Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
UNIT 2.21
Misa Arizono,1,2 Hiroko Bannai,1,3 and Katsuhiko Mikoshiba1 1
Laboratory for Developmental Neurobiology, Brain Science Institute, Saitama, Japan Interdisciplinary Institute for Neuroscience (IINS), Universit´e Bordeaux Segalen, Bordeaux, France 3 Division of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya, Japan 2
ABSTRACT This unit describes the method that we have developed to clarify endogenous mGluR5 (metabotropic glutamate receptors 5) dynamics in astrocytes by single-particle tracking using quantum dots (QD-SPT). QD-SPT has been a powerful tool to examine the contribution of neurotransmitter receptor dynamics to synaptic plasticity. Neurotransmitter receptors are also expressed in astrocytes, the most abundant form of glial cell in the brain. mGluR5s, which evoke intracellular Ca2+ signals upon receiving glutamate, contribute to the modulation of synaptic transmission efficacy and local blood flow by astrocytes. QD-SPT has previously revealed that the regulation of the lateral diffusion of mGluR5 on the plasma membrane is important for local Ca2+ signaling in astrocytes. Determining how mGluR5 dynamics are regulated in response to neuronal input would enable a better understanding of neuron-astrocyte communication in future studies. Curr. C 2014 by John Wiley & Sons, Inc. Protoc. Neurosci. 66:2.21.1-2.21.18. Keywords: astrocyte r mGluR5 r single particle tracking r quantum dot
INTRODUCTION Astrocytes, the most abundant glia in the brain that feature radiating processes, were classically known to contribute to brain homeostasis by providing neurons with energy, removing neurotransmitters, and maintaining the appropriate ion balance (Simard and Nedergaard, 2004; Magistretti, 2006). The discovery that astrocytes express neurotransmitter receptors, and that these receptors can trigger Ca2+ increase, suggested a more active role for astrocytes (Charles et al., 1991). This astrocytic Ca2+ elevation that occurred in response to receiving neurotransmitters was shown to subsequently affect synaptic transmission (Newman, 2003; Perea and Araque, 2007). Bidirectional communication between neuron and astrocytes is thought to take place at a site called the tripartite synapse, where astrocyte processes wrap synapses and participate as a third player influencing synaptic efficacy in addition to pre- and post-synapses. Among many receptors that can trigger astrocytic Ca2+ elevation, metabotropic glutamate receptors 5 (mGluR5) is reported to be particularly important in regulating synaptic transmission (Newman, 2003; Panatier et al., 2011). Although mGluR5-mediated Ca2+ increase potentiates synaptic release and enhances basal transmission, abnormally enhanced mGluR5-dependent Ca2+ signals damage neurons in epileptic brains (Ding et al., 2007). Clarifying the underlying mechanism of mGuR5-dependent Ca2+ signals in astrocytes is essential to understanding how astrocytes adjust their impact on synaptic plasticity. Several lines of evidence demonstrate that lateral diffusion of neurotransmitter receptors on the plasma membrane is a critical parameter that impacts cell-to-cell signaling, particularly at synapses (Triller and Choquet, 2008). Indeed, modification of receptor dynamics in neurons plays an important role in synaptic plasticity (Bannai et al., 2009; Imaging Current Protocols in Neuroscience 2.21.1-2.21.18, January 2014 Published online January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142301.ns0221s66 C 2014 John Wiley & Sons, Inc. Copyright
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preparation of coverslips • place coverslips in 12-well culture dishes day 0
• put 0.04% PEI into the well overnight incubation
day 1
• wash coverslips with sterile water, 20 min
preparation of coverslips • isolate rat embryo, 10 min
• dissect hippocampi, 20-60 min
• incubate with trypsin and DNase, 5 min three 30-sec washes • dissociate tissues, 3 min
• measure cell density, 3 min
• plate cells, 3 min
day 4
medium change, 5 min
day 7
transfection, 20 min • mix lipofectamine 2000 and OPTIMEM (A) • mix DNA and OPTIMEM (B) 5-min incubation • mix A and B 15-min incubation • add mixture to the cells
Figure 2.21.1
Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
Flow diagram of hippocampal culture and transfection.
Muir et al., 2010; Niwa et al., 2012). Lateral diffusion of receptors into and away from the synapse is crucial in modifying the available number of receptors at the synapse without changing the overall receptor number on the membrane. Moreover, regulation of lateral diffusion of neuronal membrane proteins underlies the functional polarity of a neuron. For example, the initial segment divides a neuron into a somato-dendrite region and an axon by blocking the diffusion, and maintaining the polarized distribution, of the membrane molecules responsible for each domain (Nakada et al., 2003). Therefore, it is conceivable that lateral diffusion of receptors on an astrocytic membrane also contributes to adjusting the receptor number required for neuron-astrocyte communication or to maintaining polarized distribution of molecules at certain astrocytic domains.
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• labeling, 15 min incubation with primary antibody, 5 min three 10-sec washes (see Fig. 2.21.4B)
incubation with secondary Fab antibody, 5 min three 10-sec washes
incubation with QD, 1 min eight 10-sec washes
• imaging, 30 min record films and images
• data analysis
Figure 2.21.2
Flow diagram of labeling protocol.
