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Summary

This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G.

Abstract

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and biochemical signals. In mammals, mechanically activated channels are essential for the detection of external and internal stimuli in processes as diverse as touch sensation, hearing, red blood cell volume regulation, basal blood pressure regulation, and the sensation of urinary bladder fullness. While the function of mechanically activated ion channels has been extensively studied in the in vitro setting using the patch-clamp technique, assessing their function in their native environment remains a difficult task, often because of limited access to the sites of expression of these channels (e.g., afferent terminals, Merkel cells, baroreceptors, and kidney tubules) or difficulties applying the patch-clamp technique (e.g., the apical surfaces of urothelial umbrella cells). This protocol describes a procedure to assess mechanically evoked Ca2+ transients using the fluorescent sensor GCaMP5G in an ex vivo urothelial preparation, a technique that could be readily adapted for the study of mechanically evoked Ca2+ events in other native tissue preparations.

Introduction

Epithelial cells in the urinary tract are subjected to mechanical forces as the urinary filtrate travels through the nephrons, and urine is pumped out of the renal pelvis and travels through the ureters to be stored in the urinary bladder. It has been long recognized that mechanical forces (e.g., shear stress and stretch) exerted by fluids on the epithelial cells that line the urinary tract regulate the reabsorption of protein in the proximal tubule and of solutes in the distal nephron1,2,3,4,5,6,7,8,9,10,11,12,13, as well as the storage of urine in the urinary bladder and micturition14,15,16,17.

The conversion of mechanical stimuli into electrical and biochemical signals, a process referred to as mechanotransduction, is mediated by proteins that respond to the deformation of cellular structures or the associated extracellular matrix18,19,20,21. Mechanically activated ion channels are unique in the sense that they transition from a closed state to an open permeable state in response to changes in membrane tension, pressure, or shear stress18,19,20,21,22. In addition, Ca2+ transients can be initiated by integrin-mediated mechanotransduction or by activation of mechanoresponsive adhesion systems at cell-cell junctions23,24,25,26. Ion channel function is usually assessed with the patch-clamp technique, which involves the formation of a gigaohm seal between the cell membrane and the patch pipette27. However, cells located in deep tissue layers with a dense extracellular matrix (e.g., kidney tubules) or surrounded by a physical barrier (e.g., glycocalyx) are difficult to access with a glass micropipette. Likewise, cells embedded or that are integral parts of tissues with poor mechanical stability (e.g., the urothelium) can not be readily studied with the patch-clamp technique. Because many mechanically activated ion channels are permeable to Ca2+, an alternative approach is to assess their activity by fluorescent microscopy using a Ca2+-sensitive dye or genetically encoded calcium indicators (GECIs) such as GCaMP. Recent efforts in protein engineering have significantly increased the dynamic range, sensitivity, and response of GECIs28,29,30, and advances in genetics have allowed their expression in specific cell populations, making them ideally suited to study mechanotransduction.

The urothelium, the stratified epithelium that covers the interior of the urinary bladder, functions as a barrier, preventing the diffusion of urinary solutes into the bladder interstitium, but also functions as a transducer, sensing bladder fullness and communicating these events to the underlying nerves and musculature16. Previous studies have shown that the communication between the urothelium and underlying tissues requires the mechanically activated ion channels Piezo1 and Piezo231. To assess mechanically induced Ca2+ transients in urothelial cells, a new technique described that uses adenoviral gene transfer to express the Ca2+ sensor GCaMP5G in urothelial cells was developed. This technique employs a mucosal sheet preparation that provides easy access to the outermost umbrella cell layer and a computer-assisted system for the simultaneous mechanical stimulation of individual cells with a closed glass micropipette and recording of changes in fluorescence over time.

Protocol

Care and handling of the animals were carried out in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee. Female, 2-4-month-old C57Bl/6J mice were used for the present study. The mice were obtained commercially (see Table of Materials).

