Calcium Imaging Studies in Enteric Glia

Abstract

Calcium (Ca²⁺) imaging is a versatile technique to study cellular activity and is a particularly important tool to study electrically passive cells such as glia. Enteric glia play important roles in gut physiology, and their activity can be captured using Ca²⁺ imaging. Ca²⁺-encoded activities command glial processes such as gliotransmission and intercellular signaling. This chapter describes a protocol that will allow users to study glial Ca²⁺ activity in ex vivo whole-mounts of circular muscle-myenteric plexus. This chapter will include comprehensive steps to execute Ca²⁺ imaging experiments, data extraction, and analysis.

1. Introduction

Calcium ions (Ca²⁺) are ubiquitous intracellular signals that regulate multiple aspects of cell survival and function, including responses to external cues, transcription, metabolism, proliferation, cell motility, and intercellular signaling. As a result, intracellular Ca²⁺ ([Ca²⁺]_i) is under tight control by mechanisms involving channels, pumps, and buffers [1]. Changes in [Ca²⁺]_i are of particular interest in neural systems where fluxes in [Ca²⁺]_i are tied to neuronal and glial processes underlying excitability, neurotransmission, gliotransmission, and synaptic signaling [2, 3]. Therefore, imaging [Ca²⁺]_i has become a core technique neurobiologists use to study cellular activity in neural networks in the brain and periphery. This is particularly important for studies of glial cells, which exhibit passive electrical currents and instead encode activity in the form of variations in [Ca²⁺]_i.

Early studies of glial activity in hippocampal explant cultures used organic Ca²⁺ indicator dyes to demonstrate that glial cells exhibit spontaneous Ca²⁺ events that propagate in patterns throughout cells and eventually form Ca²⁺ waves that spread through glial networks [4, 5]. These and subsequent observations formed the base knowledge that glia are active signaling nodes in neural networks that exhibit activity encoded by Ca²⁺-based signaling mechanisms despite lacking electrical activity similar to neurons. It is now known that glial Ca²⁺ signaling is complex and that distinct subcellular Ca²⁺ responses, intrinsic fluctuations, waves, and microdomains play roles in normal physiology regulated by cells such as astrocytes within neuronal circuits. Therefore, studying Ca²⁺ activity in glia has deepened the understanding of their roles in neural signaling, regulation of neurotransmission, neuroplasticity, neurodegeneration, and behavior [6-8].

Enteric glia are perhaps the largest population of peripheral glia and are housed with enteric neurons in the walls of the intestine. Enteric glia are active signaling cells within enteric neurocircuits that regulate enteric neurotransmission and homeostasis. Similar to astrocytes in the brain, enteric glia exhibit activity encoded by variations in [Ca²⁺]_i [9-12]. Enteric glial Ca²⁺ responses are triggered by numerous external and internal cues such as neurotransmitters, neuromodulators, immune signals, and mechanical force [13, 14]. Mechanisms downstream of glial Ca²⁺ events are involved in key gastrointestinal functions such as motility, secretion, and immune defense. Therefore, recording [Ca²⁺ ]_i in enteric glia has become a core technique to study activity among these cells in the gut.

Several techniques are available to study [Ca²⁺]_i in enteric glia. These include organic, chemical-based dyes and genetically encoded Ca²⁺ sensors. Organic Ca²⁺ indicator dyes are small molecules that are loaded into cells and exhibit changes in fluorescence upon binding to Ca²⁺ [15]. Such dyes are typically used for short-duration experiments because dyes are eventually extruded or degraded over time. Genetically encoded calcium indicators (GECIs) are sensor proteins expressed by cells with fluorescent properties in the presence of Ca²⁺ [16, 17]. GECIs are currently the most widely used method to study Ca²⁺ responses in neural networks and have broad applicability in vitro, in vivo, and ex vivo [18]. The basic GECI system is designed based on a permuted GFP with the Ca-binding protein calmodulin (GCaMP). GECIs’ expression can be targeted to specific cells and/or subcellular domains, and multiple improvements in this system have generated sophisticated GCaMPs for high-speed imaging, increased signal-to-noise ratio, and dynamic range [18-20].