Recently, we showed that regulation of mGluR5 lateral diffusion on the astrocytic membrane is a crucial factor in determining the location of the mGluR5-dependent Ca2+ signal (Arizono et al., 2012). The quantum dot (QD)–based single-particle (SPT) technique was used to visualize lateral diffusion of mGluR5. This technique has been extremely successful in revealing the role of lateral diffusion in synaptic plasticity in neurons. QDs are fluorescent semiconductor nanocrystals that are approximately 2 to 8 nm in diameter. The overall size of functionalized QDs, including polymer coating, is approximately 20 nm in diameter. By labeling endogenous mGluR5 with QDs via mGluR5 antibody, a diffusion barrier was identified that blocks the transition of mGluR5 between astrocytic processes and soma. This barrier maintains a higher density of mGluR5, and thereby provides an enhanced Ca2+ response to mGluR agonist stimulation, in the processes compared to the soma. Thus, using QDs to examine mGluR5 dynamics on the astrocytic membrane is a powerful approach to understanding how the mGluR5-dependent Ca2+ increase is regulated. In this unit, Basic Protocol 1 briefly describes a method for primary culture of hippocampal neurons and astrocytes that preserves some properties of in vivo astrocytes. By co-culturing with neurons, astrocytes can develop processes, which exist in in vivo astrocytes as the contact site with synapses, but are not formed in pure astrocyte cultures. Basic Protocol 2 describes the transfection of plasmid DNA (e.g., plasmids encoding CFP or GFP), allowing one to visualize cell morphology and thereby distinguish mGluR5s on astrocytic membranes from those on neuronal membranes. The flow diagram of Basic Protocols 1 and 2 is shown in Figure 2.21.1. Basic Protocol 3 shows how to label endogenous mGluR5s, the flow diagram for which is illustrated in Figure 2.21.2. Basic Protocols 4 and 5 describe how to record and analyze mGluR5 dynamics to calculate diffusion parameters and to examine diffusion hindrances, respectively.
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BASIC PROTOCOL 1
PRIMARY CULTURE OF HIPPOCAMPAL NEURONS AND ASTROCYTES To investigate the dynamic nature of mGluR5 in astrocytic processes contacting neuronal cells, it is essential to use an in vitro culture that preserves many of the properties of in vivo astrocytes. Because astrocytes in an astrocyte-pure culture tend to have no processes, it is preferable to use a young neuron/astrocyte culture in which astrocytes retain their processes. Below, we describe the critical parts of a basic hippocampal primary culture method that works well for both neurons and astrocytes [if more detail is needed, see Goslin et al. (1998) and Viviani (2006)]. The use of this primary culture enables one to: (1) track receptors on one focal plane and (2) examine the underlying molecular mechanism of receptor dynamics.
Materials 4% (w/v) polyethyleneimine stock solution: dilute 50% (w/v) polyethyleneimine solution (Sigma-Aldrich, cat. no. P3143) with sterile water, then filter sterilize (store up to 1 year or longer at –20°C) Hanks’ balanced salt solution (HBSS; e.g., Life Technologies, cat. no. 14170-112) 1 M HEPES, pH 7.3, filter-sterilized (prepare in lab or purchase from Life Technologies, cat. no. 15630-080). Minimum essential medium (MEM; e.g., Life Technologies, cat. no. 11090-081) B27 supplement (e.g., Life Technologies, cat. no. 17504-044, or Miltenyi Biotec, cat. no. 130-093-566) 200 mM L-glutamine, filter-sterilized (prepared in lab or purchase from Life Technologies, cat. no. 25030-081) 100 mM sodium pyruvate (e.g., Life Technologies, cat. no. 11360-070) Penicillin-streptomycin solution (penicillin, 10,000 U/ml/streptomycin, 10,000 μg/ml; e.g., Life Technologies, cat. no. 15140-122) 18- to 20-day pregnant Wistar or Sprague Dawley rat 2.5% trypsin solution (Sigma-Aldrich, cat. no. T4674; prepare 150-μl aliquots and store at −20°C) 0.5% (w/v) DNase I stock solution (e.g., Roche Diagnostics, cat. no. 10104159001) in HBSS supplemented with 120 mM MgSO4 (prepare 150-μl aliquots and store at −20°C) Neurobasal A medium (Life Technologies, cat. no. 10888-022) 18-mm-diameter round coverslips (thickness no. 1, e.g., Hecht Assistent, cat. no. 1001/18; http://www.hecht-assistent.de/) 12-well culture plates (12-well multiwell plate, BD Falcon, cat. no. 353043) Sterile hood with UV light source Improved Neubauer cell-counting chamber Additional reagents and equipment for dissection of rat embryos to isolate hippocampi (UNIT 3.2) NOTE: Before starting, this protocol must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
Day 0: Coating coverslips To have a sufficient number of cells stick on the coverslip, it is crucial to coat the glass properly. 1. Place an 18-mm-diameter glass coverslip in each well of a 12-well plate. Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
2. Dilute 4% polyethyleneimine (PEI) stock solution to 0.04% with sterile water (1 ml/well of the 12-well plate is needed).
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3. Put 1 ml of 0.04% PEI solution in each well of the 12-well plate containing a coverslip. Make sure that there are no bubbles underneath the coverslip.