1. Equipment assembly and setup

  1. Perform Ca2+ imaging with an upright microscope equipped with a high-resolution camera and a stable light source (see Table of Materials).
    1. Acquire the images with a microscope compatible software that permits direct control of the camera, light source, and piezo actuator via a USB digital I/O device (see Table of Materials).
      NOTE: Figure 1 represents a schematic of the setup. GCaMP5G has a peak excitation wavelength of 470 nm and a peak emission wavelength of 497 nm28. Use a filter cube suitable for GCaMP5G imaging.
  2. For the stimulation of individual urothelial cells in the bladder mucosa preparation (see step 2), use a piezoelectric actuator controlled by a single channel open-loop piezo controller (see Table of Materials). Mount the piezoelectric actuator in a micromanipulator.
    1. Mount the glass micropipettes in a pipette holder affixed to the piezoelectric actuator (Figure 1). Remotely operate the piezo controller by an analog/digital converter controlled by an electrophysiology data acquisition and analysis program (see Table of Materials).
      NOTE: An external trigger, in the form of a transistor-transistor logic (TTL) signal initiated by the imaging software, is used to trigger the stimulation protocol in the electrophysiology data acquisition and analysis software that moves the piezoelectric actuator (Figure 1).
  3. Ensure that the imaging software (see Table of Materials) interfaces with the digital I/O device. Configure a digital output channel in the USB digital I/O device to deliver the TTL signal to the analog/digital converter, which will initiate the protocol in the electrophysiology software that moves the piezoelectric actuator.
    1. Use a cable to connect the Start BNC port in the analog/digital converter to the ground (GND) and a screw terminal in the USB digital I/O device.
  4. Set up the recording protocol in the imaging software.
    NOTE: The following steps describe how to generate a protocol that will send a TTL signal and start collecting images at specified time intervals.
    1. Set up the acquisition protocol in the imaging software by clicking on the Experiment Manager and selecting New Experiment. A new window will open.
    2. Select the icon Time Lapse Loop and drag it to the newly opened window; set the number of cycles to 2 and the interval to the fastest setting allowed in the setup.
    3. From the icon Transmitted Shutter/Manual Shutter, select the icon NI USB-6501 and drag it into the Time Lapse Loop window, and then set the NI USB-6501 as closed. Drag an additional NI USB-6501 from the Transmitted Shutter/Manual Shutter into the Time Lapse Loop window and set it as open. To connect the two NI USB-6501 icons, drag the arrowhead on the side of the NI USB-6501 closed icon and pull until it touches the NI USB-6501 open icon. A line that connects both icons will appear.
    4. Drag another Time Lapse Loop into the Experiment Manager window and connect it to the first one by pulling the arrowhead. Set the parameters for recording. Set the number of cycles of the new Time Lapse Loop to 2400.
    5. From the Image Acquisition icon, drag the GFP filter into the recently opened Time Lapse Loop and set the exposure time to 100 ms.
    6. Set the camera image type to 8-bit, resolution to 576 x 576 (Binning 4 x 4), pixel clock to 480 MHz, and Hot pixel correction to standard.
      NOTE: The parameters can be adjusted depending on the level of expression of the fluorescent sensor, camera sensitivity, and setup configuration.
  5. Set up the Lab Bench in the electrophysiology software (see Table of Materials).
    NOTE: This will define the output signal in mV of the analog/digital converter.
    1. Go to the Configure menu in the electrophysiology software and select Lab Bench. In the resulting Output Signals window, select Analog Out #1, press the Add signal, and name it (e.g., "Piezo"). Set the Signal Unit to mV and the Scale Factor (mV/V) to 1.
  6. Generate a stimulation protocol in the electrophysiology software following the steps below.
