Imaging the Insertion of Superecliptic pHluorin-Labeled Dopamine D2 Receptor Using Total Internal Reflection Fluorescence Microscopy
UNIT 5.31
Kathryn M. Daly,1 Yun Li,1 and Da-Ting Lin1,2 1
Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 2 The Jackson Laboratory, Bar Harbor, Maine
A better understanding of mechanisms governing receptor insertion to the plasma membrane (PM) requires an experimental approach with excellent spatial and temporal resolutions. Here we present a strategy that enables dynamic visualization of insertion events for dopamine D2 receptors into the PM. This approach includes tagging a pH-sensitive GFP, superecliptic pHluorin, to the extracellular domain of the receptor. By imaging pHluorin-tagged receptors under total internal reflection fluorescence microscopy (TIRFM), we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This novel imaging approach can be applied to both secreted proteins and many membrane proteins with an extracellular domain labeled with superecliptic pHluorin, and will ultimately allow for detailed dissections of the key mechanisms governing secretion of soluble proteins or the insertion C 2015 by John Wiley & Sons, Inc. of different membrane proteins to the PM. Keywords: TIRF microscopy r superecliptic pHluorin r protein trafficking r insertion r GPCR
How to cite this article: Daly, K.M., Li, Y., and Lin, D.-T. 2015. Imaging the Insertion of Superecliptic pHluorin-Labeled Dopamine D2 Receptor Using Total Internal Reflection Fluorescence Microscopy. Curr. Protoc. Neurosci. 70:5.31.1-5.31.20. doi: 10.1002/0471142301.ns0531s70
INTRODUCTION G-protein-coupled receptors (GPCRs) compose the largest family of cell-surface receptors and are critical for mediating responses to a vast number of extracellular signaling molecules, including hormones, neurotrophic factors, chemokines, odorant molecules, and light (Alberts et al., 1998). As a result, the internalization of GPCRs has been studied extensively to elucidate their roles in physiological and pathophysiological responses in order to pinpoint potential therapeutic targets (Filmore, 2004; Drake et al., 2006; Lappano and Maggiolini, 2011). However, few details are known about other contributing pathways, such as the exocytosis or recycling pathways. Studying these pathways can complement existing information about key molecular mechanisms providing a more complete understanding of receptor trafficking and potentially a more diverse pool of available therapeutic targets. The dopamine D2 receptor is one such GPCR in which dysregulation is proven to lead to multiple psychiatric disorders. It has been shown that low DRD2 functional levels in the NAc increase the risk for addiction, both for substances of abuse and for natural reward. Current Protocols in Neuroscience 5.31.1-5.31.20, January 2015 Published online January 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142301.ns0531s70 C 2015 John Wiley & Sons, Inc. Copyright
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In addition, lower DRD2 function increases the risk for attention deficit/hyperactivity disorder (ADHD; Blum et al., 1996) and depression (Park et al., 2005; Lawford et al., 2006). Moreover, abnormally elevated function of DRD2 is thought to contribute to the positive symptoms of schizophrenia (Carlsson, 1988; Karam et al., 2010). These abnormal DRD2 functional levels associated with different psychiatric disorders are thought to originate, at least in part, from dysregulation of DRD2 trafficking. DRD2 function is characterized by its abundance on the plasma membrane (PM) (Kim and Lisman, 2001; Binda et al., 2002; Lin et al., 2002; Free et al., 2007; Tirotta et al., 2008; Genedani et al., 2010; Lin et al., 2013), it is then regulated by dynamic redistribution between intracellular compartments and the PM. This redistribution is determined by several dynamic trafficking processes, including forward trafficking from the Golgi apparatus to the PM, endocytosis from the PM, and recycling through the endosomal recycling pathways, as well as targeting towards the degradation pathways. These dynamic trafficking processes are finely balanced and ultimately determine both the number and functional level of DRD2 on the neuronal surface. Therefore, targeting DRD2 trafficking mechanisms to manipulate the number of DRD2 on the neuronal surface offers a potentially powerful therapeutic strategy for restoring the DRD2 functional level in addicted individuals. Moreover, elucidating the detailed molecular mechanisms governing dynamic trafficking of DRD2 represents an important first step toward this therapeutic goal. While some studies have underscored the importance of DRD2 endocytosis in de-sensitization of this receptor, and to some degree have identified molecular mechanisms governing DRD2 endocytosis (Kim and Lisman, 2001; Macey et al., 2004), little is known about the regulation of DRD2 recycling or insertion into the plasma membrane. The main reason for the lack of knowledge of DRD2 insertion is that current studies of DRD2 trafficking primarily use biochemical or immuno-cytochemical approaches, which lack sufficient spatial and/or temporal resolution and therefore do not allow detailed examination of the molecular mechanisms regulating dynamic trafficking of DRD2. In this unit, we present a method that allows examination of receptor insertion to the PM with excellent spatial and temporal resolution in cultured neurons. Basic Protocol 1 briefly describes a method for preparing mouse glia culture for neuronal culture conditioning and details the preparation for an interface co-culture of mouse glia and mouse striatal medium spiny neurons. Basic Protocol 2 demonstrates how to transfect cultured mouse medium spiny neurons under the interface culture conditions. Basic Protocol 3 and 4 present methods for quantitative analysis of pHluorin-labeled DRD2 dynamic insertion to the PM. BASIC PROTOCOL 1
Imaging Receptor Insertion Using Superecliptic pHluorin
INTERFACIAL NEURON-GLIA CULTURE To investigate the dynamic insertion of DRD2 in cultured medium spiny neurons, it is essential to use healthy neurons. Mixed cultures of neurons and glia on the same coverslip can provide healthy mouse neurons for live imaging purposes. However, neurons in a mixed culture are not ideal for total internal fluorescence (TIRF) imaging experiments, as under this culture condition, neurons tend to situate on top of glia, with their somata and dendritic processes beyond the evanescent field of TIRF microscopy. We therefore employed the interface culture method, which provides a low-density neuronal culture with minimal glial cells on the same coverslip. Below, we describe the critical parts of the glia culture method to support the neuron/glia interface culture. NOTE: All protocols using live animals must follow officially approved procedures for the care and use of laboratory animals as well as be submitted for review and approval by an Institutional Animal Care and Use Committee (IACUC).
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Materials Purified bovine collagen solution, Type I, 3 mg/ml (Advanced Biomatrix, cat. no. 5005-B) Dissection buffer (see recipe) Glia culture medium (see recipe) Neonatal mice Papain solution (see recipe) Phosphate-buffered saline (PBS; Life Technologies, cat. no. AM9625), 10× 0.05% trypsin-EDTA (1×) (Life Technologies, cat. no. 25300-054) 70% nitric acid Sterile deionized water Poly-L-lysine solution (see recipe) Neuronal plating media (see recipe) E15-18 pregnant mice CO2 75-cm2 culture flasks 37°C water bath Dissection microscope 15-ml conical tubes Fire-polished glass Pasteur pipets 70-μm cell strainer 37°C cell culture incubator Culture inserts (Millipore, cat. no. PICM03050) 6-well plates 25-mm round coverslips (Warner Instruments) Thomas Cover Glass Staining Outfits (Thomas Scientific) Large glass dish Isotemp oven Hemacytometer Prepare the glia culture Day 0 1. Coat 75-cm2 flasks with 1 mg/ml of collagen solution (1:3 dilution of Purified Bovine Collagen Solution in ddH2 O). Set the flasks upright and let dry overnight at room temperature in a sterile tissue culture hood. NOTE: If collagen does not dry completely, glia cells will not grow well.
2. Prepare dissection buffer and glia culture medium and store at 4°C until ready for use.
Day 1 3. On the day of glia culture, warm glia medium for at least 30 min in a 37°C water bath before dissection of mouse brains. Euthanize neonatal mice by rapid decapitation. Dissect cerebral cortexes under a dissection microscope and place dissected tissue in a 15-ml conical tube with 5 to 10 ml ice-cold dissection buffer. 4. When the dissection is complete, replace the dissection buffer with 2 ml of prepared papain solution. Allow the dissected brain tissues to digest for 20 min at 37°C. 5. Following papain digestion, rinse brain tissues with 10 ml of prewarmed glia medium. Add 2 ml of fresh glia medium to the digested brain tissues, and triturate the tissues with a fire-polished glass Pasteur pipet.
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Fully triturated tissues should appear as a slightly opaque solution. No cellular debris should be visible.