4. Incubate plates at 37°C in a humidified, 5% CO2 incubator overnight.
Day 1: Plating hippocampal neurons and astrocytes 5. Wash coated coverslips three times, each time with 1 ml of sterile water. For each wash, remove solution by aspiration, put 1 ml sterilized water in the well, then shake the 12-well plate so that the PEI solution between the coverslip and the plate can be washed out thoroughly. Make sure that the water is aspirated completely after the final wash.
6. Dry and sterilize coverslips inside hood with UV light for at least 15 min. 7. Prepare dissection medium (by combining 49 ml HBSS and 1 ml of 1 M HEPES) and plating medium (by combining 49 ml MEM, 1 ml B27, 500 μl of 200 mM glutamine, 500 μl of 100 mM sodium pyruvate, and 25 μl penicillin-streptomycin solution). Keep dissection medium on ice and keep plating medium at room temperature. The above quantities are sufficient for four 12-well plates.
8. Anesthetize a pregnant rat (E18 to E20) following the animal regulations of the institute. 9. Swiftly remove uterus containing embryos. Sacrifice dam according to approved protocol. 10. Quickly remove embryos from uterus. Remove brain from each embryo and dissect hippocampi (UNIT 3.2) in dissecting medium. Isolated brains and hippocampi should be kept in ice-cold dissection medium.
11. Treat hippocampal tissue with trypsin (150 μl of 2.5% stock solution) and DNase (140 μl of 0.5% stock solution) in 3 ml dissection medium at 37°C for 5 min. Keep the remaining 10 μl of DNase stock, which will be used at step 13.
12. Wash tissue with ice-cold HBSS three times. 13. Aspirate HBSS and suspend tissue in 1 ml plating medium containing DNase I (10 μl of 0.5% stock solution) by pipetting no more than 20 times. Measure the cell density using an improved Neubauer cell-counting chamber under a phase-contrast microscope. When automated cell counter is used, the definition of cell should be carefully determined so that only bright, white cells are counted. Make sure cell suspension looks even. Count only the bright white cells and not the dark gray or black cells.
14. Dilute cells to a density of 1.4 × 105 cells/ml with plating medium. Add 1 ml of diluted cell suspension to each well of the precoated 12-well plates.
Day 4: Medium change 15. Prepare maintenance medium (49 ml Neurobasal A, 1 ml B27, 500 μl of 200 mM glutamine, and 25 μl penicillin-streptomycin solution) and warm it to 37°C. 16. Remove culture medium from wells of plate. Do not let cells dry out. Imaging
17. Gently add 1 ml of maintenance medium. Current Protocols in Neuroscience
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BASIC PROTOCOL 2
TRANSFECTION OF PLASMID ENCODING FLUORESCENT PROTEIN INTO ASTROCYTES Astrocyte morphology in primary culture is hard to observe by transmitted light microscopy compared to morphology of neurons, because of the relatively thin, flat shape of astrocytes. Therefore, it is necessary to express a marker in these cells to distinguish receptors on the astrocytic membrane specifically. Here, we describe a simple method to express cyan fluorescent protein (CFP) in astrocytes. The primary culture should be around DIV (days in vitro) 7 to 9 to achieve transfection to astrocytes and to observe astrocytes with processes. One can use membrane-targeted CFP for good visualization of the cell morphology, but cytosolic CFP or other fluorescent proteins such as GFP can also be used. In order to distinguish CFP-expressing astrocytes by morphology, immunocytochemistry with GFAP antibodies is highly recommended for the first experiment (Fig. 2.21.3).
Materials Cultured cells on circular glass coverslips (DIV 7 to 9; see Basic Protocol 1) Purified plasmid (1 mg/ml DNA concentration) of fluorescent protein (CFP) subcloned into a mammalian expression vector (0.5 μg/well required) Opti-MEM I reduced-serum medium (Life Technologies, cat. no. 31985-070) Lipofectamine 2000 reagent (Life Technologies, cat. no. 11668-027) 1.5-ml microcentrifuge tubes (autoclaved) 1. Check condition of 1-week-old culture and mark wells with healthy cells. Avoid wells with sparse cell distribution. 2. Label two 1.5-ml tubes, one for CFP and the other for Lipofectamine 2000. 3. Add 0.5 μg of CFP plasmid (0.5 μl of 1 mg/ml DNA) per coverslip to the CFP tube and 1 μl of Lipofectamine 2000 per coverslip to the other. 4. Add 50 μl of Opti-MEM per coverslip to each tube, vortex, and incubate at room temperature for 5 min. 5. Combine the Lipofectamine 2000 and plasmid solutions, pipet up and down gently to mix, and incubate the mixture (100 μl per coverslip) for 15 min. 6. Apply mixture to cells on the coverslips droplet by droplet. Gently shake plate. Incubate cells 24 hr at 37°C.
CFP
GFAP
CFP/GFAP
Figure 2.21.3 CFP-transfected astrocyte in hippocampal culture. Transfected cell can be identified as an astrocyte by immunocytochemistry using a GFAP antibody. Note that in our culture system, astrocytes retain processes. Scale bar, 50 μm.