    1. To generate a new stimulation protocol in the electrophysiology software, go to the menu item Acquire and select New Protocol.
    2. Set Mode/Rate to Episodic Stimulation, Run/Trial to 1, Sweep/Run to 1, and Sweep Duration (s) to 150.
    3. In the Outputs menu, select channel #1 Piezo as Analog out.
    4. In the Trigger menu, set the trigger to Digitizer Start Input and Internal Timer.
    5. In the Waveform menu, select channel #1 and Analog Waveform with Epochs. Set Step A to Step, First Level to 0, Delta level to 0, First duration to 10000, and Delta duration to 0.
    6. Set Step B to Step, First level to 10, Delta Level to 0, First Duration to 1000, and Delta Duration to 0. Set Step C to Step, First Level to 0, Delta Level to 0, First Duration to 130000, and Delta Duration to 0.
    7. Save the protocol and name it Stimulation protocol.
  7. Use a BNC cable to connect Analog Output #1 in the analog/digital converter to the EXT INPUT in the front of the single-channel open-loop piezo controller (see Table of Materials).
  8. Fabricate glass micropipettes for the mechanical stimulation of umbrella cells from capillary glass tubing following the steps below.
    1. Place the capillary glass in a puller (see Table of Materials) and adjust it with the capillary retaining knobs.
    2. Position the heater unit at its full height. Adjust the first pull terminating position slider of the puller to 5.
    3. Set the first heater knob to 76.7 and the second knob to 52.7.
    4. Pull the glass capillary in two steps by pressing the start button.
    5. Close the tip of the micropipette with a microforge (see Table of Materials) with the heater adjustment knob set at 60.
      NOTE: For the present protocol, the final diameter of the micropipette tip used for poking individual cells is ~1-3 µm.
  9. Verify that the stimulating micropipette moves the distance specify in the stimulation protocol.
    NOTE: The following steps are to ensure that 12.5 s after initiating the stimulation protocol in the electrophysiology software, a voltage pulse with a duration of 1 s is generated, making the piezoelectric actuator move 20 μm. 
    1. Mount a micropipette in the holder and attach it to the piezoelectric actuator.
    2. Place the piezoelectric actuator and the attached micropipette parallel to the center of the microscope stage and within the area of view.
    3. Focus on the pipette's tip and immobilize the piezoelectric mounting rod (see Table of Materials) with tape. Under bright field illumination, adjust the camera parameters to achieve a clear image of the pipette tip.
    4. Open the Stimulation protocol in the electrophysiology software and set it to play.
      NOTE: The electrophysiology protocol will not start until the TTL signal sent from the imaging software is received.
    5. From Experiment Manager in the imaging software, press Start to initiate data acquisition.
      NOTE: This will trigger the protocol in the electrophysiology software, which will drive the piezoelectric actuator. The protocol in the imaging software will generate a file with the images of the experiment.
  10. Verify the distance traveled by the pipette following the steps below.
    1. To measure the distance traveled by the pipette during the stimulation protocol, select the Count and Measure item in the imaging software.
    2. From the Measure menu, select the Measurement and ROI option, and a new window will appear below the movie window.
    3. From the Measure menu, choose the Arbitrary Line.
    4. In the movie window, draw an arbitrary line starting at the pipette's tip. Note that the end of the arbitrary line will be adjusted later.
    5. Right click on the arbitrary line to convert the line into a region of interest (ROI), which will be visible in all movie frames.
    6. Inspect the movie and adjust the end of the arbitrary line (ROI) to the final position traveled by the pipette in response to the stimulus.
      ​NOTE: The distance traveled by the pipette in µm will appear in the Measurement and ROI window. The stimulation protocol can be modified according to user needs.

