The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.
Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.
The enteric nervous system (ENS) is organized into two ganglionated plexuses embedded within the wall of the digestive tract 1. These intramuscular neural circuits, the myenteric plexus (MP) and submucosal plexus (SMP), are composed of neurons and enteric glia (Figure 1) 2. The MP and SMP regulate gastrointestinal (GI) functions such as intestinal motility and epithelial absorption and secretion, respectively 3. Enteric glia are located in close proximity to neurons within ganglia but populations of enteric glia also exist within interconnecting fiber tracts and extra-ganglionic portions of the gut wall 3,4. Enteric glia were originally believed to provide only nutritive support to neurons. However, recent studies strongly suggest that neuron-glia interactions are essential for ENS functions 5,6. For example, data show that enteric glia “listen” to neuronal activity 7 and modulate neuronal circuits 6,8, protect enteric neurons from oxidative stress 9 and are capable of generating new enteric neurons in response to injury 10,11. The protocol presented in this technical review provides a simple and robust method to examine the complex interplay between neurons and enteric glia using in situ intracellular Ca2+ imaging.
Ca2+ is a ubiquitous signaling molecule in excitable cells and plays an essential role in synaptic signaling events in the nervous system 12. Excitation of neurons or enteric glia elicits an elevation in cytoplasmic Ca2+ concentration either by influx through Ca2+-permeable channels or Ca2+ release from intracellular calcium stores. Imaging Ca2+ transients in neurons and glia with fluorescent dyes is an established and widely used technique to study the functional organization and dynamics of the ENS 13-17. Ca2+ imaging has been shown to be an important tool in studying intact GI tissue segments to elucidate the spread of excitability through ICC pacemaker networks 18 and gut smooth muscle 19,20. It enables researchers to probe a broad spectrum of physiological parameters and provides information about both their spatial distribution and temporal dynamics. Cells can be efficiently stained in a minimally invasive manner by using membrane-permeable fluorescent indicators and optimized staining protocols 21. This offers the opportunity to monitor a large number of neurons and enteric glia in functionally preserved preparations 14-16,22, as well as in vivo 23. Whole-mount tissue preparations are bulk loaded with a high-affinity Ca2+ indicator dye such as Fluo-4 that increases its fluorescence when bound to Ca2+. Changes in fluorescence are recorded by a CCD camera and analyzed digitally 6. The advent of Ca2+ provided the opportunity to monitor neuron and glia cell interactions, responsiveness to various stimuli, and the involvement of these cell types in gastrointestinal processes in real time.
In situ Ca2+ imaging has yielded great insight into the signaling mechanisms of enteric neurons and glia and possesses several distinct advantages over cell culture models 6,24. First, in situ preparations maintain the native matrix environment of neurons and glia and leave the bulk of their connections to target tissue intact. Second, the genetics and morphology of cultured enteric glia are significantly altered compared to in vivo 6,24. Third, many heterotypic interactions are lost in primary cell culture and this limits assessing cell-cell interactions. Although cultured cells are well suited for investigation of fundamental properties, their usefulness for studying complex interactions between enteric glia and neurons is limited. Investigating neuron-glia interplay using an in situ approach is more physiologically relevant as the synaptic pathways remain intact 25. As compared to cell culture approaches, an in situ approach offers improved conditions for systematically understanding the intricate interactions between neurons and enteric glia. Furthermore, the planar organization of the ganglionated plexus in whole-mount preparations is ideal for fluorescent imaging of intracellular Ca2+ transients and this technique provides a straightforward approach for assessing neuron-glia activity in the ENS.
The methodologies described in this manuscript provide a consistent approach to effectively study neurons and enteric glia in the ENS. Although imaging neurons and enteric glia in culture has yielded a wealth of insight into the function of individual cells, studying these cells in their native, multi-cellular environment is crucial for our understanding their physiology and pathophysiology. Fluorescence microscopy is a crucial technique for assessing multidirectional interactions of cells in the ENS. Loading cells of the ENS with selective fluorescent markers and image acquisition with high-resolution microscopy permits quantitative observations of cellular activity in the ENS. Imaging live tissue samples of the ENS is performed relatively quickly and generates large amounts of in-depth functional and spatial data. Mouse myenteric and submucosal plexus preparations used in these experiments allow for molecular and genetic manipulation approaches. Ca2+ imaging in whole-mount preparations provides a useful tool for the assessment of neuron-glia interactions.
