to accomplish their specific tasks. ICC-MY are stellate in shape and form a network of interconnected cells via gap junctions51,52. Ca2+ signals in ICC-MY manifest as brief and spatially localized Ca2+ release events occurring at multiple sites asynchronously through the ICC-MY network as visualized within a FOV (imaged with a 60X objective)38. These asynchronous signals are organized temporally into 1 second clusters that, when tabulated together, amount to a net 1 s cellular rise in Ca2+. These signals propagate cell-to-cell within the ICC network and therefore organize Ca2+ signaling, generated from sub-cellular sites, into a tissue wide Ca2+ wave. Temporal clustering and summation of Ca2+ signals in ICC-MY has been termed Ca2+ transient clusters (CTCs)38. CTCs occur rhythmically (e.g. quite similar durations and similar periods between CTCs) 30 times per minute in the mouse. Conversely, ICC-DMP are spindle shaped cells, some with secondary processes, that distribute between SMCs and varicose nerve processes and do not independently form a network51,52. ICC-DMP form gap junctions with SMCs, however, and function within this greater syncytium, known as the SIP syncytium53. Ca2+ signals occur at multiple sites along the lengths of cells, but these transients are not entrained or temporally clustered, as observed in ICC-MY37. Ca2+ signals in ICC-DMP occur in a stochastic manner, with variable intensities, durations and spatial characteristics. The protocols below, using the example of Ca2+ signaling in ICC-MY and ICC-DMP, describe techniques to analyze complex signaling in specific types of cells in situ. We utilized the inducible Cre-Lox p&#160;system to express GCaMP6f exclusively in ICC, after inducing activation of Cre-Recombinase (Cre) driven by an ICC specific promoter (Kit).

<table>
	<tbody>
		<tr>
			<td>Image J software</td>
			<td>NIH</td>
			<td>1.5a</td>
			<td>Image J software</td>
		</tr>
		<tr>
			<td>Volumetry</td>
			<td>GWH</td>
			<td>Volumetry 8Gd</td>
			<td>Custom made analysis software</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter, Deerfield, IL, USA</td>
			<td>NDC 10019-360-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>GCaMP6F mice</td>
			<td>Jackson Laboratory</td>
			<td>Ai95 (RCL-GCaMP6f)-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Kit-Cre mice</td>
			<td>Gifted From Dr. Dieter Saur</td>
			<td>c-Kit+/Cre-ERT2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-Aaper</td>
			<td>SDA 2B-6</td>
			<td></td>
		</tr>
	</tbody>
</table>

applications of spatio-temporal mapping and particle analysis techniques to quantify intracellular ca2+ signaling in situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Using Kit-Cre-GCaMP6F mice (Figure 1), dynamic Ca2+ signaling behaviors of ICC in the gastrointestinal tract can be imaged in situ. With confocal microscopy, high-resolution images of specific populations of ICC can be acquired without contaminating signals from other populations of ICC within the same tissue but in anatomically distinct planes of focus (Figure 2A)37,<sup class="xref"...

Watch this Scientific Journal Video about Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ at JoVE.com