This chapter provides a detailed protocol for Ca²⁺ imaging in enteric glia in live whole-mount preparations of the myenteric plexus using GECIs. Following this protocol will allow investigators to study glial responses evoked by numerous modes of activation in cellular to network level resolution.

2. Materials

2.1. Animals

For WntCre2; GCaMP5g-tdT mouse generation, commercially available mouse lines were acquired and crossed in-house. Transgenic mice expressing the genetically encoded Ca2+ indicator GCaMP5g under the transcriptional control of the Wnt1 promoter (Wnt1Cre2+/−;PC::G5-tdT+/−; hereafter, referred to as WntCre2; GCaMP5g-tdT) were generated by crossing Wnt1Cre2 mice (129S4.Cg-E2f1Tg(Wnt1-cre)2Sor/J (https://www.jax.org/strain/022137; The Jackson Laboratory; RRID: IMSR_JAX:022137) with PC::G5-tdTomato (PC::G5-tdT) mice (catalog #02447, The Jackson Laboratory; B6;129S6-Polr2aTn (pb-CAG-GCaMP5g,-tdTomato)Tvrd/J; RRID: IMSR_JAX:024477). Overall, the WntCre2; GCaMP5g-tdT animal model allows Ca²⁺ activity to be monitored in enteric glia and neurons (Wnt1+ cells). The tdT reporter is highly expressed in enteric glia, enabling high-precision detection of enteric glia in the processing and quantification steps.

2.2. Tissue Extraction and Dissection

1. Modified Krebs solution: 121 mM NaCl, 5.9 mM KCl, 21.2 mM NaCO₃, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 1.2 mM NaHPO₄, 8 mM D-glucose, 10 mM HEPES, and 1 mM sodium pyruvate.

2. Fine surgical tools: Tweezers numbers 5, 7, and 5/45, scissors, micro scissors.

3. Dissection station (C-DS Diascopic Stand S).

4. Sylgard-coated Petri dishes.

5. Insect pins.

2.3. Tissue Incubation

Incubator.

2.4. Ca²⁺ Imaging Setup

1. Wide-field water-immersion objective lens (Olympus XLUMPLFLN20xW, 1.0 numerical aperture) on an upright Olympus BX51WI fixed-stage microscope (Center Valley).

2. Lumencor solid-state LED light source.

3. Photometrics Prime BSI camera.

4. Perfusion system.

5. Automatic temperature controller connected to perfusion pencil system (Warner instruments).

6. Sylgard-coated live imaging engineered dishes.

7. Peri-Star Pro peristaltic pump.

8. 1 mL syringe with valve.

9. Micromanipulator: ROI200, MPC200 (Sutter instruments).

10. Glass micropipette.

2.5. Acquisition

Metamorph.

2.6. Analysis

Fiji software.

3. Methods

3.1. Tissue Extraction and Mucosal Dissection

1. After euthanasia, open the lower abdominal cavity and expose the gastrointestinal tract with the aid of surgical scissors (see Note 1).

2. “Unroll” the small intestine with the help of forceps. Cecum identification makes it possible to reach the colon and the ileum. For ileum preparation, cut the intestine distal to the stomach and proximal to the cecum. Preparation of the large intestine involves cutting the colon above the cecum and below the rectum.

3. Immediately transfer tissue samples to a container of ice-cold Krebs solution and keep on ice. Sylgard-coated dishes are used as a flat, flexible base for fine dissection. Fill the Sylgard dissecting dish with Krebs solution and then transfer the intact piece of intestine (~3–6 cm). The following steps must be performed under a stereomicroscope (C-DS Diascopic Stand S, Nikon).

4. Stretch and secure the intestinal end with insect pins with the mesenteric border facing the operator—this way, this morphological mark will guide the opening of the gut (see Note 2).

5. Use micro scissors to make a straight cut along the mesenteric border from the distal to the proximal end of the intestinal tube.

