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&#8217;s Health) grant K12 HD065879 (B. Gulbransen) and start-up funds from Michigan State University (B. Gulbransen).

NOTE: The following procedures involving tissue from laboratory animals are consistent with the AVMA Guidelines for the Euthanasia of Animals 2013 and were approved in advance by the Michigan State University IACUC.
1. Tissue Preparation
<ol>
	<li>Anesthetize research animal in a chamber containing 2.5% isoflurane in oxygen or by placing 3-5 ml of liquid isoflurane onto an absorbent material on the floor of the chamber, ensuring that a physical barrier prevents animals from direct contact with the isoflurane. Test for the depth of anesthesia by pinching the footpad. 
	NOTE: The depth of anesthesia is deemed appropriate when there is no withdrawal reflex of the hind limb. Once appropriately anesthetized, euthanize the mouse by cervical dislocation</li>
	<li>Place the animal in a supine position and clean abdominal skin with 70% ethanol. Use forceps to pinch abdominal skin at midline and use surgical scissors to make a 6 cm medial incision along the linea alba to expose internal digestive organs.</li>
	<li>Use blunt forceps to locate and expose the ileum inside the peritoneum. Cut the ileal/colon mesentery with scissors and begin unraveling the intestine.</li>
	<li>Once the length of the intestine is adequately unraveled, cut the intestine distal to the stomach and proximal to the cecum for an ileum preparation. For a large intestine preparation, cut the colon distal to the cecum and proximal to the rectum.</li>
	<li>Quickly remove the intestinal segment and place it in a beaker with DMEM/F12 media supplemented with 3 &#956;M nicardipine hydrochloride and 1 &#956;M scopolamine hydrochloride (hereafter referred to as &#8220;media&#8221;) on ice. The addition of these inhibitors facilitates microdissection and subsequent imaging by paralyzing the gut smooth muscle.</li>
	<li>Cut segment of interest (e.g., jejunum, ileum, distal or proximal colon) based on established anatomical markers. Typically utilize tissue from the distal ileum or distal colon. However, use the same basic procedure to isolate, load and image myenteric neurons and glia in all intestinal regions.</li>
	<li>Remove a small segment (4-6 cm) of desired intestine segment and place in a Sylgard&#8211;coated petri dish filled with chilled media.</li>
	<li>Secure the proximal and distal ends of the intestinal segment with insect pins and open the intestinal tube by making a straight, lengthwise cut along the mesenteric border.</li>
	<li>Pin tissue flat under light tension with mucosa side up and carefully dissect away mucosal layer using fine forceps (#5 and #5/45 work best) and very fine spring scissors. 
	NOTE: The removal of the mucosa can be quite traumatic for the ENS if not done properly. For quality preparations, take care to limit abrupt removal of the mucosa by peeling or scraping. The best practice is to lift the mucosa and cut underneath with fine scissors.</li>
	<li>Cut the tissue into smaller preparations (approximately 0.5 cm2) and pin into imaging dishes (4 corners with circular muscle layer facing up) placed on ice with fresh media.</li>
	<li>Carefully dissect away circular muscle by teasing apart with fine tweezers to expose the myenteric plexus. Avoid excessive stretching.</li>
	<li>Place imaging dish back on ice and change solution with fresh media.</li>
	<li>Prepare 2 ml of enzyme mix per dish [Dispase 1 U/ml (4.48 mg/8 ml), Collagenase Type II 150 U/ml (5.45 mg/8 ml) in media].</li>
	<li>Remove dishes from ice and add the enzyme mix from step 1.13.</li>
	<li>Incubate dishes at RT for 15 min with 5% CO2 / 95% air.</li>
	<li>Wash tissue preparations with media 3 times and re-pin corners.</li>
</ol>
2. Loading Fluo-4 Dye
NOTE: Avoid photobleaching by working with limited light while handling fluorescent dyes and tissue loaded with indicator dyes.
<ol>
	<li>Prepare 4 &#956;M Fluo-4 loading solution.
	<ol>
		<li>Add 1.5 ml media and 1.2 &#956;l of a 250 mM probenecid stock to a 1.5 &#956;l aliquot of 4 mM Fluo-4 stock. 4 mM Fluo-4 stock is prepared by adding 11.4 &#956;l of pluronic F-127 (20% in DMSO; supplemented with 0.25% cremaphor-EL) to 50 &#956;g of Fluo-4, AM.</li>
	</ol>
	</li>
	<li>Incubate preps in Fluo-4 loading solution for 45 min in a dark incubator at 37 &#176;C.</li>
	<li>Remove from incubator and wash preparations with media 3 times.</li>
	<li>Exchange media for media containing 200 &#956;M probenecid and incubate 15 min&#160;at 37 &#176;C before imaging. 