2. In situ transduction and isolation of the bladder mucosa

  1. Transduce female mouse bladders in situ with an adenovirus encoding the cDNA of CGaMP5G according to the procedure described in the virus transduction protocol31.
    1. When transducing urothelial cells for Ca2+ imaging experiments, instill the bladders with 50 µL of the solution containing 2 x 107 infectious viral particles (IVP).
      NOTE: This technique tends to restrict the expression of CGaMP5G to the umbrella cell layer. Alternatively, experiments could be conducted with urinary bladders harvested from transgenic mice expressing CGaMP5G in urothelial cells (i.e., GCaMP5G expression driven by a uroplakin-2 promotor) or any other cell type of interest.
  2. 24-72 h after transduction, euthanize the mice by CO2 asphyxiation and perform a thoracotomy by opening the chest cavity with scissors to cause the lungs to collapse.
  3. Canulate the urethra with a 24 G catheter. Expose the bladder by an abdominal incision of ~1.5 cm through the skin and muscle, and use a 6.0 suture (see Table of Materials) to secure the catheter to the urethra.
  4. Harvest the bladder and urethra32 and affix to a silicone rubber holder pad (see Table of Materials) bathed in recording solution containing (in mM): 135 NaCl, 5.0 KCl, 1 MgCl2, 2.5 CaCl2, 10 glucose, 10 HEPES, pH 7.4, and bubbled up with 100% O2 (Figure 2A).
  5. Separate the bladder mucosa from the underlying muscular layer with fine forceps following previously published reports32 (Figure 2B).
  6. Cut open the bladder mucosa and pin it down with the urothelium facing up to a silicone elastomer insert in the bottom of a 35 mm diameter tissue cultured dish (Figure 2C).

3. Mechanical stimulation of individual urothelial cells and Ca2+ imaging

  1. Mount the tissue cultured dish with the pinned bladder mucosa in the microscope stage equipped with a culture dish incubator with resistive heating elements (Figure 2E). Perfuse (following the manufacturer's instructions, see Table of Materials) the cell culture dish continuously at a rate of 1.7 mL/min with recording solution warmed at ~37 °C with an in-line heater.
  2. Maintain the temperature of the tissue dish incubator and solutions at ~37 °C with a dual channel bipolar temperature controller model (see Table of Materials). Equilibrate the tissue in the chamber with continuous perfusion for at least 15 min before conducting further experimental procedures.
  3. To record mechanically induced Ca2+ transients, submerge the micropipette in the solution bathing the pinned bladder mucosa in the tissue dish. Move the micropipette to the center of the field with the help of a low magnification scanning objective (4x) using bright field illumination.
    1. Move the micropipette near the surface of the urothelial tissue by coordinately moving the micromanipulator in the vertical plane and adjusting the focus.
    2. Switch the objective to one with a higher magnification (20x) suitable for immunofluorescence with a high numerical aperture (NA).
    3. Set the micromanipulator to Fine and move the micropipette near the top of the target cell.
    4. Change the field of view to camera and press Live in the imaging software.
      NOTE: This must turn on the reflected shutter and allow the observation of the fluorescent signal emitting from the tissue in the computer.
    5. Adjust the focus to visualize the top of the cell and adjust the pipette's position if necessary.
    6. Open the Stimulation protocol in the electrophysiology software and set it to play.
      NOTE: The protocol in the electrophysiology software will not start until the TTL signal sent from the imaging software is received.
    7. From Experiment Manager in the imaging software, press Start to initiate data acquisition. This will trigger the protocol in the electrophysiology software, which will drive the piezoelectric actuator and generate a file with the images of the experiment. The stimulation protocol can be modified according to the user's needs.

4. Data analysis

  1. Quantify the fluorescence intensity over time following the steps below.
    1. Open the image file in the imaging software and select the Count and Measure window. Select the polygon tool and draw a ROI on the boundaries of the cell that was poked.
    2. Go to the Measure window, select Intensity Profile, set the measurement to Over time, Results to Average, Background Subtraction to none, and then press Execute. The mean fluorescence intensity will be computed over time (Figure 3).
    3. To export the intensity profile data, click on the Excel icon in the Intensity Profile Results window. This will let the user choose the destination folder and file name and save the data in a .xlsx format.
  2. Perform data analysis using scientific graphing and data analysis software (see Table of Materials).
    NOTE: The amplitude of the Ca2+ peak evoked by poking is expressed as the change in fluorescence intensity (ΔF/F), where F is the fluorescence intensity of GCaMP5G at time 0, and ΔF is the difference between the fluorescence intensity maxima and the basal at time 0. The decay of the Ca2+ response can also be calculated (not shown).