6. Remove the feces and hold the tissue on the Sylgard-dish using insect pins. The expected orientation is shown in Fig. 1a, with the mucosal layer facing upwards.

7. Pin the tissue flat by creating a uniformly applied vector force stretching the tissue with the same tension and orientation (Fig. 1b).

8. In the most proximal region, start removing the mucosal layer using fine forceps (#5 and #5/45) (Fig. 1d). It is recommended to leave a thin layer of intestinal mucosa on the laterals of the preparation, as this helps to identify smooth muscle orientation (Fig. 1e-f).

Fig. 1.

Schematic illustration of the mucosal dissection for whole-mount circular muscle-myenteric plexus preparations. (a) Upward exposure of the mucosal layer after cutting the mesenteric border. In (b), the organization of the insect’s pins for stretching the intestine. Note: the pins are placed in parallel pairs. Grab the edge of the intestine, and pull it slightly outward and upward, like the arrows representing vector forces. This pattern stretches the intestine with well-distributed tension as seen in (c). To remove the mucosa, define a starting point as the dotted line in (d) and remove pieces of mucosa along this defined line. Finally, dissect gently and very carefully up to the most distal portion. It is expected that at the end of this process, a considerable area of the mucosa will have been dissected, exposing the submucosal plexus

3.2. Smooth Longitudinal Muscle Microdissection

1. Cut small proportional squares of mucosa-free colon samples (~0.5 cm²) and transfer them to the live imaging dish filled with ice-cold Krebs solution (see Note 3) (Fig. 2a).

2. Flip the tissue orientation to reach the smooth longitudinal muscle (Fig. 2b).

3. Start pinning at the four corners with mini-insect pins and then gradually stretch and pin the preparation using the same vector technique as for dissecting the mucosa (Fig. 2c).

4. Identify muscle layer orientation and use fine forceps (#5) with a delicate pinching and pulling movement to remove the longitudinal smooth muscle bundles (see Note 4) (Fig. 2c).

5. Return the live imaging dish containing circular muscle-myenteric plexus preparation (CMMP) to ice until tissue incubation or any tissue treatment is performed. After dissection, you will have a sample containing the following layers from top to bottom: myenteric plexus, circular muscle, and submucosal plexus.

6. Acclimate the cells to the optimal temperature and conditions for cellular processes (e.g., 37 °C/5%CO₂) (see Note 5).

Fig. 2.

Longitudinal muscle dissection. (a) Define areas of ~0.5cm², as illustrated by the gray dotted line, and cut pieces for posterior longitudinal muscle dissection. (b) Flip the tissue to expose the serosa and longitudinal smooth muscle. At this stage, the expected orientation of the layers on the live imaging dish, from top to bottom, is described on the right. (c) Define a starting point (gray dots) and stop (orange dots) for dissecting the longitudinal muscle bundles, and continue dissecting until reaching the opposite side. From the new starting point (orange dotted line), repeat the same movement until a new starting point (green dotted line). Dissect to the bottom to generate the whole-mount circular muscle-myenteric plexus preparation. The dissection method described above is a suggestion; however, other methods of dissection achieve the same goals

3.3. Live Imaging Dish Setup

In this step, CMMP preparations were transferred to a microscope with an external gravity perfusion system setup (see Note 6). The perfusion system allows for a constant supply of warmed solutions. Warmed solutions from the perfusion system flow into the live imaging dishes through an inflow and exit using the outflow created by a peristaltic pump (see Note 7).

1. Fill the perfusion system compartments (e.g., syringes) with Krebs and desired treatment solutions such as agonists, antagonists, and blockers.

2. Turn on Krebs solution perfusion (2–3 mL/min was the inflow rate applied here)

3. Start Krebs solution perfusion for acclimatization for a few minutes.

4. Immerse the water-dipping objective lens in the Krebs solution where the tissue is located. With the help of the bright field, locate the depth at which the myenteric plexus is located. The myenteric plexus has a plexiform structure, with its ganglia interconnected with nerve fibers. Define and center the ganglion of interest to be studied.

