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

Summary

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.

Abstract

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.

Introduction

Ca²⁺ is a ubiquitous intracellular messenger which controls a wide range of cellular processes, such as muscle contraction^1,2, metabolism³, cell proliferation^3,4,5, stimulation of neurotransmitter release at nerve terminals^6,7, and activation of transcription factors in the nucleus.⁷ Intracellular Ca²⁺ signals often take the form of transient elevations in cytosolic Ca²⁺, and thes....

Protocol

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

1. 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).
   NOTE: GCaMP6f was used due to its reported efficiency in reporting localized, brief intracellular Ca²⁺ signals in situ a....

Results

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,

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ImageJ software	NIH	1.5a	ImageJ software
Volumetry	GWH	Volumetry 8Gd	Custom made analysis software
Isoflurane	Baxter, Deerfield, IL, USA	NDC 10019-360-60
Tamoxifen	Sigma	T5648
GCaMP6F mice	Jackson Laboratory	Ai95 (RCL-GCaMP6f)-D
Kit-Cre mice	Gifted From Dr. Dieter Saur	c-Kit+/Cre-ERT2
Ethanol	Pharmco-Aaper	SDA 2B-6