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.

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		<tr height="22">
			<td height="22" style="height:22px;width:391px;">BubbleStop Syringe Heater&#160;</td>
			<td style="width:245px;">AutoMate Scientific</td>
			<td style="width:128px;">10-4-35-G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">CaCl2</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">C3306</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Collagenase, Type II, powder</td>
			<td style="width:245px;">Gibco</td>
			<td style="width:128px;">17101-015</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Dispase</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:128px;">42613-33-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Dissection tools</td>
			<td style="width:245px;">Roboz&#160;</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">DMSO</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:128px;">D5879</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Fixed-stage microscope</td>
			<td style="width:245px;">Olympus&#160;</td>
			<td style="width:128px;">BX51WI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Fluo-4 AM dye</td>
			<td style="width:245px;">Invitrogen</td>
			<td style="width:128px;">F-14201</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Glucose</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">G8270</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Insect pins</td>
			<td style="width:245px;">Fine Science Tools</td>
			<td style="width:128px;">Minutien Pins</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">iQ Live Cell Imaging Software</td>
			<td style="width:245px;">Andor</td>
			<td style="width:128px;">Andor iQ3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">KCl</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">P3911</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">MgCl2</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">M9272</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">NaCl</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">NaH2PO4</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">S8282</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">NaHCO3</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">S6014</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Neo sCMOS camera</td>
			<td style="width:245px;">Andor</td>
			<td style="width:128px;">Neo 5.5 sCMOS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Nicardipine</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">N7510</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Perfusion chamber</td>
			<td style="width:245px;">Custom</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Peristaltic pump</td>
			<td style="width:245px;">Harvard Apparatus</td>
			<td style="width:128px;">Model 720</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Pluronic F-127&#160;</td>
			<td style="width:245px;">Invitrogen</td>
			<td style="width:128px;">P3000MP</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Probenecid</td>
			<td style="width:245px;">Molecular Probes</td>
			<td style="width:128px;">P36400</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Scopolamine</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:128px;">S1013&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Sutter Lambda DG-4</td>
			<td style="width:245px;">Sutter</td>
			<td style="width:128px;">DG-4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Sylgard&#160;</td>
			<td style="width:245px;">Dow Corning</td>
			<td style="width:128px;">184</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:391px;">Temperature Controller</td>
			<td style="width:245px;">Warner Instruments</td>
			<td style="width:128px;">TC-344C</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

Research

JoVE Journal

Neuroscience

17.0K 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

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.