5. If not already, move to the red channel to acquire a tdT picture. This picture will be used as a tool for region of interest identification using the animal model described here.

6. Move to the green channel to visualize the cells expressing the GECI GCaMP5.

3.4. Experimental Procedures

This method allows the evaluation of ENS-evoked responses by broad activation of myenteric ganglia.

3.4.1. Bath Application

1. Using the Krebs solution, record a baseline period before stimulation (~30 s).

2. After the baseline period, begin perfusing the desired treatment drugs by switching to the correct channel in the perfusion system.

3. Record until the evoked activity has ended (or following experimental goals).

The compartments of a perfusion system can contain different treatments, such as agonists and antagonists. The application time of these varies, depending on the nature of the target receptors/channels. Depending on the nature of individual experiments, the timing of stimulation and responses may vary because of variables such as the calcium indicator and speed of the perfusion system. For enteric glia stimulation, ADP is a well-documented driver of glial and neuronal calcium activity [21]. Timing and concentration will vary depending on experimental goals. Using ADP 100 μM stimuli for 30 s triggers strong enteric glia and neuron calcium responses.

Several aspects inherent to this activity can be quantified in the Fiji software.

3.4.2. Puffing Setup for Local Drug Application

1. Backfill the glass micropipette with the drug of interest.

2. Position the micropipette in the micromanipulator.

3. Move the micropipette using the micromanipulator (ROI 200, MPC 200) over the sample (see Notes 8 and 9).

4. Reach the target ganglion for stimulation. Raise the objective lens so that the micropipette is positioned below it. Position the micropipette close to the objective lens and find the end of the micropipette. Keep the micropipette tip in focus.

5. Ideally, advance the focus to find the tissue. Then, bring the micropipette into focus. Repeat these steps until to bring the tissue to focus.

6. Position the micropipette tip as close as possible to the target ganglion. A light touch on the muscle helps to check the depth of the micropipette and whether its position is optimal. This positioning must ensure that the drug application will be carried out on the target ganglion and that it will be washed due to the constant bath application (see Note 11).

7. Record a baseline period before local drug application.

8. Open the valve to deliver a volume of drug onto the enteric ganglion. Manual or automatic applications may apply.

9. Close the syringe valve to stop drug application.

10. Record until the evoked activity is ended (or following experimental goals) (Fig. 3a and b).

Fig. 3.

Representative images of Ca²⁺ imaging. (a) High tdT reporter expression in enteric glia. (b) Baseline period before ADP bath application. (B′-B″) Evoked calcium response over time. (c) tdT picture with overlayed regions of interest (ROI) generated by the automatic detection method described in this chapter. Calibration bar: 50 μM. In (d), the peak of Ca²⁺ activity is with the glial ROIs. (e) Zoomed in on the ganglion with glial and neuron ROIs in the overlay. (f) Ca²⁺ traces of enteric glia (cell 36) and neuron (cell 1) activity over time due to ADP stimulation. Note that glia were recruited first, followed by enteric neuron activity. (g) Enteric glial cells’activities over time. (a–c) Calibration bar: 50 μM; (e) calibration bar: 25 μM

Multiple drugs have been used for enteric glial activation and verification of calcium activity, such as ADP (3 mM) and Lysophosphatidic acid (1 μM and 10 μM), while transgenic animals expressing DREADDs sensitive to N-clozapine in GFAP⁺ cells are a specific tool to activate enteric glia only and follow calcium responses in the enteric network.

3.5. Calcium Imaging Parameters

1. To excite GCaMP5g, pass light through a 485/20-nm band-pass filter and filter the emission with a 515-nm long-pass filter.

2. For the tdTomato channel, excite by passing the light through a 535/20-nm band-pass filter and filter the reflected tdTomato (tdT) signals through a 610/75-nm band-pass emission filter before detection.

3. Image acquisition may be controlled by several commercial or open-source platforms, depending on availability and preference. In this example, Metamorph software was used.