6. Add 8 ml of fresh glia medium to the dissociated tissues. Filter the dissociated tissue suspension with a 70-μm cell strainer. Seed cells into 75-cm2 flasks (usually two to three postnatal pups typically seed one 75-cm2 flask) and place into a 37°C cell culture incubator.
Day 2 7. Aspirate the medium from the 75-cm2 flasks. Rinse the flasks with 10 ml PBS. Then add 10 to 15 ml of fresh glia medium to each flask. Glia in 75-cm2 flasks should be confluent in 12 days.
Prepare glial culture inserts Day 12 8. Place culture inserts into 6-well plates, and coat them with collagen (1:3 dilution of Purified Bovine Collagen Solution in ddH2 O). Allow culture inserts to air-dry overnight in a biosafety cabinet. This step can be performed a day in advance such that culture inserts are ready when glial cells reach confluency.
Plate glia on culture inserts Day 13 9. Following overnight drying, add 2 ml of glia culture medium into each well of the 6-well plate underneath each culture insert. 10. Rinse confluent glial cells in the 75-cm2 flask with 10 ml PBS, and add 5 ml of 0.05% trypsin to each 75-cm2 flask. Incubate trypsin for 5 min at 37°C. If the glial cells are still attached to the bottom of the flask after incubation, replace trypsin with 5 ml of glia culture medium and aspirate the medium gently with a pipet to remove cells from flask surface. If the glial cells have detached from the flask after incubation, add 5 ml of glia culture medium to the flask. Gently aspirate to remove remaining cells from the flask surface. Transfer into a 50-ml conical tube and centrifuge for 3 min at 1000 rpm in a clinical table top centrifuge. Remove the supernatant and resuspend the cell pellet in 5 ml fresh glia culture medium. Serum present in the glia culture medium will neutralize the trypsin.
11. Once glial cells are dissociated from the 75-cm2 flasks, seed 2 ml onto each of the culture inserts (one 75-cm2 flask per 6-well plate, 2 ml each insert). These glia culture inserts will be used to condition the neuronal cultures.
Prepare for neuronal culture Day 14 12. Load dry 25-mm round coverslips onto Thomas Cover Glass Staining Outfits, or comparable coverslip rack and soak coverslips in 70% nitric acid in large glass dish under the chemical fume hood overnight. Do not soak coverslip in water before soaking in nitric acid. Imaging Receptor Insertion Using Superecliptic pHluorin
Day 15 13. Remove coverslips from the nitric acid, Soak coverslips in sterile deionized water under a fume hood. Exchange water and repeat to wash coverslips at least three times.
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When removing the coverslip rack from the nitric acid, suspend the rack over the acid container for a few minutes or until no discernable volume of acid remains. Use small volumes of deionized water and more frequent washes to rinse remaining HNO3 from coverslips and rack. Be aware: the combining of nitric acid with water results in an exothermic reaction that may generate heat and fumes.
14. When finished rinsing coverslips, discard water and bake coverslips in Isotemp Oven overnight at 200°C. This will serve to sterilize coverslip, as well as to remove any trace amounts of HNO3 .
Day 16 15. Take coverslips out of the oven and air cool in the culture hood. Place one coverslip per well of a 6-well culture plate in the tissue culture biosafety cabinet and then pipet 1 ml of 1 mg/ml poly-L-lysine solution (PLL) to coat the coverslip with PLL. Place the 6-well plate with coverslip/PLL in the tissue culture incubator overnight. Make sure the coverslip does not float on top of PLL solution.
Day 17 16. Wash the PLL-coated coverslip at least 3 times with autoclaved deionized water in the tissue culture biosafety cabinet. Subsequently, incubate the coverslip in 2 ml neuronal plating medium overnight in tissue culture incubator.
Prepare and plate neuronal culture Day 18 17. Obtain at least 12 ml of neuronal plating medium from stock (2 ml per coverslip). Add 2% (v/v) B27 supplement to the neuronal plating medium and warm to 37°C. 18. Remove the plating medium in the 6-well plate and add 2 ml of fresh plating medium with B-27 to each well containing a glass coverslip. 19. Euthanize pregnant mice (E15-18) with CO2 , and embryonic mice by decapitation. Dissect the striatum from the embryonic brain and place into a 15-ml conical tube with 5 to 10 ml ice-cold dissection buffer. 20. When the dissection is complete, replace the dissection buffer with 2 ml of prepared papain solution. Allow the dissected brain tissues to digest for 20 min at 37°C. 21. Following papain digestion, rinse the brain tissues with 10 ml prewarmed neuronal culture medium and then add 2 ml of fresh neuronal culture medium to the digested brain tissues. Triturate the tissues with a fire-polished glass Pasteur pipet. 22. Add 8 ml of fresh neuronal culture medium to the dissociated tissues. Filter the dissociated tissue suspension with a 70-μm cell strainer. 23. Following dissociation, count the number of cells in the neuronal suspension with a hemacytometer. Seed the neurons on each coverslip at a density of 0.34 × 106 cells per well of a 6-well plate, and place overnight in a 37°C incubator. NOTE: On the day of plating, neurons are considered days in vitro (DIV) 0.
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Day 19 25. The next day (DIV 1), move neuron coverslips into the 6-well plate containing the glia culture inserts. Place neuron coverslips underneath the glia culture inserts, one coverslip per glial insert in each well. 26. Replace 1 ml of culture medium from glial insert and 1 ml culture medium from neuron chamber with fresh prewarmed neuronal culture medium (with B-27) every 3 to 4 days until they are ready for transfection and imaging experiments. Typically, 7 to 8 days for striatal mouse neurons BASIC PROTOCOL 2
NEURONAL TRANSFECTION WITH LIPOFECTAMINE 2000 USING PLASMID CDNA Neuronal cultures are transfected 24 to 72 hr before imaging using Lipofectamine 2000 reagent. Expected transfection efficiencies are usually low (1%), but capable of consistently transfecting five to ten neurons per coverslip. This is considered sufficient for studying live-cell insertion events.
Materials Lipofectamine 2000 (Life Technologies, cat. no. 11668-019) Neurobasal medium (Life Technologies, cat. no. 21103-049) Plasmid cDNA superecliptic pHluorin Drd2 (see Li et al., 2012) Neuronal culture medium + B27 (see recipe) Neuronal-glia culture (see Basic Protocol 1) 1.5-ml microcentrifuge tubes 50-ml conical tubes 37°C incubator Day 26 1. Dilute 2 μl Lipofectamine 2000 in 100 μl fresh neurobasal medium in a 1.5-ml microcentrifuge tube, and 1 μg of DNA in 100 μl of fresh neurobasal medium in a separate 1.5-ml microcentrifuge tube. Then, carefully combine the diluted DNA with the diluted Lipofectamine 2000. Incubate the DNA and Lipofectamine mixture for 20 min at room temperature after mixing. NOTE: Neuronal transfection is performed using Lipofectamine 2000. For each neuronal culture (one culture per coverslip), 2 μl Lipofectamine 2000 and 1 μg of DNA are used. Fresh neurobasal medium with no supplement is used to dilute Lipofectamine and DNA. If transfecting multiple coverslips, scale up the amount of Lipofectamine 2000 and DNA accordingly.
2. While the DNA and Lipofectamine mixture is incubating, transfer 1 ml of the medium from underneath each glia culture insert, and 1 ml from inside each insert to a sterilized 50-ml conical tube. Add the same volume of fresh neuronal culture medium + B27 to the saved medium and incubate at 37°C. This medium will be used for neuronal culture following transfection.
3. Following the 20-min Lipofectamine/DNA incubation, remove glia culture inserts momentarily from the 6-well plates. Add 200 μl Lipofectamine/DNA mixture to each coverslip with neurons. Return the glia culture inserts to each well, above the neurons. Incubate neurons with the Lipofectamine/DNA mixture for 2 to 4 hr at 37°C. Imaging Receptor Insertion Using Superecliptic pHluorin
Generation of bubbles under the membrane of the culture inserts should be avoided.
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4. After the 2 to 4 hr incubation period, remove glia inserts. Aspirate the old culture media with the Lipofectamine/DNA mixture from each well. Add 2 ml of the medium saved into the 50-ml conical tube (step 2) to the neurons (2 ml per well). Return glia culture inserts to each well. Replace 2 ml of the glia medium from each insert with the medium saved in the 50-ml conical tube (step 2). 5. Incubate neurons an additional 24 to 72 hr to allow expression of transfected cDNA before imaging.