Results

The present protocol describes a technique to assess mechanically evoked Ca2+ transients in umbrella cells using the fluorescent Ca2+ sensor GCaMP5G. Adenoviral transduction was employed to express GCaMP5G in urothelial cells due to its high efficiency and because it produces an elevated level of expression. Fluorescent images of stained cryosections from a transduced bladder are shown in Figure 2D. For these experiments, GCaMP5G expression is highest in the umbrella ce...

Discussion

All organisms, and seemingly most cell types, express ion channels that respond to mechanical stimuli20,33,34,35,36,37. The function of these mechanically activated channels has been predominantly assessed with the patch-clamp technique. However, due to accessibility issues, patch-clamp studies of mechanically activated ion c...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by NIH grants R01DK119183 (to G.A. and M.D.C.) and S10OD028596 (to G.A.) and by the Cell Physiology and Model Organisms Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30DK079307).

Materials

NameCompanyCatalog NumberComments
20x ObjectiveOlympusUMPlanFL N
24 G ¾” catheterMedline Suresite IV slide 
4x ObjectiveOlympusUPlanFL N
Analog/digital converterMolecular DevicesDigidata 1440A
Anti-GFP antibodyAbcam Ab6556
Beam splitterChromaT495lpxr
Bipolar temperature controller Warner InstrumentsTC-344B
CaCl2Fluka21114-1L1 M solution
cellSens softwareOlympusImaging software
CMOS cameraHamamatsuORCA fusion
Donkey anti-rabbit conjugated to Alexa Fluor 488 Jackson ImmunoResearch711-545-152
ExcelMicrosoft Corporation
Filter Chroma ET470/40X
Glass capillaries Corning 8250 glassWarner Instruments G85150T-4
GlucoseSigmaG8270
HEPES SigmaH4034
Inline heater Warner InstrumentsSH-27B
KClSigma793590
Light sourceSutter InstrumentsLambda XL 
Manifold pump tubingFisherbrand14-190-510ID 1.52 mm
Manifold pump tubingFisherbrand14-190-533ID 2.79 mm
MgCl2SigmaM9272
Mice Jackson Lab6642-4 months old female C57BL/6J
MicroforgeNarishige MF-830
MicromanipulatorSutter InstrumentsMP-285
MicroscopeOlympusBX51W
Mounting media with DAPIInvitrogenS36964 Slowfade Diamond Antifade with DAPI
NaCl SigmaS7653
pClamp softwareMolecular DevicesVersion 10.4Patch-clamp electrophysiology data acquisition and analysis software
Peristaltic pumpGilsonMinipuls 3
Piezoelectric actuatorThorlabsPAS005
Pipette holderWorld Precision Instruments
Pipette pullerNarishigePP-830
Quick exchange heated base with perfusion and adapter ring kitWarner InstrumentsQE-1Quick exchange platform fits 35 mm dish  
Rhodamine-phalloidin InvitrogenR415
Sigma-PlotSystat Software IncVersion 14.0Scientific graphing and data analysis software  
Silicone elastomerDowSylgard 184
Single channel open-loop piezo controllerThorlabsMDT694B
Square grid holder padTed Pella10520
SutureAD SurgicalS-S618R136-0 Sylk
Teflon mounting rodCustom madeUse to mount the piezoelectric actuator in the micromanipulator
TubingFisher Scientific14171129Tygon S3 ID 1/16 IN, OD 1/8 IN
USB Digital I/O device National InstrumentsNI USB-6501

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