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LABELING mGluR5s WITH QDs The quantum dot (QD) labeling procedure for receptors on astrocytic membranes is similar to that for neuronal membranes (Bannai et al., 2006). Cells are first incubated with a primary antibody that recognizes the extracellular domain, then with a biotinylated secondary Fab antibody, and finally with streptavidin-conjugated quantum dots. The labeling conditions optimized for neuronal receptors cannot readily be used for astrocytic receptors, since conditions depend on the amount of receptor on the membrane, which differs between neurons and astrocytes. The labeling conditions must be optimized for the cell of interest. Make sure to check the specificity of the primary antibody to the extracellular domain. This is done by immunocytochemistry on fixed, non-permeabilized cells. After the specificity has been confirmed, perform live staining with QDs and optimize (yet minimize) the concentration of the primary and secondary antibody. This step is crucial in preventing the cross-linking of molecules and overlapping of QD trajectories. Once the conditions have been optimized, check the specificity of labeling again by omitting the primary antibody.
BASIC PROTOCOL 3
Figures 2.21.2 and 2.21.5 provide an overview of this protocol.
Materials Cultured cells on circular glass coverslips (DIV 8 to 10, >24 hr after transfection; see Basic Protocol 2) Imaging medium (see recipe) Primary antibody (rabbit) against extracellular part of mGluR5 (Alomone Labs, cat. no. AGC-007): 10-μl aliquots of antibody reconstituted according to the manufacturer’s instructions should be kept at −20°C and at 4°C after thawing (avoid freeze-thaw cycles) Secondary antibody: biotinylated Fab fragment of goat anti–rabbit IgG (Jackson Immunoresearch, cat. no. 111-066-047) 1 μM Qdot 605 or 625 streptavidin conjugate (Life Technologies, cat. no. Q10101MP or A10196) 1× QD binding buffer (see recipe) Sucrose Heating block 10-cm-diameter dish (e.g., Cell Culture Dishes, 100 × 20 mm style; BD Falcon, cat. no. 353003) Vacuum pump 1. Check expression of the CFP in astrocytes. Ensure that you have flat star-shaped cells with thick processes expressing CFP. If the primary culture (Basic Protocol 1) and transfection (Basic Protocol 2) procedures were successful, at least 10 transfected cells per coverslip should be observed.
2. Make a moist chamber for staining. Put Parafilm sheet (approximately 10 × 5 cm) on a heating block set at 37°C and use moist tissue paper to form a ring 9 cm in diameter. Put a 10-cm-diameter dish atop the heating block as a lid (Fig. 2.21.4A). 3. Transfer a coverslip containing transfected cells from the culture dish to the center of the moist chamber on the Parafilm sheet. Parafilm prevents the liquid from spreading and allows staining in a small volume (100 μl for an 18-mm glass coverslip).
4. Wash cells briefly by sucking medium with a vacuum pump from one corner of the coverslip and then immediately adding 250 μl of imaging medium at the other corner (Fig. 2.21.4B).
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A
lid (10-cm dish)
moist tissue paper coverslip
Parafilm
heating block (37°C)
B
imaging medium to suction pump
Figure 2.21.4 Moist chamber used for live staining and how to wash the coverslip. (A) Moist chamber consists of a 10-cm dish placed over a coverslip located in the center. Wet tissue paper surrounds the coverslip, and it is all on a Parafilm sheet on a heating block. (B) Incubation medium is removed with a sucking pipet connected to a vacuum pump, and is immediately followed by the gentle addition of imaging medium. One wash takes 10 sec.
Make sure that the coverslip does not dry out. All washes and incubations with antibodies should be performed using fresh imaging medium.
5. Incubate cells for 5 min in mGluR5 antibody solution (1.9 μg/ml). The concentration of antibody should be optimized for each antibody and for the culture conditions. Significant labeling compared to the control should be achieved, but must be well below the saturation level (Fig. 2.21.5A).
6. Wash cells three times and place in biotinylated secondary antibody (10 μg/ml) for 5 min; Fig. 2.21.5B). 7. After three brief washes, incubate cells for 1 min in 0.5 to 2 nM Qdot 625 streptavidin conjugate in 1× QD binding buffer supplemented with 215 mM sucrose (Fig. 2.21.5C). The sucrose increases the osmolarity of the buffer to a physiological level (300 mosmol). The QD suspension should be vortexed beforehand to avoid aggregation.
8. Wash cells about eight times. Wash cells thoroughly after QD incubation to remove unbound QDs. Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
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A
B
mGluR5 antibody
biotinylated Fab fragment
C
QD
Figure 2.21.5 Labeling mGluR5s with QDs. Endogenous mGluR5 is labeled first by the mGluR5 antibody at a non-saturating concentration (A), followed by incubation with biotinylated Fab fragment (B), and is subsequently labeled with QDs (C). Cells are washed after each step to remove unbound antibodies or QDs.
RECORDING AND ANALYZING mGluR5 DYNAMICS TO CALCULATE DIFFUSION PARAMETERS Diffusion coefficients and mode of diffusion (confined or free diffusing) are important measures to understand the properties of receptor diffusion (Kusumi et al., 1993). These parameters are useful for characterizing the diffusion quantitatively, and these diffusion properties can be compared between different conditions or different molecules. To evaluate diffusion behavior of mGluR5, mGluR5-QD images should be acquired at 13 Hz or faster. The trajectory of mGluR5-QDs is reconstructed from the image sequence of a single QD identified by its fluorescence intermittency (Fig. 2.21.6A; Arizono et al., 2012). This procedure consists of two main steps. First, fluorescent spots are detected by cross-correlating the image with a Gaussian model of the point spread function. A least-squares Gaussian fit is applied (around the local maximum above a threshold) to determine the center of each spot with a spatial accuracy of 5 to 10 nm. Secondly, QD trajectories are assembled automatically or manually by linking the centers of fluorescent spots that are likely from the same QD from frame to frame.