	NOTE: Probenecid is a drug that inhibits multidrug resistance transporters in neurons. Addition of this drug inhibits the ability of neurons to extrude dyes and enhances neuronal labeling. Bulk-loading of Ca2+ indicator dyes in the absence of probenecid produces mainly glial cell loading. The addition of probenecid allows for visualization of neuronal and glial responses.</li>
	<li>Prepare Modified Krebs buffer.
	<ol>
		<li>Make a modified Krebs buffer such that the final concentrations (in mM) of the components are as followed: 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 10 HEPES, 21.2 NaHCO3, 1 pyruvic acid and 8 glucose (pH adjusted to 7.4 with NaOH). Add 3 &#956;M nicardipine and 1 &#956;M scopolamine to inhibit muscle contractions during Ca2+ imaging and whole-mount dissections.</li>
	</ol>
	</li>
</ol>
3. Imaging and Analysis
NOTE: Use at least a basic imaging rig with a fluorescent light source, microscope, a quality CCD camera and appropriate acquisition software. Vary the addition of other components depending on the light source and specific application. A filter wheel and shutter must be used with a traditional xenon arc light source. However, LED light sources and illumination systems do not require those components.
<ol>
	<li>Position the recording chamber under the microscope and using a gravity flow perfusion system with multiple heated syringe reservoirs establish a continuous perfusion rate of 2&#8211;3 ml/min of 37 &#176;C Krebs buffer. Make sure to prevent air bubble formation in both the input and suction line connected to a vacuum trap.</li>
	<li>Bring the desired plexus into focus under bright field illumination. Avoid overexposing tissue, which may lead to photo bleaching.</li>
	<li>Examine the Fluo-4 loading within ganglia and select healthy ganglia for imaging. Unhealthy/damaged ganglia will exhibit autofluorescence or punctate morphology and should not be used for imaging.</li>
	<li>Once ganglion is selected, divert light path to camera and obtain live image with image acquisition software. Ensure that ganglion is in focus and set image acquisition rate and exposure times. 
	NOTE: Image acquisition rates and times will vary depending on the events investigators wish to record. For most experiments, images are traditionally acquired at 0.5-1 Hz for glial cells and up to 2-10 Hz for neurons because glial Ca2+ responses are not as rapid as Ca2+ transients in neurons.</li>
	<li>Begin experiment and establish baseline activity for 30 sec.</li>
	<li>Apply pre-warmed drugs of interest such as receptor agonists and antagonists using the gravity flow perfusion system at a rate of 2-3 ml/min. Follow application of agonists/antagonists by returning to a perfusion of normal buffer and allow a wash/recovery period of at least 10 min. 
	NOTE: The drug application times will vary depending on the individual compound and experimental design. In general, a 20-30 sec application of agonist is sufficient to activate G-protein coupled receptors in neurons and glial cells. However, ligand-gated ion channels (such as nicotinic acetylcholine receptors) require application times of no more than 5-10 sec. Further duration exposures will cause ligand gated ion channels to rapidly desensitize. Antagonists should be applied for approximately 3-15 min to ensure complete blockade of receptor pathways. However, this is a gross generalization and investigators should always optimize any experimental drug in their particular paradigm.</li>
	<li>Stop recording and view time-lapse movie of the experiment. Carefully select regions of interest (ROI&#8217;s) using the appropriate image analysis software.</li>
	<li>Use software to normalize and compare ROI fluorescent intensity against its initial baseline fluorescent value. Changes in normalized fluorescence are directly proportional to changes in [Ca2+].
	<ol>
		<li>Use a modification of a method described previously 26 using &#916;F/F = ((F1&#8722; F0)/F0)ROI &#8722; ((F1&#8722; F0)/F0)background, where F1 is the fluorescence at any given point and F0 is the baseline fluorescence, to improve the assessment accuracy. This modification aids in reducing noise from fluorescence changes in tissue preparations that exhibit movement of the muscle layer underlying the myenteric plexus.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 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 &#8220;listen&#8221; 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.