TOTAL INTERNAL REFLECTION FLUORESCENCE IMAGING This protocol describes the general procedure to image the insertion of pHluorin-labeled DRD2 (pH-DRD2) to the PM using an objective-based TIRF microscope. All steps of this protocol (post-warm up) should be performed in a dark, humidity controlled (optional) room.
BASIC PROTOCOL 3
NOTE: All users should be properly trained in laser safety and other relevant safety procedures before using a laser based TIRF microscope.
Materials Artificial cerebrospinal fluid (aCSF) (see recipe) Lens cleaning solution Transfected neurons (see Basic Protocol 2) Immersion oil (n = 1.52) 37°C water bath TIRF microscope setup including: Inverted microscope (e.g., Zeiss AxioObserver D1) Light source 488-nm excitation laser (e.g., Newport Excelsior 200 mW) Epi-fluorescent light source (e.g., Zeiss metal halide lamp HXP 120V) Laser beam shutter and controller (e.g., Vincent Associates Uniblitz LS6 shutter, VCM–D1 controller) Filter cubes in microscope (e.g., Chroma Technology Corporation) EGFP: excitation filter (ET470/40×) dichroic mirror (Z488RDC), emission filter (ET525/50 m) A-Plan 100× Objective lens (e.g., Zeiss, NA 1.46) sCMOS camera (e.g., Hamamatsu Photonics ORCA Flash 4.0 v2) Image acquisition software (μManager) Heating insert for live imaging (specific model depends on microscope platform used) Live Cell Imaging Chamber (specific model depends on microscope platform used) Lens paper NOTE: The imaging system described here and in Figure 5.31.1 is the setup used by the authors’ laboratory. In principle, any commercial TIRF imaging platform with sufficient laser excitation and either a sCMOS or Electron Multiplying Charge Coupled Device (EMCCD) camera should provide comparable imaging performance.
Day 28 1. At least 30 min prior to imaging experiments, warm the aCSF in a 37°C water bath. Place the heating insert for live imaging on the microscope stage and turn on the
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Figure 5.31.1 Hardware configuration for TIRFM. Any inverted fluorescence microscope can be adapted to perform total internal fluorescence microscopy. In this instance, a manual Zeiss AxioObserver microscope was used as the main imaging platform for this TIRF microscope. An excitation laser (Newport Excelsior 488 nm 200 mW) was then coupled to a proprietary Zeiss TIRF slider via an optical fiber (KineFLEX–P–2–S–488–640–0.7–FCP–P2) in a laser port on the microscope body. The laser beam position is controlled via a fine adjustment knob then reflected out of the TIRF slider by a polarized beam splitter (POL BS) and onto the back aperture of a TIRF objective lens (Zeiss α–plan 100X objective lens NA = 1.46) by a dichroic mirror (DCM) inside a filter cube (FC). Resulting GFP fluorescence was selected with an emission filter mounted in the filter cube and relayed to a digital scientific complementary metal-oxide-semiconductor (sCMOS) camera (ORCA-Flash4.0 V2 Hamamatsu). In addition, a standard metal halide lamp (lamp) was coupled to the rear port on the microscope body for epi-fluorescence imaging. During epi-fluorescence imaging the excitation laser was blocked with a laser beam shutter (S) integrated between the laser head and fiber launcher.
Imaging Receptor Insertion Using Superecliptic pHluorin
heating insert, laser, epi-fluorescence light source, and camera to allow components to reach a stable operating state. 2. Clean the TIRF objective and bottom of sample coverslip with lens paper and lens cleaning solution.
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Figure 5.31.2 Healthy medium spiny neuron DIV 7. (A) Transfected neuron before photobleaching. (B) Transfected neuron after photobleaching. Note the improved contrast allowing visualization of insertion events. Examples of insertion events are boxed in red.
3. Launch image acquisition software (μManager). Set camera exposure, binning, region of interest (ROI), and gain if applicable. Typical settings for this experiment using a sCMOS camera were as follows:100 msec exposure.
4. Confirm that the TIRF setup is properly aligned and that illumination is uniform. A thin layer of fluorescent dye such as Alexa Fluor488 (Life Technologies) can be spread across a coverslip to check for uniformity. Uniform illumination should result in uniform fluorescence; compare pixel intensity across the field of view and adjust the critical angle as necessary.
5. Assemble a coverslip with transfected neurons into the live-imaging chamber. Place prewarmed aCSF in the chamber after the chamber is assembled. Mounting of a neuronal coverslip into the live-imaging chamber will vary with chamber type. In this authors’ setup a custom perfusion chamber was used to facilitate assembly of the chamber. It is important that this step be completed as quickly as possible to avoid damage to live cells. In practice any imaging chamber with coverglass bottom will work for this purpose.
6. Add a small drop of immersion oil to the objective lens. Place the sample chamber onto the heating insert on the microscope stage, and identify transfected neurons visually using an epi-fluorescent light source. IMPORTANT NOTE: It is critical that no bubbles be present in the immersion oil for successful TIRFM.
7. After a healthy transfected neuron has been identified, photobleach the cell surface by switching to TIRF illumination for approximately one minute or until all pre-existing pHluorin fluorescence on the plasma membrane is eliminated and single insertion events are visible (Fig. 5.31.2). pHluorin-receptors on the plasma membrane exhibit significant fluorescence at the beginning of the TIRF imaging experiment, which interferes with visualization of individual insertion events.
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8. Once the pre-existing pHluorin fluorescence has been bleached, set the acquisition software experiment duration to capture the following insertion events in situ. Record as a series of tiff images or a tiff image stack. In this experiment, neurons were treated with 1μM dopamine and recordings were performed immediately following or 20 min after dopamine stimulation. Each recording was performed for 1 min at a 10 Hz acquisition rate (100ms exposure) and contained a total of 600 images that were saved on a hard drive for analysis.
9. Repeat as necessary on subsequent transfected cells until all neurons have been recorded or until cell viability becomes questionable. BASIC PROTOCOL 4
MANUAL DATA ANALYSIS FOR TIRFM VISUALIZATION OF INSERTION EVENTS The manual analysis of insertion recordings is simple and requires no additional resources, but takes a significant amount of user input and time. Consequently, this method is not very practical for large sample experiments. The following protocol describes the procedure used to manually process, identify, and analyze insertion events with ImageJ. Results inherently provide spatial information about the direction of insertion as well as calculated insertion frequency in events per unit area over time, and relative number of molecules involved in individual insertion events.
Materials Recombinant GFP (Clontech) Artificial Cerebrospinal Fluid (aCSF) (see recipe) TIRF microscope setup (see Basic Protocol 3) Computer Image processing software (ImageJ) Microsoft Excel Quantify fluorescence intensity of single recombinant GFP 1. Dilute GFP to 100 ng/ml using aCSF buffer. 2. Image single GFP molecules with acquisition settings identical to experimental conditions. See Basic Protocol 3 for TIRFM imaging. 3. Open recordings with ImageJ by either dragging the dataset onto an open ImageJ window or through the menu options (File > open or File > import . . . > image sequence). Select a region of interest around a single GFP molecule using the rectangle tool. Single molecules should appear as diffraction-limited spots or point spread functions (PSF) with a diameter of 200 to 250 nm. In addition, photobleaching is assumed to be a one-step process for single GFP molecules. Therefore, a PSF can be considered a single molecule if its fluorescence intensity is eliminated rather than gradually reduced.
4. In the menu options, select Analyze > measure to calculate the area, integrated density (IntDen), and mean intensity of the region of interest (ROI). A results window should appear displaying various measurements made within the ROI selected. If mean intensity or integrated density is not displayed, open “set measurements” in Analyze > set measurements. Check the box to select the missing measurement.
5. Click and drag the center of the ROI region and move ROI to a new PSF. Imaging Receptor Insertion Using Superecliptic pHluorin
It is important that the ROI remain the same area for the most accurate results. Resizing the rectangular selection can be avoided by zooming in on the image and only clicking the center of the selection box to drag it to a new area. It may be helpful to note the dimensions of the ROI in the event that a new region is accidentally selected.
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t = 0 msec
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Figure 5.31.3 Identifying insertion events in medium spiny neuron. (A) Insertion events will appear as small bright bursts of fluorescence. (B) Fluorescence from insertion events is subsequently bleached within several seconds. Both the size and the time required for pHluorin-tagged receptors to bleach are helpful parameters in identifying or verifying insertion events.