4. Define acquisition speed. Based on the animal model used here, a GECI GCaMP5, the acquisition setup was 2 frames/s for 5 min (see Note 10).

3.6. Analysis: Basic Data Extraction Protocol Based on Cell Reporter and Automatic Detection of Regions of Interest

With the high expression of tdT reporter in enteric glia in the animal model reported here, the tdT image of the ganglion identifies enteric glial cells (Fig. 3a and c). Neurons are identified and delineated based on their morphology and/or evoked activity (Fig. 3c and e). Using Fiji software, tdT image processing includes the following steps:

3.6.1. tdT Image Processing

1. Open tdT image.

2. Define a region of interest (ROI) that includes the ganglion that was stimulated and click T. This defined ROI will be added to the ROI Manager.

3. ROI Manager: click More, Save. Later, this ROI will be applied to the recordings to define the quantification areas.

4. Then, click Image → Crop to obtain the image of the ganglion with the cropped ROI.

5. Click Process → FFT → Band-pass filter. The free Fourier transform (FFT) tool should have a setup to filter larger objects to 40 pixels and small objects to 3 pixels, with 5% directional tolerance permitted.

6. Click Adjust → Threshold. The resulting filtered image should be auto-thresholded using the Intermodes threshold algorithm.

7. The resulting binarized image should be analyzed: click Analyze → Analyze particles. The setup should include size: 400-pixel units in size to infinity and with the circularity of 0.0–1.0. The software is expected to create an ROI Manager window containing all ROIs based on tdT image processing. Otherwise, all ROIs should be manually added to the ROI Manager. In the ROI Manager, click More → Save.

3.6.2. Recordings Processing

1. The initially selected and saved ROI of the tdT image must be applied to the respective video, and this area cropped.

2. Apply ROI by dragging the file to Fiji, and it will automatically open in ROI Manager.

3. Apply your saved ROI over the video. Click Image → Crop (see Note 12). Several Fiji plugins, such as “BatchFolderProcessing” and “BatchVideoAlingment,” can be applied to the recording to decrease background and movement.

3.6.3. Data Extraction

To determine which parameter will be extracted from your recordings, click Analyze → Set measurements → Mean Gray Value. Following are the steps to extract pixel values from your pre-determined ROIs:

1. Determine the baseline period and the activity start period (e.g., frames 1–60 as baseline; 61–400 activity).

2. Open the ROI file containing the glial ROIs automatically detected in Subheading 3.6.1.

3. Apply all glial ROIs (Fig. 3d). Align any if necessary by clicking “Show label” in the ROI Manager and moving the ROI.

4. In ROI Manager, click on More → Multimeasure. A result window will be generated with all frames and values for all your ROIs (e.g., cells) throughout the time.

5. For neuronal ROI generation, manually create neuronal ROIs using the cropped tdT or even the recording, by identifying and delineating neuronal cells (Fig. 3e). Then, follow steps 4 and 5 detailed in Subheading 3.6.3.

3.6.4. Signal-to-Baseline Ratio

Create a signal-to-baseline ratio, also known as ΔF/F₀.

This formula measures the variation of emitted fluorescence through time. F₀ represents the baseline fluorescence, while ΔF is the moment-by-moment variation from that baseline.

$$\Delta \mathrm{F}∕\mathrm{F}0=(\mathrm{F}-\mathrm{F}0)∕{\mathrm{F}}_0$$

Multiple analyses can be applied to study and compare calcium activity over time and population recruitment [11, 22, 23] (Fig. 3). To have representative videos, color (“Lookup tables”) and temporal color codes (Image → Hyperstack → Temporal color code) can be applied to (Video 1, see Electronic Supplementary Material). solution into the newly opened abdominal cavity. This will help to separate the adhesion between the tissues and prevent the organs from drying out. A refresh of ice-cold Krebs solution throughout the protocol is required.