<table><tbody><tr><td>BubbleStop Syringe Heater&amp;nbsp;</td><td>AutoMate Scientific</td><td>10-4-35-G</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C3306</td><td></td></tr><tr><td>Collagenase, Type II, powder</td><td>Gibco</td><td>17101-015</td><td></td></tr><tr><td>Dispase</td><td>Sigma-Aldrich</td><td>42613-33-2</td><td></td></tr><tr><td>Dissection tools</td><td>Roboz&amp;nbsp;</td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Fixed-stage microscope</td><td>Olympus&amp;nbsp;</td><td>BX51WI</td><td></td></tr><tr><td>Fluo-4 AM dye</td><td>Invitrogen</td><td>F-14201</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Insect pins</td><td>Fine Science Tools</td><td>Minutien Pins</td><td></td></tr><tr><td>iQ Live Cell Imaging Software</td><td>Andor</td><td>Andor iQ3</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S8282</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>Neo sCMOS camera</td><td>Andor</td><td>Neo 5.5 sCMOS</td><td></td></tr><tr><td>Nicardipine</td><td>Sigma</td><td>N7510</td><td></td></tr><tr><td>Perfusion chamber</td><td>Custom</td><td></td><td></td></tr><tr><td>Peristaltic pump</td><td>Harvard Apparatus</td><td>Model 720</td><td></td></tr><tr><td>Pluronic F-127&amp;nbsp;</td><td>Invitrogen</td><td>P3000MP</td><td></td></tr><tr><td>Probenecid</td><td>Molecular Probes</td><td>P36400</td><td></td></tr><tr><td>Scopolamine</td><td>Sigma</td><td>S1013&amp;nbsp;</td><td></td></tr><tr><td>Sutter Lambda DG-4</td><td>Sutter</td><td>DG-4</td><td></td></tr><tr><td>Sylgard&amp;nbsp;</td><td>Dow Corning</td><td>184</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments</td><td>TC-344C</td><td></td></tr></tbody></table>

in situ ca2+ imaging of the enteric nervous system

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.