6. Calculate the average background IntDen for each single GFP. Drag the ROI to a background area and record several intensity measurements. Be sure to measure background levels at the same time point as the insertion event since the background integrated density decreases as the cell is photobleached.
7. Repeat measurements as necessary to generate a statistically sound dataset. Save results window as separate text file. 8. Import results into Microsoft Excel. Subtract the background from the integrated density of each ROI. The fluorescence intensity for a single GFP is then the calculated average of the new background-subtracted IntDen values.
Identifying and processing insertion events 9. Open an experimental recording dataset in ImageJ. Typically, a 600 tif image stack.
10. Navigate the recording frame by frame and identify insertion events. Refer to Figure 5.31.3. 11. For each insertion event, select a region of interest around an insertion event and plot fluorescence change over time (Image > Stacks > Plot Z-axis Profile). Use this plot to determine the time point (frame number) signifying the beginning of the event (peak maximum intensity). In addition, this plot can be used to verify an insertion event based on the peak shape. Note the maximum fluorescence at the beginning of the event exponentially decays over several seconds due to photobleaching.
12. Measure both the background fluorescence level at the beginning of the insertion event and the insertion event. Subtract background IntDen from event IntDen. Store in a spreadsheet as amplitude. Measure integrated density by selecting Analyze > measure. Avoid resizing the ROI selection between measurements.
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Calculate relative number of receptors for experimental recordings 13. Divide integrated density of ROI for a single insertion event by the previously calculated average fluorescence intensity of a single GFP molecule. The relative number of pHluorin-labeled receptors present in any given insertion event is directly related to the intensity or amplitude (z profile) of the emitted fluorescence. In this experiment, the intensity representing a single pHluorin-labeled receptor is assumed to be approximately the same as the intensity of a single recombinant GFP.
Calculate insertion frequency of receptors for experimental recordings 14. Once all the insertion events have been identified for a single recording, create two maximum intensity projections (Image > Stacks > Z project: max intensity). One projection is for reference, the other will become a binary image to calculate total area.
15. Create a mask image of the neuron by manually adjusting the threshold level (Image > Adjust > Threshold). Using the duplicate max intensity projection as a reference, adjust the minimum threshold level such that the red mask covers the neuron. The threshold maximum should be set to the highest possible integer. 16. Measure the integrated density of the mask image (Analyze > measure) and then divide by the pixel intensity value of the mask. Store the result as the total surface area in pixels. Note that no ROI is needed to measure the entire image. In addition, note that the integrated density is equivalent to the sum of all the pixels since it is the product of the area measured and the mean pixel value. Normalized frequency (insertion events per unit area over time) can then be calculated by dividing the total number of events in a recording by the surface area. ALTERNATE PROTOCOL
DATA ANALYSIS FOR TIRFM VISUALIZATION OF INSERTION EVENTS WITH WOMBAT SOFTWARE Due to the long processing times associated with manual image analysis, a program was developed to automate the identification and characterization of insertion events. Results are presented as a text document. NOTE: Wombat insertion analysis program is only compatible with Mac operating systems.
Additional Materials (also see Basic Protocol 4) Wombat insertion analysis software Microsoft Excel or similar spreadsheet software (optional) 1. Quantify the fluorescence of a single GFP molecule (steps 1 through 8 in Basic Protocol 4). 2. Organize raw insertion recordings. All recordings to be analyzed should be organized into subfolders; one recording per folder. All subfolders should be located in a single batch analysis folder. Wombat software preserves folder names and lists quantitative results with respect to each subfolder when analysis is complete. To avoid confusing datasets choose a descriptive name for each subfolder such as ‘treatment_cell#’. Imaging Receptor Insertion Using Superecliptic pHluorin
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Figure 5.31.4 Wombat insertion analysis software and representative results. (Left) Wombat user interface post analysis. (Right) Raw results reported by Wombat are saved to a text document which includes the number of events in a recording, cell surface area, normalized frequency, amplitude for each event, and the half-life (frame) of each event.
3. Open the Wombat software. Set the input directory by browsing to the batch analysis folder. Similarly choose the output directory to store results. Finally, set the amplitude threshold above background intensity levels (Fig. 5.31.4). The amplitude threshold is the main parameter used to identify insertion events by extracting intensity values greater than the threshold for further characterization. Note that lower amplitude thresholds may extend the processing time.
4. Run Wombat insertion analysis. Depending on the number of recordings being analyzed, and number of insertion events present, Wombat insertion analysis may take several hours to complete. When finished, the Wombat log will read “idle” and the progress bar will disappear. A “Batchfoldername_results.txt” text document will be saved in the folder selected as output directory.
5. Import (Mac) or open results from Wombat into Microsoft Excel. 6. Calculate relative number of receptors for experimental recordings. Divide integrated density of ROI for a single insertion event by the previously calculated average fluorescence intensity of a single GFP molecule. 7. Perform statistical calculations on raw data to determine effects, if any, from treatments.
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps unless otherwise specified.
Artificial cerebrospinal fluid (aCSF) 119 mM NaCl 2.5 mM KCl 30 mM glucose 2 mM CaCl2 1 mM MgCl2 25 mM HEPES (Life Technologies, cat. no. 15630-080)
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Adjust pH to 7.4 using NaOH or HCl Filter sterilize using a 0.22-μm filter Store up to 6 months at 4°C Dissection buffer 100 ml of 10× HBSS (Life Technologies, cat. no. 14185-052) 10 ml penicillin/streptomycin (Life Technologies, cat. no. 15140-122) 10 ml of 100 mM Na pyruvate (Life Technologies, cat. no. 11360-070) 30 ml of 1 M glucose Adjust pH to 7.4 using NaOH Filter sterilize using a 0.22-μm filter Store up to 6 months at 4°C DNase I solution 10 mg/1 ml dissection buffer Divide into 20-μl aliquots into 1.5-ml microcentrifuge tubes Store up to 1 year at −20°C Glia culture medium 500 ml DMEM (Life Technologies, cat. no. 10313-021) 5 ml penicillin/streptomycin (Life Technologies, cat. no. 15140-122) 5 ml of 100× GlutaMAX (Life Technologies, cat. no. 35050-061) 50 ml fetal bovine serum (FBS; Biowest, cat. no. US1520) Filter sterilize using a 0.22-μm filter Store up to 6 months at 4°C Neuronal culture medium 500 ml neurobasal medium (Life Technologies, cat. no. 21103-049) 5 ml of 100× penicillin/streptomycin (Life Technologies, cat. no. 15140-122) 5 ml of 100× GlutaMAX (Life Technologies, cat. no. 35050-061) Filter sterilize using a 0.22-μm filter Divide into aliquots into 50-ml tubes Store up to 6 months at 4°C Add 2% B-27 (v/v) immediately before use in culture Store up to 7 days at 4°C Neuronal plating medium Neuronal culture medium (see recipe) 5% (v/v) HI horse serum (Life Technologies, cat. no. 26050-070) Store up to 6 months at 4°C Papain solution 100 μl Papain, 20 mg/ml slurry (Worthington Biochemical Corp.) 20 μl of 1% DNase I (Sigma, cat. no. DN25-10MG) 2 ml dissection buffer (see recipe) Prepare fresh Poly-L-lysine solution
Imaging Receptor Insertion Using Superecliptic pHluorin
1 mg/ml poly-L-lysine (Sigma, cat. no. P2636-1G) Dissolve in 1 liter of 100 mM borate buffer (pH 8.5) Filter sterilize using a 0.22-μm filter Divide into aliquots into 25-ml tubes Store up to 1 year at −20°C
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COMMENTARY Background Information Internalization of G-protein-coupled receptors has traditionally been characterized by radioligand-binding assays. While these methods yield powerful quantitative data and are capable of high-throughput measurements, they do not reveal the spatial or temporal information necessary to fully deduce molecular mechanisms nor are they easily applied to studying internal molecular pathways such as recycling or insertion to the plasma membrane (PM) (Evans, 2004). Total internal reflection fluorescence microscopy (TIRFM) as an optical method provides excellent spatial and temporal information that can be used to dynamically study insertion of G protein-coupled receptors (GPCRs) to the PM when imaging a superecliptic pHluorin labeled receptor. As a pH-sensitive GFP derivative, superecliptic pHluorin is nonfluorescent at pH