4. Notes

1. A careful and delicate tissue extraction and manipulation are essential to maintain the ENS structure and connection with its target cells (smooth muscle). Dispense a small volume of Krebs solution into the newly opened abdominal cavity. This will help to separate the adhesion between the tissues and prevent the organs from drying out. A refresh of ice-cold Krebs solution throughout the protocol is required.

2. This cut, following the orientation of the mesenteric border line, guarantees the maintenance of the structure and orientation of the smooth muscle bundles, essential for microdissection.

3. The proportionality of tissue size will guarantee a good distribution of vector forces and stretching of the preparation. It is recommended to bring the dissection dishes to a vacuum chamber to eliminate any bubbles inside the Sylgard after a few uses. Inserting pins pushes air bubbles into the Sylgard, which can compromise calcium imaging acquisition. Mini insect-pins are custom cuts from a tungsten wire.

4. Refresh the ice-cold Krebs solution. Pull bundles to one point and then restart a new movement to another lower point, and so on. This will ensure that all longitudinal muscles will be microdissected, exposing the myenteric plexus. After longitudinal dissection, the tissue has likely moved or loosened. Repin the corners and where else you deem necessary so that the CMMP is well exposed and stretched.

5. The incubation time may vary, depending on the possible treatment or physiological manipulation that you wish to test on the calcium activity of cells that express GECI. In an example of a control situation, the live dish containing CMMP is incubated for 20–30 min at 37 °C/5%CO₂ before live imaging. Keep incubation and treatment times consistent between CMMP preparations to reduce variability during the execution of the experiment. In this sense, plan intervals to organize the next stage of the imaging setup and start calcium live imaging at the appropriate time.

6. Different kinds of microscope stages can be used (such as fixed or mechanical). This technique demands an upright microscope for the correct sample orientation for proper perfusion over the dishes to perform calcium imaging.

7. Test the perfusion system, inflow and outflow connections, outflow pump, and all other components that should work properly during live imaging. Commonly, perfusion systems get bubbles between uses, so constant flow in the perfusion system must be ensured. Preferably, test these steps with some advance.

8. This tool allows the positioning and movement of the micropipette over the CMMP preparation. The pipette micromanipulator is connected to a 1 mL syringe with a valve that can be manually or automatically manipulated to administer the contents of the micropipette over the CMMP preparation. The manual application of the drug can be carried out through very slight manual positive pressure applied to the syringe apparatus, creating an application in a picoliter range. Another option is using an automatic pump with a defined volume and pressure. The local puffing technique is extremely delicate since the glass micropipette has a tip with a few μM openness. The movement of the filled micropipette must be as slow and delicate as possible, to ensure that during the positioning of the micropipette on the preparation, there is no mechanical stimulation, mechanical shock, or content leaking from the micropipette. Furthermore, the micropipette micromanipulator has different travel speeds and directions (left, right; forward and backward; up and down). These commands will guide the movement and positioning of the micropipette in the ganglion.

9. Depending on the quality of the micropipette (e.g., tip is not properly forged, aligned, symmetrical, or not broken), drug leak is possible. This can occur spontaneously without any need for pressure on the syringe or during local application to the ganglion. This demonstrates that there is a problem with the micropipette, and it must be replaced.

10. Several aspects of the calcium indicator will determine the acquisition speed, such as sensor signal strength, stability, transition kinetics, and molecule interactions [24]. Combining characteristics of the indicator with attributes of the biological events and cell types to be verified will determine optimal times for recording acquisition speed. The model used as an example here is GCaMP5, an indicator with a high dynamic range and high fluorescence emission after Ca²⁺ binding. GCaMP5 features such as lower Ca²⁺-free fluorescence, high Ca²⁺-bound fluorescence, and high Ca²⁺ affinity favor the acquisition of rapid events upon stimulation [19]. The calcium imaging recording setup (frames/s, total time) described here is 2 frames/s for 5 min. For a detailed evaluation of temporal responses, acquisition at different rates can be applied and will depend on the microscope features, software, etc.

Supplementary Material

Supplementary Information The online version contains supplementary material available at 10.1007/978-1-0716-4795-0_12.