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

The capacity to acquire large amounts of data from calcium imaging experiments has drastically improved, especially with tissue imaging experiments. Our protocols describe appropriate analysis techniques to quantify and describe these data sets. Our protocols generate a range of spatial, temporal, and intensity values that are intrinsic to calcium signaling in entire tissues and this can be lost in more rudimentary-based analysis.One hour before the imaging transfer a small intestine harvested from a mouse with interstitial cells of Cajal or ICC that specifically express a genetically-encoded calcium indicator to the stage of a spinning disk confocal microscope. And continuously perfuse the tissue with 37 degrees Celsius Krebs-Ringer bicarbonate or KRB solution. Using a 60 to 100 X magnification locate the stellate-shaped myenteric plexus ICC or ICC-MY between the circular and longitudinal smooth muscle layers of the small intestine.Then locate the spindle-shaped deep muscular plexus ICC or ICC-DMP in a single plane between the circular smooth muscle layer and the submucosal plexus and save the movies as a stack of TIFF images. To create spatiotemporal maps or STMs of the ICC-DMP calcium activity open the image stack in ImageJ, and click Image, Type and 32-bit to modify the image quality. To create a line scan right-click on the line selection tool and select segmented line.Using single left clicks draw a line along the mid access of an individual ICC-DMP, double-clicking when the entire cell has been outlined. Select Image, Stacks and Reslice. A pop-up window will appear.Select Rotate 90 degrees to orient the line scan from left to right so that it can be calibrated and read against time on the x-axis with space on the y-axis and click OK.The spatiotemporal map will appear. To accurately measure the amplitude of the calcium signals from the map, select rectangular selection and draw a region of interest around the area within the STM that displays the most uniform and least intense area of fluorescence. Click Analyze and Histogram to obtain a mean value of the intensity within the selected region of interest and click Edit, Selection, Select All to select the entire STM.Next, click Process, Math and Divide and enter the mean value of the intensity with the selected STM region of interest in the pop-up box. The STM will turn black. Correct this by clicking Image, Adjust, Brightness/Contrast, and Auto in the pop-up window.The line scan will now be calibrated for the amplitude with the intensity of the fluorescence expressed as the fluorescence divided by the zero fluorescence. The STM will display the number of frames in the TIFF stack on the x-axis and the number of pixels representing the length of the cell on the y-axis. To quantify the temporal and spatial data from the calcium signals click Image and Properties and enter the appropriate values to fully calibrate the STM for space and time in the pop-up window.To analyze the individual calcium events click Straight Line and click and drag on the STM to draw a straight horizontal line through the center of a calcium event parallel to the x-axis. Then click Analyze and Plot Profile. A box will appear showing the plot profile of the calcium event.For particle-based analysis of the ICC-MY calcium signaling data, open the movie file in Volumetry and right-click to select STK Filter, Differentiate. Right-click again to input a value to differentiate. Press Enter and left-click to initiate the differentiation.To create particles, use the middle mouse button to scroll through the movie to select a 20 to 40 frame section that includes a quiescent period, followed by the occurrence of a calcium transient cluster and 20 to 40 frames that follow right after the cluster. When all of the frames have been selected right-click in the movie window to access STK OPS Ramp DS particle info and click to run a particle analysis routine that progressively ramps the threshold from the maximum to the minimum intensity. To ensure a successful particle analysis, ensure that you incorporate as much pre-calcium transient cluster non-activity, and as much post-calcium transient cluster non-activity as you can.This will help to boost the signal-to-noise ratio. The analysis will be displayed graphically in the plot window as plot of noise and in the traces window as three color traces with the green trace representing the number of particles, the red trace representing the average particle size, and the blue trace representing the absolute intensity threshold. To histogram balance the plot of particle noise press H on the keyboard.Then press the right bracket key to cycle through color schemes until the background is white with the color traces on top. Press F on the keyboard one time to bring up a measuring tool. Then press F a second time to mark a single vertical line in the scroll bar of the traces window on the plot at the intersection where the colored plots begin to shift to the right-hand side.Within the traces window use the middle mouse button to scroll from the inflection point of the red trace to the marked white vertical line. After noting the displayed Y average, click the middle mouse button again inside the traces window to deselect the blue trace. In the movie window click C to bring up a color wheel, and D to adjust the threshold.Valid particles will now be shown in red, and those that saturate will be white. Use the left mouse button to scroll until the white areas are removed and use the middle mouse button to adjust the yellow numerical value in the color wheel to the Y average value to assign everything in red as an active calcium transient particle at the determined threshold point. To save the data as a coordinate-based particle file right-click to access STK 3D, save particle zero, and left-click to save the file.To create particle activity heat maps open the saved particle file and right-click in the movie window to access particle STKops, StatMap Flag and enter flag assignments to analyze. Click to apply the analysis and a heat map showing the total particles for the entire length of recording with different colors representing the percent occurrence throughout the recording. Right-click to save the heat map as a TIFF file.Then right-click in the movie window to access particle measure, particle stats STK equals, and enter a flag assignment for the analysis. A series of traces will be generated in the trace window. STM analysis provides the ability to monitor and record the spatial characteristics of calcium signaling, such as the spatial spread and propagation velocity.This information can be amassed to provide a rather complete view of calcium signaling behaviors within cells in their native environments. Using particle analysis, quantitative information about calcium signaling can be calculated by measuring the particle area and the particle count, which together can be used to determine the spatial ranges of calcium signal activation within a specified field of view. Particle analysis also allows the in-depth quantification of subcellular calcium signaling through examination of the location and firing probabilities of calcium firing sites.By allocating the particles into different flags based on their temporal characteristics, the initiating particles can be accurately mapped. And a wealth of hard data acquired on the number of initiation sites, the size of the site in pixels and micrometers, probability of each initiation site firing either once or multiple times during each calcium transient cluster, and the percentage of firing sites that fire during each calcium transient cluster cycle can be obtained. Consistency is the key to success in this protocol.All data sets and all movies must be treated exactly the same to ensure reliable, repeatable results. This is especially true when dealing with particle files. Our protocols allow a deep characterization of calcium signaling in situ, and a range of statistical methods, when used appropriately, can assess differences between a range of spatial and temporal parameters.These analysis techniques in our protocol have allowed researchers to ask and answer previously unaddressable questions relating to intestinal pacemaking, and intestinal innervation, as well as the neuronal control of the lower urinary tract.

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