UNIT 5.31
Kathryn M. Daly,1 Yun Li,1 and Da-Ting Lin1,2 1
Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 2 The Jackson Laboratory, Bar Harbor, Maine
A better understanding of mechanisms governing receptor insertion to the plasma membrane (PM) requires an experimental approach with excellent spatial and temporal resolutions. Here we present a strategy that enables dynamic visualization of insertion events for dopamine D2 receptors into the PM. This approach includes tagging a pH-sensitive GFP, superecliptic pHluorin, to the extracellular domain of the receptor. By imaging pHluorin-tagged receptors under total internal reflection fluorescence microscopy (TIRFM), we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This novel imaging approach can be applied to both secreted proteins and many membrane proteins with an extracellular domain labeled with superecliptic pHluorin, and will ultimately allow for detailed dissections of the key mechanisms governing secretion of soluble proteins or the insertion C 2015 by John Wiley & Sons, Inc. of different membrane proteins to the PM. Keywords: TIRF microscopy r superecliptic pHluorin r protein trafficking r insertion r GPCR
How to cite this article: Daly, K.M., Li, Y., and Lin, D.-T. 2015. Imaging the Insertion of Superecliptic pHluorin-Labeled Dopamine D2 Receptor Using Total Internal Reflection Fluorescence Microscopy. Curr. Protoc. Neurosci. 70:5.31.1-5.31.20. doi: 10.1002/0471142301.ns0531s70
INTRODUCTION G-protein-coupled receptors (GPCRs) compose the largest family of cell-surface receptors and are critical for mediating responses to a vast number of extracellular signaling molecules, including hormones, neurotrophic factors, chemokines, odorant molecules, and light (Alberts et al., 1998). As a result, the internalization of GPCRs has been studied extensively to elucidate their roles in physiological and pathophysiological responses in order to pinpoint potential therapeutic targets (Filmore, 2004; Drake et al., 2006; Lappano and Maggiolini, 2011). However, few details are known about other contributing pathways, such as the exocytosis or recycling pathways. Studying these pathways can complement existing information about key molecular mechanisms providing a more complete understanding of receptor trafficking and potentially a more diverse pool of available therapeutic targets. The dopamine D2 receptor is one such GPCR in which dysregulation is proven to lead to multiple psychiatric disorders. It has been shown that low DRD2 functional levels in the NAc increase the risk for addiction, both for substances of abuse and for natural reward. Current Protocols in Neuroscience 5.31.1-5.31.20, January 2015 Published online January 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142301.ns0531s70 C 2015 John Wiley & Sons, Inc. Copyright
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In addition, lower DRD2 function increases the risk for attention deficit/hyperactivity disorder (ADHD; Blum et al., 1996) and depression (Park et al., 2005; Lawford et al., 2006). Moreover, abnormally elevated function of DRD2 is thought to contribute to the positive symptoms of schizophrenia (Carlsson, 1988; Karam et al., 2010). These abnormal DRD2 functional levels associated with different psychiatric disorders are thought to originate, at least in part, from dysregulation of DRD2 trafficking. DRD2 function is characterized by its abundance on the plasma membrane (PM) (Kim and Lisman, 2001; Binda et al., 2002; Lin et al., 2002; Free et al., 2007; Tirotta et al., 2008; Genedani et al., 2010; Lin et al., 2013), it is then regulated by dynamic redistribution between intracellular compartments and the PM. This redistribution is determined by several dynamic trafficking processes, including forward trafficking from the Golgi apparatus to the PM, endocytosis from the PM, and recycling through the endosomal recycling pathways, as well as targeting towards the degradation pathways. These dynamic trafficking processes are finely balanced and ultimately determine both the number and functional level of DRD2 on the neuronal surface. Therefore, targeting DRD2 trafficking mechanisms to manipulate the number of DRD2 on the neuronal surface offers a potentially powerful therapeutic strategy for restoring the DRD2 functional level in addicted individuals. Moreover, elucidating the detailed molecular mechanisms governing dynamic trafficking of DRD2 represents an important first step toward this therapeutic goal. While some studies have underscored the importance of DRD2 endocytosis in de-sensitization of this receptor, and to some degree have identified molecular mechanisms governing DRD2 endocytosis (Kim and Lisman, 2001; Macey et al., 2004), little is known about the regulation of DRD2 recycling or insertion into the plasma membrane. The main reason for the lack of knowledge of DRD2 insertion is that current studies of DRD2 trafficking primarily use biochemical or immuno-cytochemical approaches, which lack sufficient spatial and/or temporal resolution and therefore do not allow detailed examination of the molecular mechanisms regulating dynamic trafficking of DRD2. In this unit, we present a method that allows examination of receptor insertion to the PM with excellent spatial and temporal resolution in cultured neurons. Basic Protocol 1 briefly describes a method for preparing mouse glia culture for neuronal culture conditioning and details the preparation for an interface co-culture of mouse glia and mouse striatal medium spiny neurons. Basic Protocol 2 demonstrates how to transfect cultured mouse medium spiny neurons under the interface culture conditions. Basic Protocol 3 and 4 present methods for quantitative analysis of pHluorin-labeled DRD2 dynamic insertion to the PM. BASIC PROTOCOL 1
Imaging Receptor Insertion Using Superecliptic pHluorin
INTERFACIAL NEURON-GLIA CULTURE To investigate the dynamic insertion of DRD2 in cultured medium spiny neurons, it is essential to use healthy neurons. Mixed cultures of neurons and glia on the same coverslip can provide healthy mouse neurons for live imaging purposes. However, neurons in a mixed culture are not ideal for total internal fluorescence (TIRF) imaging experiments, as under this culture condition, neurons tend to situate on top of glia, with their somata and dendritic processes beyond the evanescent field of TIRF microscopy. We therefore employed the interface culture method, which provides a low-density neuronal culture with minimal glial cells on the same coverslip. Below, we describe the critical parts of the glia culture method to support the neuron/glia interface culture. NOTE: All protocols using live animals must follow officially approved procedures for the care and use of laboratory animals as well as be submitted for review and approval by an Institutional Animal Care and Use Committee (IACUC).
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Materials Purified bovine collagen solution, Type I, 3 mg/ml (Advanced Biomatrix, cat. no. 5005-B) Dissection buffer (see recipe) Glia culture medium (see recipe) Neonatal mice Papain solution (see recipe) Phosphate-buffered saline (PBS; Life Technologies, cat. no. AM9625), 10× 0.05% trypsin-EDTA (1×) (Life Technologies, cat. no. 25300-054) 70% nitric acid Sterile deionized water Poly-L-lysine solution (see recipe) Neuronal plating media (see recipe) E15-18 pregnant mice CO2 75-cm2 culture flasks 37°C water bath Dissection microscope 15-ml conical tubes Fire-polished glass Pasteur pipets 70-μm cell strainer 37°C cell culture incubator Culture inserts (Millipore, cat. no. PICM03050) 6-well plates 25-mm round coverslips (Warner Instruments) Thomas Cover Glass Staining Outfits (Thomas Scientific) Large glass dish Isotemp oven Hemacytometer Prepare the glia culture Day 0 1. Coat 75-cm2 flasks with 1 mg/ml of collagen solution (1:3 dilution of Purified Bovine Collagen Solution in ddH2 O). Set the flasks upright and let dry overnight at room temperature in a sterile tissue culture hood. NOTE: If collagen does not dry completely, glia cells will not grow well.
2. Prepare dissection buffer and glia culture medium and store at 4°C until ready for use.
Day 1 3. On the day of glia culture, warm glia medium for at least 30 min in a 37°C water bath before dissection of mouse brains. Euthanize neonatal mice by rapid decapitation. Dissect cerebral cortexes under a dissection microscope and place dissected tissue in a 15-ml conical tube with 5 to 10 ml ice-cold dissection buffer. 4. When the dissection is complete, replace the dissection buffer with 2 ml of prepared papain solution. Allow the dissected brain tissues to digest for 20 min at 37°C. 5. Following papain digestion, rinse brain tissues with 10 ml of prewarmed glia medium. Add 2 ml of fresh glia medium to the digested brain tissues, and triturate the tissues with a fire-polished glass Pasteur pipet.
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Fully triturated tissues should appear as a slightly opaque solution. No cellular debris should be visible.
6. Add 8 ml of fresh glia medium to the dissociated tissues. Filter the dissociated tissue suspension with a 70-μm cell strainer. Seed cells into 75-cm2 flasks (usually two to three postnatal pups typically seed one 75-cm2 flask) and place into a 37°C cell culture incubator.