BASIC PROTOCOL 4
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A
B
C
process
** * soma 0
2
4
% Confined
100
6⫻10⫺2
D (m2/sec)
75
***
50 25 0
process
soma
Figure 2.21.6 Analysis of diffusion parameters. (A) Representative trajectories of mGluR5-QDs in the process (top) and soma (bottom) recorded for 15.2 sec. Scale bar: 1 μm. (B) Median diffusion coefficients of mGluR5QDs (arrowhead) plus (hatched bar) and minus (filled bar) interquartile range (IQR) at the process and soma. Leftward shift of the diffusion coefficient in the soma indicates slower diffusion of mGluR5-QDs. ***: p < 0.005, Mann–Whitney’s U-test. (C) Percentage of mGluR5-QDs showing confined lateral diffusion (average ± SEM, n = 27 cells). ***: p < 0.005, (paired t-test). 1268 QDs were analyzed for processes. 253 QDs were analyzed for soma.
Materials Cells on coverslips labeled with QDs (Basic Protocol 3) Imaging medium (see recipe) Recording chamber that allows recording in imaging medium (e.g., Ludin chamber; Life Imaging Service, http://www.lis.ch/) Inverted fluorescent microscope (e.g., IX70, Olympus) equipped with a Plan Apo 60× objective lens (NA 1.42) (e.g., Olympus) Cooled charge-coupled device (CCD) camera (e.g., ORCA II-ER, Hamamatsu Photonics) or EM-CCD camera (e.g., ImagEM, Hamamatsu Photonics) Appropriate filter sets (e.g., excitation: 470 to 490 nm, emission: 515 to 550 nm for CFP or GFP signal and excitation: 420 to 490 nm, emission: 595 to 615 nm for QD signal) Light-emitting diode (LED) illumination system (e.g., precisExcite, CoolLED 490 nm for CFP or GFP signal and 440 nm for QD signal) or mercury/xenon lamp (e.g., Olympus) PC and software for image acquisition (e.g., MetaMorph, Molecular Devices) 1. Mount coverslip in recording chamber, add imaging medium (>200 μl) to chamber, and place on microscope stage. 2. Set light source and filters for CFP, look for a CFP transfected astrocyte, take a snapshot, and save image. 3. Change filters for QD and carry out real-time recording. Obtain QD images with an integration of 75 msec for at least 200 consecutive frames. 4. Save recorded images on computer hard drive. 5. Merge CFP image and the sequential images of QD. Throughout analysis, analyze only signals that overlap with the CFP signal (Fig. 2.21.7). 6. Reconstruct trajectory from image sequence of a single QD identified by its fluorescence intermittency using analysis software for SPT. Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
We use custom-made analysis software for SPT “TI Workbench” written by Dr. Takafumi Inoue, Waseda University, Japan (Bannai et al., 2009). For TI Workbench, contact the following e-mail address: [email protected].
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mGluR5-QD
CFP
mGluR5-QD/CFP
Figure 2.21.7 mGluR5-s on CFP-positive astrocyte. An appropriate density of mGluR5-QDs on the astrocytic membrane. Only the mGluR5-QDs that overlap with CFP signal are analyzed. Scale bar, 10 μm.
Another option for SPT analysis is G-track from G-Angstrom Inc. (http://www.gangstrom.com/eng/products/gtrack.php).
7. Calculate diffusion parameters from trajectories reconstructed from sequential recordings of mGluR5-QDs as described in the following steps.
Calculating the diffusion coefficient 8. Calculate mean square displacement (MSD) as the function of time for each trajectory of mGluR5-QD by applying the relation:
MSD (nτ ) =
N −n 1 (x ((i + n) τ ) − x (iτ ))2 + (y ((i + n) τ ) − y (iτ ))2 N −n i=1
Equation 2.21.1
where τ is the acquisition time, N is the total number of frames, and n and i are positive integers, with n determining the time increment (Saxton and Jacobson, 1997). 9. Calculate the diffusion coefficients (D) by fitting first four points of MSD versus time curves with the equation: MSD (nτ ) = 4Dnτ + b Equation 2.21.2
An mGluR5-QD with diffusion coefficient (D)
UNIT 2.21
Misa Arizono,1,2 Hiroko Bannai,1,3 and Katsuhiko Mikoshiba1 1
Laboratory for Developmental Neurobiology, Brain Science Institute, Saitama, Japan Interdisciplinary Institute for Neuroscience (IINS), Universit´e Bordeaux Segalen, Bordeaux, France 3 Division of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya, Japan 2
ABSTRACT This unit describes the method that we have developed to clarify endogenous mGluR5 (metabotropic glutamate receptors 5) dynamics in astrocytes by single-particle tracking using quantum dots (QD-SPT). QD-SPT has been a powerful tool to examine the contribution of neurotransmitter receptor dynamics to synaptic plasticity. Neurotransmitter receptors are also expressed in astrocytes, the most abundant form of glial cell in the brain. mGluR5s, which evoke intracellular Ca2+ signals upon receiving glutamate, contribute to the modulation of synaptic transmission efficacy and local blood flow by astrocytes. QD-SPT has previously revealed that the regulation of the lateral diffusion of mGluR5 on the plasma membrane is important for local Ca2+ signaling in astrocytes. Determining how mGluR5 dynamics are regulated in response to neuronal input would enable a better understanding of neuron-astrocyte communication in future studies. Curr. C 2014 by John Wiley & Sons, Inc. Protoc. Neurosci. 66:2.21.1-2.21.18. Keywords: astrocyte r mGluR5 r single particle tracking r quantum dot