Proper use of this technique allows investigators to accurately measure intracellular Ca2+ [Ca2+]i transients in enteric neurons and glia in whole-mount tissue preparations. A representative example of an agonist-evoked Ca2+ responses in glia within a myenteric ganglion from the mouse colon is shown in Video 1. The following results are meant to illustrate some representative results we have obtained using this method. First, Figure 2 illustrates the results of an experiment measuring enteric glial [Ca2+]i changes in response to stimulation by ATP within guinea pig colonic longitudinal myenteric muscle plexus (LMMP) preparations. Specifically, this figure shows the method for proper analysis of the experimental protocol listed above including the outline of the analyzed myenteric ganglion and asterisks denoting the location of enteric neurons. These results also illustrate the optimal dose of one hundred micromolar ATP on the mobilization of [Ca2+]i in guinea pig myenteric glia. This response may be used to calibrate enteric glial stimulation and normalize responses to test stimuli. Next, Figure 3 elucidates how to properly select regions of interest (ROIs) surrounding glial cells, shown with surrounding yellow circles. These results also show the desired fluorescence changes under basal conditions and in response to pharmacological stimuli. Finally, Figure 4 demonstrates the spatial considerations for choosing enteric glia and neurons for [Ca2+]i responses in whole-mount preparations.
<img alt="Figure 1" src="/files/ftp_upload/52506/52506fig1highres.jpg" /> 
Figure 1.&#160;Organization of the ENS. The ENS contains two major ganglionated plexuses. The myenteric plexus is located between the longitudinal and circular muscle layers. The submucosal plexus is situated between the mucosa and the circular muscle layer. The ENS is solely comprised of neurons and enteric glia. Nerve fiber tracts connect the ganglia. <a href="https://www.jove.com/files/ftp_upload/52506/52506fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52506/52506fig2highres.jpg" /> 
Figure 2.&#160;Enteric glia in the guinea pig colonic myenteric plexus respond to ATP in situ. (A) Fluo-4 fluorescence in a myenteric ganglion (outlined by dashed line) under basal conditions. Arrows point to thick interganglionic fiber tracts. (B) Upon stimulation with 100 &#956;mol/L ATP, glial cells, but not neurons, rapidly increase Fluo-4 fluorescence indicating an increase in [Ca2+]i. Note that responding cells are small and surround the much larger neurons (dark spaces marked by asterisks). (C) Enteric glia respond to ATP in a dose-dependent manner with 1 mmol/L eliciting maximal responses 24. <a href="https://www.jove.com/files/ftp_upload/52506/52506fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52506/52506fig3highres.jpg" /> 
Figure 3.&#160;Murine S-100-GFP+ cells in the colonic myenteric plexus respond to ATP in situ. (A) S-100-GFP+ glial cells (green) in a mouse colonic myenteric ganglion (outlined by dashed line). Six regions of interest (ROIs) surrounding glial cells within the ganglia are shown as yellow circles. Arrows indicate thick fiber tracts leading into the ganglion. Asterisks denote location of 2 enteric neurons. (B) Same ganglion showing Rhod-2 fluorescence under basal conditions. (C) Following stimulation with 100 &#956;mol/L ATP, glial cells respond with increased [Ca2+]i as shown by increased Rhod-2 fluorescence. (D) Traces corresponding to each ROI shown in A&#8211;C. (E) Averaged response (mean &#177; SEM) of the 6 ROIs shown in D 24. <a href="https://www.jove.com/files/ftp_upload/52506/52506fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52506/52506fig4highres.jpg" /> 
Figure 4. In situ imaging of enteric neuron-to-glia communication. (A) Representative images (pseudocolored) from a Ca2+ imaging experiment where a whole-mount preparation of the myenteric plexus was challenged with the neuronal P2X7 receptor agonist BzATP (100 &#956;M, 30 sec). Note that the neuronal agonist causes an increase in Fluo4 fluorescence in the neuron (A&#8217;) prior to the surrounding enteric glial cells (A&#8221;). (B) Analysis of the change in fluorescence over time in glia (blue) and neurons (red) following application of the neuronal agonist, BzATP. (C) Neuronal and glial responses to BzATP in normal buffer (solid lines) and in buffer containing low Ca2+ and Mg2+ (dashed lines) to potentiate neuronal P2X7 receptors 13. <a href="https://www.jove.com/files/ftp_upload/52506/52506fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1. Agonist-evoked Ca2+ response in enteric glia in situ. This video shows a myenteric ganglion from the mouse distal colon loaded with the Ca2+ indicator dye, Fluo-4. The glial cell agonist, ADP, is added to the bath when indicated. ADP elicits an increase in intracellular Ca2+ in enteric glia as observed by the transient elevation in Fluo-4 fluorescence. <a href="https://www.jove.com/files/ftp_upload/52506/JOVE.mp4" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about In Situ Ca2+ Imaging of the Enteric Nervous System at JoVE.com

In Situ Ca2+ Imaging of the Enteric Nervous System

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.

In Situ Ca2+ Imaging of the Enteric Nervous System

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 ...

in-situ-ca2-imaging-of-the-enteric-nervous-system

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.1K Views.  Michigan State University. 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. 

Video: In Situ Ca2+ Imaging of the Enteric Nervous System

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Biochemistry

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Calcium Imaging: A Method to Visualize Neural Activity in Live C. elegans

Source: Turek, M. et al. <a href="https://www.jove.com/video/52742/agarose-microchambers-for-long-term-calcium-imaging-caenorhabditis">Agarose Microchambers for Long-term Calcium Imaging of&#160;Caenorhabditis elegans.</a>&#160;J. Vis. Exp.&#160;(2015).
This video introduces the concepts behind calcium imaging and includes an example protocol for time-lapse imaging of worms contained in molded agarose wells.

Source: Turek, M. et al. Agarose Microchambers for Long-term Calcium Imaging of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp.&#160;(2015).
This video ...

In Situ Ca²⁺ Imaging of the Enteric Nervous System

Summary

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Chapters in this video

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

In vivo Neuronal Calcium Imaging in C. elegans

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

In vivo Calcium Imaging in Mouse Inferior Olive

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Subcellular Imaging of Neuronal Calcium Handling In Vivo

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

Calcium Imaging: A Method to Visualize Neural Activity in Live C. elegans