Day 2 7. Aspirate the medium from the 75-cm2 flasks. Rinse the flasks with 10 ml PBS. Then add 10 to 15 ml of fresh glia medium to each flask. Glia in 75-cm2 flasks should be confluent in 12 days.
Prepare glial culture inserts Day 12 8. Place culture inserts into 6-well plates, and coat them with collagen (1:3 dilution of Purified Bovine Collagen Solution in ddH2 O). Allow culture inserts to air-dry overnight in a biosafety cabinet. This step can be performed a day in advance such that culture inserts are ready when glial cells reach confluency.
Plate glia on culture inserts Day 13 9. Following overnight drying, add 2 ml of glia culture medium into each well of the 6-well plate underneath each culture insert. 10. Rinse confluent glial cells in the 75-cm2 flask with 10 ml PBS, and add 5 ml of 0.05% trypsin to each 75-cm2 flask. Incubate trypsin for 5 min at 37°C. If the glial cells are still attached to the bottom of the flask after incubation, replace trypsin with 5 ml of glia culture medium and aspirate the medium gently with a pipet to remove cells from flask surface. If the glial cells have detached from the flask after incubation, add 5 ml of glia culture medium to the flask. Gently aspirate to remove remaining cells from the flask surface. Transfer into a 50-ml conical tube and centrifuge for 3 min at 1000 rpm in a clinical table top centrifuge. Remove the supernatant and resuspend the cell pellet in 5 ml fresh glia culture medium. Serum present in the glia culture medium will neutralize the trypsin.
11. Once glial cells are dissociated from the 75-cm2 flasks, seed 2 ml onto each of the culture inserts (one 75-cm2 flask per 6-well plate, 2 ml each insert). These glia culture inserts will be used to condition the neuronal cultures.
Prepare for neuronal culture Day 14 12. Load dry 25-mm round coverslips onto Thomas Cover Glass Staining Outfits, or comparable coverslip rack and soak coverslips in 70% nitric acid in large glass dish under the chemical fume hood overnight. Do not soak coverslip in water before soaking in nitric acid. Imaging Receptor Insertion Using Superecliptic pHluorin
Day 15 13. Remove coverslips from the nitric acid, Soak coverslips in sterile deionized water under a fume hood. Exchange water and repeat to wash coverslips at least three times.
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When removing the coverslip rack from the nitric acid, suspend the rack over the acid container for a few minutes or until no discernable volume of acid remains. Use small volumes of deionized water and more frequent washes to rinse remaining HNO3 from coverslips and rack. Be aware: the combining of nitric acid with water results in an exothermic reaction that may generate heat and fumes.
14. When finished rinsing coverslips, discard water and bake coverslips in Isotemp Oven overnight at 200°C. This will serve to sterilize coverslip, as well as to remove any trace amounts of HNO3 .
Day 16 15. Take coverslips out of the oven and air cool in the culture hood. Place one coverslip per well of a 6-well culture plate in the tissue culture biosafety cabinet and then pipet 1 ml of 1 mg/ml poly-L-lysine solution (PLL) to coat the coverslip with PLL. Place the 6-well plate with coverslip/PLL in the tissue culture incubator overnight. Make sure the coverslip does not float on top of PLL solution.
Day 17 16. Wash the PLL-coated coverslip at least 3 times with autoclaved deionized water in the tissue culture biosafety cabinet. Subsequently, incubate the coverslip in 2 ml neuronal plating medium overnight in tissue culture incubator.
Prepare and plate neuronal culture Day 18 17. Obtain at least 12 ml of neuronal plating medium from stock (2 ml per coverslip). Add 2% (v/v) B27 supplement to the neuronal plating medium and warm to 37°C. 18. Remove the plating medium in the 6-well plate and add 2 ml of fresh plating medium with B-27 to each well containing a glass coverslip. 19. Euthanize pregnant mice (E15-18) with CO2 , and embryonic mice by decapitation. Dissect the striatum from the embryonic brain and place into a 15-ml conical tube with 5 to 10 ml ice-cold dissection buffer. 20. When the dissection is complete, replace the dissection buffer with 2 ml of prepared papain solution. Allow the dissected brain tissues to digest for 20 min at 37°C. 21. Following papain digestion, rinse the brain tissues with 10 ml prewarmed neuronal culture medium and then add 2 ml of fresh neuronal culture medium to the digested brain tissues. Triturate the tissues with a fire-polished glass Pasteur pipet. 22. Add 8 ml of fresh neuronal culture medium to the dissociated tissues. Filter the dissociated tissue suspension with a 70-μm cell strainer. 23. Following dissociation, count the number of cells in the neuronal suspension with a hemacytometer. Seed the neurons on each coverslip at a density of 0.34 × 106 cells per well of a 6-well plate, and place overnight in a 37°C incubator. NOTE: On the day of plating, neurons are considered days in vitro (DIV) 0.
24. To prepare glia culture inserts for conditioning the neuronal cultures, remove the glia medium from the glia culture inserts and add fresh plating medium (2 ml in the insert and 2 ml underneath the insert). Molecular Neuroscience
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Day 19 25. The next day (DIV 1), move neuron coverslips into the 6-well plate containing the glia culture inserts. Place neuron coverslips underneath the glia culture inserts, one coverslip per glial insert in each well. 26. Replace 1 ml of culture medium from glial insert and 1 ml culture medium from neuron chamber with fresh prewarmed neuronal culture medium (with B-27) every 3 to 4 days until they are ready for transfection and imaging experiments. Typically, 7 to 8 days for striatal mouse neurons BASIC PROTOCOL 2
NEURONAL TRANSFECTION WITH LIPOFECTAMINE 2000 USING PLASMID CDNA Neuronal cultures are transfected 24 to 72 hr before imaging using Lipofectamine 2000 reagent. Expected transfection efficiencies are usually low (1%), but capable of consistently transfecting five to ten neurons per coverslip. This is considered sufficient for studying live-cell insertion events.
Materials Lipofectamine 2000 (Life Technologies, cat. no. 11668-019) Neurobasal medium (Life Technologies, cat. no. 21103-049) Plasmid cDNA superecliptic pHluorin Drd2 (see Li et al., 2012) Neuronal culture medium + B27 (see recipe) Neuronal-glia culture (see Basic Protocol 1) 1.5-ml microcentrifuge tubes 50-ml conical tubes 37°C incubator Day 26 1. Dilute 2 μl Lipofectamine 2000 in 100 μl fresh neurobasal medium in a 1.5-ml microcentrifuge tube, and 1 μg of DNA in 100 μl of fresh neurobasal medium in a separate 1.5-ml microcentrifuge tube. Then, carefully combine the diluted DNA with the diluted Lipofectamine 2000. Incubate the DNA and Lipofectamine mixture for 20 min at room temperature after mixing. NOTE: Neuronal transfection is performed using Lipofectamine 2000. For each neuronal culture (one culture per coverslip), 2 μl Lipofectamine 2000 and 1 μg of DNA are used. Fresh neurobasal medium with no supplement is used to dilute Lipofectamine and DNA. If transfecting multiple coverslips, scale up the amount of Lipofectamine 2000 and DNA accordingly.
2. While the DNA and Lipofectamine mixture is incubating, transfer 1 ml of the medium from underneath each glia culture insert, and 1 ml from inside each insert to a sterilized 50-ml conical tube. Add the same volume of fresh neuronal culture medium + B27 to the saved medium and incubate at 37°C. This medium will be used for neuronal culture following transfection.
3. Following the 20-min Lipofectamine/DNA incubation, remove glia culture inserts momentarily from the 6-well plates. Add 200 μl Lipofectamine/DNA mixture to each coverslip with neurons. Return the glia culture inserts to each well, above the neurons. Incubate neurons with the Lipofectamine/DNA mixture for 2 to 4 hr at 37°C. Imaging Receptor Insertion Using Superecliptic pHluorin
Generation of bubbles under the membrane of the culture inserts should be avoided.
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4. After the 2 to 4 hr incubation period, remove glia inserts. Aspirate the old culture media with the Lipofectamine/DNA mixture from each well. Add 2 ml of the medium saved into the 50-ml conical tube (step 2) to the neurons (2 ml per well). Return glia culture inserts to each well. Replace 2 ml of the glia medium from each insert with the medium saved in the 50-ml conical tube (step 2). 5. Incubate neurons an additional 24 to 72 hr to allow expression of transfected cDNA before imaging.