INTRODUCTION Astrocytes, the most abundant glia in the brain that feature radiating processes, were classically known to contribute to brain homeostasis by providing neurons with energy, removing neurotransmitters, and maintaining the appropriate ion balance (Simard and Nedergaard, 2004; Magistretti, 2006). The discovery that astrocytes express neurotransmitter receptors, and that these receptors can trigger Ca2+ increase, suggested a more active role for astrocytes (Charles et al., 1991). This astrocytic Ca2+ elevation that occurred in response to receiving neurotransmitters was shown to subsequently affect synaptic transmission (Newman, 2003; Perea and Araque, 2007). Bidirectional communication between neuron and astrocytes is thought to take place at a site called the tripartite synapse, where astrocyte processes wrap synapses and participate as a third player influencing synaptic efficacy in addition to pre- and post-synapses. Among many receptors that can trigger astrocytic Ca2+ elevation, metabotropic glutamate receptors 5 (mGluR5) is reported to be particularly important in regulating synaptic transmission (Newman, 2003; Panatier et al., 2011). Although mGluR5-mediated Ca2+ increase potentiates synaptic release and enhances basal transmission, abnormally enhanced mGluR5-dependent Ca2+ signals damage neurons in epileptic brains (Ding et al., 2007). Clarifying the underlying mechanism of mGuR5-dependent Ca2+ signals in astrocytes is essential to understanding how astrocytes adjust their impact on synaptic plasticity. Several lines of evidence demonstrate that lateral diffusion of neurotransmitter receptors on the plasma membrane is a critical parameter that impacts cell-to-cell signaling, particularly at synapses (Triller and Choquet, 2008). Indeed, modification of receptor dynamics in neurons plays an important role in synaptic plasticity (Bannai et al., 2009; Imaging Current Protocols in Neuroscience 2.21.1-2.21.18, January 2014 Published online January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142301.ns0221s66 C 2014 John Wiley & Sons, Inc. Copyright
2.21.1 Supplement 66
preparation of coverslips • place coverslips in 12-well culture dishes day 0
• put 0.04% PEI into the well overnight incubation
day 1
• wash coverslips with sterile water, 20 min
preparation of coverslips • isolate rat embryo, 10 min
• dissect hippocampi, 20-60 min
• incubate with trypsin and DNase, 5 min three 30-sec washes • dissociate tissues, 3 min
• measure cell density, 3 min
• plate cells, 3 min
day 4
medium change, 5 min
day 7
transfection, 20 min • mix lipofectamine 2000 and OPTIMEM (A) • mix DNA and OPTIMEM (B) 5-min incubation • mix A and B 15-min incubation • add mixture to the cells
Figure 2.21.1
Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
Flow diagram of hippocampal culture and transfection.
Muir et al., 2010; Niwa et al., 2012). Lateral diffusion of receptors into and away from the synapse is crucial in modifying the available number of receptors at the synapse without changing the overall receptor number on the membrane. Moreover, regulation of lateral diffusion of neuronal membrane proteins underlies the functional polarity of a neuron. For example, the initial segment divides a neuron into a somato-dendrite region and an axon by blocking the diffusion, and maintaining the polarized distribution, of the membrane molecules responsible for each domain (Nakada et al., 2003). Therefore, it is conceivable that lateral diffusion of receptors on an astrocytic membrane also contributes to adjusting the receptor number required for neuron-astrocyte communication or to maintaining polarized distribution of molecules at certain astrocytic domains.
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• labeling, 15 min incubation with primary antibody, 5 min three 10-sec washes (see Fig. 2.21.4B)
incubation with secondary Fab antibody, 5 min three 10-sec washes
incubation with QD, 1 min eight 10-sec washes
• imaging, 30 min record films and images
• data analysis
Figure 2.21.2
Flow diagram of labeling protocol.
Recently, we showed that regulation of mGluR5 lateral diffusion on the astrocytic membrane is a crucial factor in determining the location of the mGluR5-dependent Ca2+ signal (Arizono et al., 2012). The quantum dot (QD)–based single-particle (SPT) technique was used to visualize lateral diffusion of mGluR5. This technique has been extremely successful in revealing the role of lateral diffusion in synaptic plasticity in neurons. QDs are fluorescent semiconductor nanocrystals that are approximately 2 to 8 nm in diameter. The overall size of functionalized QDs, including polymer coating, is approximately 20 nm in diameter. By labeling endogenous mGluR5 with QDs via mGluR5 antibody, a diffusion barrier was identified that blocks the transition of mGluR5 between astrocytic processes and soma. This barrier maintains a higher density of mGluR5, and thereby provides an enhanced Ca2+ response to mGluR agonist stimulation, in the processes compared to the soma. Thus, using QDs to examine mGluR5 dynamics on the astrocytic membrane is a powerful approach to understanding how the mGluR5-dependent Ca2+ increase is regulated. In this unit, Basic Protocol 1 briefly describes a method for primary culture of hippocampal neurons and astrocytes that preserves some properties of in vivo astrocytes. By co-culturing with neurons, astrocytes can develop processes, which exist in in vivo astrocytes as the contact site with synapses, but are not formed in pure astrocyte cultures. Basic Protocol 2 describes the transfection of plasmid DNA (e.g., plasmids encoding CFP or GFP), allowing one to visualize cell morphology and thereby distinguish mGluR5s on astrocytic membranes from those on neuronal membranes. The flow diagram of Basic Protocols 1 and 2 is shown in Figure 2.21.1. Basic Protocol 3 shows how to label endogenous mGluR5s, the flow diagram for which is illustrated in Figure 2.21.2. Basic Protocols 4 and 5 describe how to record and analyze mGluR5 dynamics to calculate diffusion parameters and to examine diffusion hindrances, respectively.
