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Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution.
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode.
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.
Careful pinning of the tissue is necessary in order to minimize spontaneous movement of the tissue (a property of imaging of smooth muscle cells from relatively intact tissue) and to facilitate the location of a suitable site for imaging (as a flat piece of tissue may be surveyed in two dimensions, rather than three).
Exposure to excitation illumination will photobleach the indicator, so avoid using for more than a few seconds at a time.
The details of how the confocal microscope is ‘set up’ are specific to each microscope type, but the key considerations are: speed (minimum of 5 Hz is required for most Ca2+ transients); resolution and size of field (both of which are traded-off with speed). Some of the following protocol is specific to a Leica SP2 upright confocal microscope. A typical protocol is: 100 frame series, acquired at 5 Hz (256 x 256 pixels; approx. 100 x 100 μm) or 13.5 Hz (256 x 64 pixels; approx. 100 x 25 μm). The scanning head set to move bi-directionally (to maximize acquisition speed) and acquire at 1000 Hz per line.
Although movement of the imaged site may be minimized with good pinning and evenly-stretched tissue, some smooth muscle organs exhibit spontaneous contractions that cannot be abolished by careful pinning alone. Here we describe the use of a freely available algorithm that aligns or matches frames within a series.
For each ‘site’ imaged, factors will vary that affect the detection of Ca2+ transients. By measuring certain properties of each site (‘particle parameters’ - detailed below), the process of detection is facilitated. Thus for each site, one calculates ‘threshold for subtracted’, ‘threshold for ratio’ and ‘cell edge threshold’.
Oregon Green 488 BAPTA-1 AM was chosen as the Ca2+ indicator because it (i) is bright compared to other indicators (e.g. Fluo-4 and Calcium Green)1; (ii) is sensitive to physiological changes in Ca2+ concentration with a Kd of similar magnitude1 to the intracellular Ca2+ concentration [Ca2+]i (e.g. of vas deferens2); (iii) loads many smooth muscle cells, enabling smooth muscle cell coupling to be monitored. However, Oregon Green 488 BAPTA-1 AM is a non-ratioable Ca2+ indicator, and as such cannot easily be used to determine the absolute [Ca2+]i. Furthermore, it may also buffer Ca2+ if present at high concentrations and becomes saturated with high amplitude changes in [Ca2+]i.
The spontaneous contractility of smooth muscle organs may be reduced pharmacologically, such as by blocking the movement of Ca2+ through L-type Ca2+ channels (e.g. by: nifedipine3; diltiazem4). Note that these drugs will greatly reduce the amplitude of whole-cell Ca2+ flashes / transients.
Contraction of the vas deferens results from a combination of primarily purinergic and noradrenergic neurotransmission. Focal Ca2+ transients in this tissue are, however, insensitive to noradrenergic receptor blockade (e.g. α1 adrenoceptor, by prazosin5) so movement may be reduced without affecting focal Ca2+ transients.
Alternatively, contraction may be prevented ‘downstream’ through the inhibition of myosin light chain kinase e.g. by wortmannin6.
Monitoring Ca2+ fluorescence at submicron resolution is a powerful technique to study the post-junctional effects of neurotransmitter release5,7 and release of Ca2+ from internal stores (e.g. ‘sparks’8). The analysis of Ca2+ transients using the methodology outlined here is cost-effective compared to commercially-available image analysis packages and allows tailor-made analysis through custom-written Java plugins for ImageJ.
Efforts were made to minimize the number of animals used and their suffering; all experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and European Communities Council Directive 86/09/EEC.
The Wellcome Trust provided financial support (VIP Award to J.S.Y. and 074128 Research Fellowship to K.L.B.). Medical Research Council Studentship to R.J.A.
Leica_SP2_Stacker, an algorithm used to import TIFFs for ‘reconstruction’ into a series, is based on Leica_tiff_sequence, a plugin for reading Leica SP2 TIFFs in ImageJ, developed by Tony Collins and used with his permission.
(http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html)StackReg and TurboReg, algorithms used to correct for movement of tissue, are based on Th venaz and colleagues (1998)9.
Excel_writer.jar was written by Kurt De Vos (http://rsb.info.nih.gov/ij/plugins/excel-writer.html).
JNCT0.08JoVE.ijm, the particle detection algorithm, is based on an algorithm written by K.L. Brain and detailed previously5.