TOTAL INTERNAL REFLECTION FLUORESCENCE IMAGING This protocol describes the general procedure to image the insertion of pHluorin-labeled DRD2 (pH-DRD2) to the PM using an objective-based TIRF microscope. All steps of this protocol (post-warm up) should be performed in a dark, humidity controlled (optional) room.
BASIC PROTOCOL 3
NOTE: All users should be properly trained in laser safety and other relevant safety procedures before using a laser based TIRF microscope.
Materials Artificial cerebrospinal fluid (aCSF) (see recipe) Lens cleaning solution Transfected neurons (see Basic Protocol 2) Immersion oil (n = 1.52) 37°C water bath TIRF microscope setup including: Inverted microscope (e.g., Zeiss AxioObserver D1) Light source 488-nm excitation laser (e.g., Newport Excelsior 200 mW) Epi-fluorescent light source (e.g., Zeiss metal halide lamp HXP 120V) Laser beam shutter and controller (e.g., Vincent Associates Uniblitz LS6 shutter, VCM–D1 controller) Filter cubes in microscope (e.g., Chroma Technology Corporation) EGFP: excitation filter (ET470/40×) dichroic mirror (Z488RDC), emission filter (ET525/50 m) A-Plan 100× Objective lens (e.g., Zeiss, NA 1.46) sCMOS camera (e.g., Hamamatsu Photonics ORCA Flash 4.0 v2) Image acquisition software (μManager) Heating insert for live imaging (specific model depends on microscope platform used) Live Cell Imaging Chamber (specific model depends on microscope platform used) Lens paper NOTE: The imaging system described here and in Figure 5.31.1 is the setup used by the authors’ laboratory. In principle, any commercial TIRF imaging platform with sufficient laser excitation and either a sCMOS or Electron Multiplying Charge Coupled Device (EMCCD) camera should provide comparable imaging performance.
Day 28 1. At least 30 min prior to imaging experiments, warm the aCSF in a 37°C water bath. Place the heating insert for live imaging on the microscope stage and turn on the
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Figure 5.31.1 Hardware configuration for TIRFM. Any inverted fluorescence microscope can be adapted to perform total internal fluorescence microscopy. In this instance, a manual Zeiss AxioObserver microscope was used as the main imaging platform for this TIRF microscope. An excitation laser (Newport Excelsior 488 nm 200 mW) was then coupled to a proprietary Zeiss TIRF slider via an optical fiber (KineFLEX–P–2–S–488–640–0.7–FCP–P2) in a laser port on the microscope body. The laser beam position is controlled via a fine adjustment knob then reflected out of the TIRF slider by a polarized beam splitter (POL BS) and onto the back aperture of a TIRF objective lens (Zeiss α–plan 100X objective lens NA = 1.46) by a dichroic mirror (DCM) inside a filter cube (FC). Resulting GFP fluorescence was selected with an emission filter mounted in the filter cube and relayed to a digital scientific complementary metal-oxide-semiconductor (sCMOS) camera (ORCA-Flash4.0 V2 Hamamatsu). In addition, a standard metal halide lamp (lamp) was coupled to the rear port on the microscope body for epi-fluorescence imaging. During epi-fluorescence imaging the excitation laser was blocked with a laser beam shutter (S) integrated between the laser head and fiber launcher.
Imaging Receptor Insertion Using Superecliptic pHluorin
heating insert, laser, epi-fluorescence light source, and camera to allow components to reach a stable operating state. 2. Clean the TIRF objective and bottom of sample coverslip with lens paper and lens cleaning solution.
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Figure 5.31.2 Healthy medium spiny neuron DIV 7. (A) Transfected neuron before photobleaching. (B) Transfected neuron after photobleaching. Note the improved contrast allowing visualization of insertion events. Examples of insertion events are boxed in red.
3. Launch image acquisition software (μManager). Set camera exposure, binning, region of interest (ROI), and gain if applicable. Typical settings for this experiment using a sCMOS camera were as follows:100 msec exposure.
4. Confirm that the TIRF setup is properly aligned and that illumination is uniform. A thin layer of fluorescent dye such as Alexa Fluor488 (Life Technologies) can be spread across a coverslip to check for uniformity. Uniform illumination should result in uniform fluorescence; compare pixel intensity across the field of view and adjust the critical angle as necessary.
5. Assemble a coverslip with transfected neurons into the live-imaging chamber. Place prewarmed aCSF in the chamber after the chamber is assembled. Mounting of a neuronal coverslip into the live-imaging chamber will vary with chamber type. In this authors’ setup a custom perfusion chamber was used to facilitate assembly of the chamber. It is important that this step be completed as quickly as possible to avoid damage to live cells. In practice any imaging chamber with coverglass bottom will work for this purpose.
6. Add a small drop of immersion oil to the objective lens. Place the sample chamber onto the heating insert on the microscope stage, and identify transfected neurons visually using an epi-fluorescent light source. IMPORTANT NOTE: It is critical that no bubbles be present in the immersion oil for successful TIRFM.
7. After a healthy transfected neuron has been identified, photobleach the cell surface by switching to TIRF illumination for approximately one minute or until all pre-existing pHluorin fluorescence on the plasma membrane is eliminated and single insertion events are visible (Fig. 5.31.2). pHluorin-receptors on the plasma membrane exhibit significant fluorescence at the beginning of the TIRF imaging experiment, which interferes with visualization of individual insertion events.
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8. Once the pre-existing pHluorin fluorescence has been bleached, set the acquisition software experiment duration to capture the following insertion events in situ. Record as a series of tiff images or a tiff image stack. In this experiment, neurons were treated with 1μM dopamine and recordings were performed immediately following or 20 min after dopamine stimulation. Each recording was performed for 1 min at a 10 Hz acquisition rate (100ms exposure) and contained a total of 600 images that were saved on a hard drive for analysis.
9. Repeat as necessary on subsequent transfected cells until all neurons have been recorded or until cell viability becomes questionable. BASIC PROTOCOL 4
MANUAL DATA ANALYSIS FOR TIRFM VISUALIZATION OF INSERTION EVENTS The manual analysis of insertion recordings is simple and requires no additional resources, but takes a significant amount of user input and time. Consequently, this method is not very practical for large sample experiments. The following protocol describes the procedure used to manually process, identify, and analyze insertion events with ImageJ. Results inherently provide spatial information about the direction of insertion as well as calculated insertion frequency in events per unit area over time, and relative number of molecules involved in individual insertion events.
Materials Recombinant GFP (Clontech) Artificial Cerebrospinal Fluid (aCSF) (see recipe) TIRF microscope setup (see Basic Protocol 3) Computer Image processing software (ImageJ) Microsoft Excel Quantify fluorescence intensity of single recombinant GFP 1. Dilute GFP to 100 ng/ml using aCSF buffer. 2. Image single GFP molecules with acquisition settings identical to experimental conditions. See Basic Protocol 3 for TIRFM imaging. 3. Open recordings with ImageJ by either dragging the dataset onto an open ImageJ window or through the menu options (File > open or File > import . . . > image sequence). Select a region of interest around a single GFP molecule using the rectangle tool. Single molecules should appear as diffraction-limited spots or point spread functions (PSF) with a diameter of 200 to 250 nm. In addition, photobleaching is assumed to be a one-step process for single GFP molecules. Therefore, a PSF can be considered a single molecule if its fluorescence intensity is eliminated rather than gradually reduced.
4. In the menu options, select Analyze > measure to calculate the area, integrated density (IntDen), and mean intensity of the region of interest (ROI). A results window should appear displaying various measurements made within the ROI selected. If mean intensity or integrated density is not displayed, open “set measurements” in Analyze > set measurements. Check the box to select the missing measurement.
5. Click and drag the center of the ROI region and move ROI to a new PSF. Imaging Receptor Insertion Using Superecliptic pHluorin
It is important that the ROI remain the same area for the most accurate results. Resizing the rectangular selection can be avoided by zooming in on the image and only clicking the center of the selection box to drag it to a new area. It may be helpful to note the dimensions of the ROI in the event that a new region is accidentally selected.
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t = 0 msec
t = 100 msec
t = 200 msec
t = 300 msec
t = 400 msec
t = 500 msec
t = 600 msec
t = 700 msec
Figure 5.31.3 Identifying insertion events in medium spiny neuron. (A) Insertion events will appear as small bright bursts of fluorescence. (B) Fluorescence from insertion events is subsequently bleached within several seconds. Both the size and the time required for pHluorin-tagged receptors to bleach are helpful parameters in identifying or verifying insertion events.