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PRIMARY CULTURE OF HIPPOCAMPAL NEURONS AND ASTROCYTES To investigate the dynamic nature of mGluR5 in astrocytic processes contacting neuronal cells, it is essential to use an in vitro culture that preserves many of the properties of in vivo astrocytes. Because astrocytes in an astrocyte-pure culture tend to have no processes, it is preferable to use a young neuron/astrocyte culture in which astrocytes retain their processes. Below, we describe the critical parts of a basic hippocampal primary culture method that works well for both neurons and astrocytes [if more detail is needed, see Goslin et al. (1998) and Viviani (2006)]. The use of this primary culture enables one to: (1) track receptors on one focal plane and (2) examine the underlying molecular mechanism of receptor dynamics.
Materials 4% (w/v) polyethyleneimine stock solution: dilute 50% (w/v) polyethyleneimine solution (Sigma-Aldrich, cat. no. P3143) with sterile water, then filter sterilize (store up to 1 year or longer at –20°C) Hanks’ balanced salt solution (HBSS; e.g., Life Technologies, cat. no. 14170-112) 1 M HEPES, pH 7.3, filter-sterilized (prepare in lab or purchase from Life Technologies, cat. no. 15630-080). Minimum essential medium (MEM; e.g., Life Technologies, cat. no. 11090-081) B27 supplement (e.g., Life Technologies, cat. no. 17504-044, or Miltenyi Biotec, cat. no. 130-093-566) 200 mM L-glutamine, filter-sterilized (prepared in lab or purchase from Life Technologies, cat. no. 25030-081) 100 mM sodium pyruvate (e.g., Life Technologies, cat. no. 11360-070) Penicillin-streptomycin solution (penicillin, 10,000 U/ml/streptomycin, 10,000 μg/ml; e.g., Life Technologies, cat. no. 15140-122) 18- to 20-day pregnant Wistar or Sprague Dawley rat 2.5% trypsin solution (Sigma-Aldrich, cat. no. T4674; prepare 150-μl aliquots and store at −20°C) 0.5% (w/v) DNase I stock solution (e.g., Roche Diagnostics, cat. no. 10104159001) in HBSS supplemented with 120 mM MgSO4 (prepare 150-μl aliquots and store at −20°C) Neurobasal A medium (Life Technologies, cat. no. 10888-022) 18-mm-diameter round coverslips (thickness no. 1, e.g., Hecht Assistent, cat. no. 1001/18; http://www.hecht-assistent.de/) 12-well culture plates (12-well multiwell plate, BD Falcon, cat. no. 353043) Sterile hood with UV light source Improved Neubauer cell-counting chamber Additional reagents and equipment for dissection of rat embryos to isolate hippocampi (UNIT 3.2) NOTE: Before starting, this protocol must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
Day 0: Coating coverslips To have a sufficient number of cells stick on the coverslip, it is crucial to coat the glass properly. 1. Place an 18-mm-diameter glass coverslip in each well of a 12-well plate. Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
2. Dilute 4% polyethyleneimine (PEI) stock solution to 0.04% with sterile water (1 ml/well of the 12-well plate is needed).
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3. Put 1 ml of 0.04% PEI solution in each well of the 12-well plate containing a coverslip. Make sure that there are no bubbles underneath the coverslip.
4. Incubate plates at 37°C in a humidified, 5% CO2 incubator overnight.
Day 1: Plating hippocampal neurons and astrocytes 5. Wash coated coverslips three times, each time with 1 ml of sterile water. For each wash, remove solution by aspiration, put 1 ml sterilized water in the well, then shake the 12-well plate so that the PEI solution between the coverslip and the plate can be washed out thoroughly. Make sure that the water is aspirated completely after the final wash.
6. Dry and sterilize coverslips inside hood with UV light for at least 15 min. 7. Prepare dissection medium (by combining 49 ml HBSS and 1 ml of 1 M HEPES) and plating medium (by combining 49 ml MEM, 1 ml B27, 500 μl of 200 mM glutamine, 500 μl of 100 mM sodium pyruvate, and 25 μl penicillin-streptomycin solution). Keep dissection medium on ice and keep plating medium at room temperature. The above quantities are sufficient for four 12-well plates.
8. Anesthetize a pregnant rat (E18 to E20) following the animal regulations of the institute. 9. Swiftly remove uterus containing embryos. Sacrifice dam according to approved protocol. 10. Quickly remove embryos from uterus. Remove brain from each embryo and dissect hippocampi (UNIT 3.2) in dissecting medium. Isolated brains and hippocampi should be kept in ice-cold dissection medium.