In advanced experimental paradigms, several probes can be combined to obtain information about different events within the cells. Fluorescence microscopy can record images with enhanced contrast of specific molecules, if an appropriate fluorescent label is used. Fluo-4 was chosen because it possesses a large dynamic range. Sufficient incubation time is vital when using the AM dyes in ENS. Dye concentration and loading method may need to be adjusted to achieve best results. Ideal preparations should be loaded with sufficient dye to visualize changes in fluorescence but not so much so that the dye chelates the target ions and interferes with intracellular signaling. Exposure to fluorescent light should be limited to prevent phototoxicity in cells and photobleaching of dyes.
Investigators must be careful with several steps of this experiment, especially solution and tissue preparation. Particular care has to be taken during processing and dissection of ENS tissue in order to maintain cellular functions. The GI tract contains several layers and tissue varieties, which pose challenges for dissection and imaging quality in these whole-mount preparations 27. Furthermore, the interconnecting fiber tracts of the MP are wider and ganglia are larger than those of the SMP 2. The neuronal density of the myenteric plexus is higher compared to that of the submucosal plexus 28. Slow and imprecise dissections will have detrimental effects on the quality of the plexus preparations and thus the overall success of the experiments. Therefore, clean/undamaged tools, practice and manual dexterity are critical to this procedure.
In whole-mount tissue preparations, careful consideration should be taken when drawing the regions of interest (ROI) to correctly assess the kinetics and degree of observed change in fluorescence intensity of the desired cell type. As the ganglia are located on a contractile muscle layer, motion artifacts caused by gut motility are a primary concern during in situ imaging. Thus, suppressing these motion disturbances through re-pinning tissue preparations after incubation with enzymes and the addition of pharmacological inhibition (nicardipine/scopolamine) to buffers permits clear and reliable image acquisition. Aside from pharmacology and mechanical approaches to prevent tissue movement, recent studies illustrate the application of advanced software methodologies and cell type response characteristics to correct for residual tissue movement in the recordings and improve the accuracy of analysis 29. Barring these technical hurdles, this method provides physiologically relevant conditions to assess morphologic and quantitative characteristics of neurons and enteric glia in the ENS.
The authors have nothing to disclose.
This work was supported by grants from the Pharmaceutical Research and Manufacturers Association of America (PhRMA) Foundation (to B. Gulbransen), National Institutes of Health (Building Interdisciplinary Research Careers in Women’s Health) grant K12 HD065879 (B. Gulbransen) and start-up funds from Michigan State University (B. Gulbransen).
Name | Company | Catalog Number |
BubbleStop Syringe Heater | AutoMate Scientific | 10-4-35-G |
CaCl2 | Sigma | C3306 |
Collagenase, Type II, powder | Gibco | 17101-015 |
Dispase | Sigma-Aldrich | 42613-33-2 |
Dissection tools | Roboz | |
DMSO | Sigma-Aldrich | D5879 |
Fixed-stage microscope | Olympus | BX51WI |
Fluo-4 AM dye | Invitrogen | F-14201 |
Glucose | Sigma | G8270 |
Insect pins | Fine Science Tools | Minutien Pins |
iQ Live Cell Imaging Software | Andor | Andor iQ3 |
KCl | Sigma | P3911 |
MgCl2 | Sigma | M9272 |
NaCl | Sigma | S9888 |
NaH2PO4 | Sigma | S8282 |
NaHCO3 | Sigma | S6014 |
Neo sCMOS camera | Andor | Neo 5.5 sCMOS |
Nicardipine | Sigma | N7510 |
Perfusion chamber | Custom | |
Peristaltic pump | Harvard Apparatus | Model 720 |
Pluronic F-127 | Invitrogen | P3000MP |
Probenecid | Molecular Probes | P36400 |
Scopolamine | Sigma | S1013 |
Sutter Lambda DG-4 | Sutter | DG-4 |
Sylgard | Dow Corning | 184 |
Temperature Controller | Warner Instruments | TC-344C |