6. Calculate the average background IntDen for each single GFP. Drag the ROI to a background area and record several intensity measurements. Be sure to measure background levels at the same time point as the insertion event since the background integrated density decreases as the cell is photobleached.
7. Repeat measurements as necessary to generate a statistically sound dataset. Save results window as separate text file. 8. Import results into Microsoft Excel. Subtract the background from the integrated density of each ROI. The fluorescence intensity for a single GFP is then the calculated average of the new background-subtracted IntDen values.
Identifying and processing insertion events 9. Open an experimental recording dataset in ImageJ. Typically, a 600 tif image stack.
10. Navigate the recording frame by frame and identify insertion events. Refer to Figure 5.31.3. 11. For each insertion event, select a region of interest around an insertion event and plot fluorescence change over time (Image > Stacks > Plot Z-axis Profile). Use this plot to determine the time point (frame number) signifying the beginning of the event (peak maximum intensity). In addition, this plot can be used to verify an insertion event based on the peak shape. Note the maximum fluorescence at the beginning of the event exponentially decays over several seconds due to photobleaching.
12. Measure both the background fluorescence level at the beginning of the insertion event and the insertion event. Subtract background IntDen from event IntDen. Store in a spreadsheet as amplitude. Measure integrated density by selecting Analyze > measure. Avoid resizing the ROI selection between measurements.
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Calculate relative number of receptors for experimental recordings 13. Divide integrated density of ROI for a single insertion event by the previously calculated average fluorescence intensity of a single GFP molecule. The relative number of pHluorin-labeled receptors present in any given insertion event is directly related to the intensity or amplitude (z profile) of the emitted fluorescence. In this experiment, the intensity representing a single pHluorin-labeled receptor is assumed to be approximately the same as the intensity of a single recombinant GFP.
Calculate insertion frequency of receptors for experimental recordings 14. Once all the insertion events have been identified for a single recording, create two maximum intensity projections (Image > Stacks > Z project: max intensity). One projection is for reference, the other will become a binary image to calculate total area.
15. Create a mask image of the neuron by manually adjusting the threshold level (Image > Adjust > Threshold). Using the duplicate max intensity projection as a reference, adjust the minimum threshold level such that the red mask covers the neuron. The threshold maximum should be set to the highest possible integer. 16. Measure the integrated density of the mask image (Analyze > measure) and then divide by the pixel intensity value of the mask. Store the result as the total surface area in pixels. Note that no ROI is needed to measure the entire image. In addition, note that the integrated density is equivalent to the sum of all the pixels since it is the product of the area measured and the mean pixel value. Normalized frequency (insertion events per unit area over time) can then be calculated by dividing the total number of events in a recording by the surface area. ALTERNATE PROTOCOL
DATA ANALYSIS FOR TIRFM VISUALIZATION OF INSERTION EVENTS WITH WOMBAT SOFTWARE Due to the long processing times associated with manual image analysis, a program was developed to automate the identification and characterization of insertion events. Results are presented as a text document. NOTE: Wombat insertion analysis program is only compatible with Mac operating systems.
Additional Materials (also see Basic Protocol 4) Wombat insertion analysis software Microsoft Excel or similar spreadsheet software (optional) 1. Quantify the fluorescence of a single GFP molecule (steps 1 through 8 in Basic Protocol 4). 2. Organize raw insertion recordings. All recordings to be analyzed should be organized into subfolders; one recording per folder. All subfolders should be located in a single batch analysis folder. Wombat software preserves folder names and lists quantitative results with respect to each subfolder when analysis is complete. To avoid confusing datasets choose a descriptive name for each subfolder such as ‘treatment_cell#’. Imaging Receptor Insertion Using Superecliptic pHluorin
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Figure 5.31.4 Wombat insertion analysis software and representative results. (Left) Wombat user interface post analysis. (Right) Raw results reported by Wombat are saved to a text document which includes the number of events in a recording, cell surface area, normalized frequency, amplitude for each event, and the half-life (frame) of each event.
3. Open the Wombat software. Set the input directory by browsing to the batch analysis folder. Similarly choose the output directory to store results. Finally, set the amplitude threshold above background intensity levels (Fig. 5.31.4). The amplitude threshold is the main parameter used to identify insertion events by extracting intensity values greater than the threshold for further characterization. Note that lower amplitude thresholds may extend the processing time.
4. Run Wombat insertion analysis. Depending on the number of recordings being analyzed, and number of insertion events present, Wombat insertion analysis may take several hours to complete. When finished, the Wombat log will read “idle” and the progress bar will disappear. A “Batchfoldername_results.txt” text document will be saved in the folder selected as output directory.
5. Import (Mac) or open results from Wombat into Microsoft Excel. 6. Calculate relative number of receptors for experimental recordings. Divide integrated density of ROI for a single insertion event by the previously calculated average fluorescence intensity of a single GFP molecule. 7. Perform statistical calculations on raw data to determine effects, if any, from treatments.
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps unless otherwise specified.
Artificial cerebrospinal fluid (aCSF) 119 mM NaCl 2.5 mM KCl 30 mM glucose 2 mM CaCl2 1 mM MgCl2 25 mM HEPES (Life Technologies, cat. no. 15630-080)
Molecular Neuroscience
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Adjust pH to 7.4 using NaOH or HCl Filter sterilize using a 0.22-μm filter Store up to 6 months at 4°C Dissection buffer 100 ml of 10× HBSS (Life Technologies, cat. no. 14185-052) 10 ml penicillin/streptomycin (Life Technologies, cat. no. 15140-122) 10 ml of 100 mM Na pyruvate (Life Technologies, cat. no. 11360-070) 30 ml of 1 M glucose Adjust pH to 7.4 using NaOH Filter sterilize using a 0.22-μm filter Store up to 6 months at 4°C DNase I solution 10 mg/1 ml dissection buffer Divide into 20-μl aliquots into 1.5-ml microcentrifuge tubes Store up to 1 year at −20°C Glia culture medium 500 ml DMEM (Life Technologies, cat. no. 10313-021) 5 ml penicillin/streptomycin (Life Technologies, cat. no. 15140-122) 5 ml of 100× GlutaMAX (Life Technologies, cat. no. 35050-061) 50 ml fetal bovine serum (FBS; Biowest, cat. no. US1520) Filter sterilize using a 0.22-μm filter Store up to 6 months at 4°C Neuronal culture medium 500 ml neurobasal medium (Life Technologies, cat. no. 21103-049) 5 ml of 100× penicillin/streptomycin (Life Technologies, cat. no. 15140-122) 5 ml of 100× GlutaMAX (Life Technologies, cat. no. 35050-061) Filter sterilize using a 0.22-μm filter Divide into aliquots into 50-ml tubes Store up to 6 months at 4°C Add 2% B-27 (v/v) immediately before use in culture Store up to 7 days at 4°C Neuronal plating medium Neuronal culture medium (see recipe) 5% (v/v) HI horse serum (Life Technologies, cat. no. 26050-070) Store up to 6 months at 4°C Papain solution 100 μl Papain, 20 mg/ml slurry (Worthington Biochemical Corp.) 20 μl of 1% DNase I (Sigma, cat. no. DN25-10MG) 2 ml dissection buffer (see recipe) Prepare fresh Poly-L-lysine solution
Imaging Receptor Insertion Using Superecliptic pHluorin
1 mg/ml poly-L-lysine (Sigma, cat. no. P2636-1G) Dissolve in 1 liter of 100 mM borate buffer (pH 8.5) Filter sterilize using a 0.22-μm filter Divide into aliquots into 25-ml tubes Store up to 1 year at −20°C
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COMMENTARY Background Information Internalization of G-protein-coupled receptors has traditionally been characterized by radioligand-binding assays. While these methods yield powerful quantitative data and are capable of high-throughput measurements, they do not reveal the spatial or temporal information necessary to fully deduce molecular mechanisms nor are they easily applied to studying internal molecular pathways such as recycling or insertion to the plasma membrane (PM) (Evans, 2004). Total internal reflection fluorescence microscopy (TIRFM) as an optical method provides excellent spatial and temporal information that can be used to dynamically study insertion of G protein-coupled receptors (GPCRs) to the PM when imaging a superecliptic pHluorin labeled receptor. As a pH-sensitive GFP derivative, superecliptic pHluorin is nonfluorescent at pH