11. Treat hippocampal tissue with trypsin (150 μl of 2.5% stock solution) and DNase (140 μl of 0.5% stock solution) in 3 ml dissection medium at 37°C for 5 min. Keep the remaining 10 μl of DNase stock, which will be used at step 13.
12. Wash tissue with ice-cold HBSS three times. 13. Aspirate HBSS and suspend tissue in 1 ml plating medium containing DNase I (10 μl of 0.5% stock solution) by pipetting no more than 20 times. Measure the cell density using an improved Neubauer cell-counting chamber under a phase-contrast microscope. When automated cell counter is used, the definition of cell should be carefully determined so that only bright, white cells are counted. Make sure cell suspension looks even. Count only the bright white cells and not the dark gray or black cells.
14. Dilute cells to a density of 1.4 × 105 cells/ml with plating medium. Add 1 ml of diluted cell suspension to each well of the precoated 12-well plates.
Day 4: Medium change 15. Prepare maintenance medium (49 ml Neurobasal A, 1 ml B27, 500 μl of 200 mM glutamine, and 25 μl penicillin-streptomycin solution) and warm it to 37°C. 16. Remove culture medium from wells of plate. Do not let cells dry out. Imaging
17. Gently add 1 ml of maintenance medium. Current Protocols in Neuroscience
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TRANSFECTION OF PLASMID ENCODING FLUORESCENT PROTEIN INTO ASTROCYTES Astrocyte morphology in primary culture is hard to observe by transmitted light microscopy compared to morphology of neurons, because of the relatively thin, flat shape of astrocytes. Therefore, it is necessary to express a marker in these cells to distinguish receptors on the astrocytic membrane specifically. Here, we describe a simple method to express cyan fluorescent protein (CFP) in astrocytes. The primary culture should be around DIV (days in vitro) 7 to 9 to achieve transfection to astrocytes and to observe astrocytes with processes. One can use membrane-targeted CFP for good visualization of the cell morphology, but cytosolic CFP or other fluorescent proteins such as GFP can also be used. In order to distinguish CFP-expressing astrocytes by morphology, immunocytochemistry with GFAP antibodies is highly recommended for the first experiment (Fig. 2.21.3).
Materials Cultured cells on circular glass coverslips (DIV 7 to 9; see Basic Protocol 1) Purified plasmid (1 mg/ml DNA concentration) of fluorescent protein (CFP) subcloned into a mammalian expression vector (0.5 μg/well required) Opti-MEM I reduced-serum medium (Life Technologies, cat. no. 31985-070) Lipofectamine 2000 reagent (Life Technologies, cat. no. 11668-027) 1.5-ml microcentrifuge tubes (autoclaved) 1. Check condition of 1-week-old culture and mark wells with healthy cells. Avoid wells with sparse cell distribution. 2. Label two 1.5-ml tubes, one for CFP and the other for Lipofectamine 2000. 3. Add 0.5 μg of CFP plasmid (0.5 μl of 1 mg/ml DNA) per coverslip to the CFP tube and 1 μl of Lipofectamine 2000 per coverslip to the other. 4. Add 50 μl of Opti-MEM per coverslip to each tube, vortex, and incubate at room temperature for 5 min. 5. Combine the Lipofectamine 2000 and plasmid solutions, pipet up and down gently to mix, and incubate the mixture (100 μl per coverslip) for 15 min. 6. Apply mixture to cells on the coverslips droplet by droplet. Gently shake plate. Incubate cells 24 hr at 37°C.
CFP
GFAP
CFP/GFAP
Figure 2.21.3 CFP-transfected astrocyte in hippocampal culture. Transfected cell can be identified as an astrocyte by immunocytochemistry using a GFAP antibody. Note that in our culture system, astrocytes retain processes. Scale bar, 50 μm.
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LABELING mGluR5s WITH QDs The quantum dot (QD) labeling procedure for receptors on astrocytic membranes is similar to that for neuronal membranes (Bannai et al., 2006). Cells are first incubated with a primary antibody that recognizes the extracellular domain, then with a biotinylated secondary Fab antibody, and finally with streptavidin-conjugated quantum dots. The labeling conditions optimized for neuronal receptors cannot readily be used for astrocytic receptors, since conditions depend on the amount of receptor on the membrane, which differs between neurons and astrocytes. The labeling conditions must be optimized for the cell of interest. Make sure to check the specificity of the primary antibody to the extracellular domain. This is done by immunocytochemistry on fixed, non-permeabilized cells. After the specificity has been confirmed, perform live staining with QDs and optimize (yet minimize) the concentration of the primary and secondary antibody. This step is crucial in preventing the cross-linking of molecules and overlapping of QD trajectories. Once the conditions have been optimized, check the specificity of labeling again by omitting the primary antibody.
BASIC PROTOCOL 3
Figures 2.21.2 and 2.21.5 provide an overview of this protocol.
Materials Cultured cells on circular glass coverslips (DIV 8 to 10, >24 hr after transfection; see Basic Protocol 2) Imaging medium (see recipe) Primary antibody (rabbit) against extracellular part of mGluR5 (Alomone Labs, cat. no. AGC-007): 10-μl aliquots of antibody reconstituted according to the manufacturer’s instructions should be kept at −20°C and at 4°C after thawing (avoid freeze-thaw cycles) Secondary antibody: biotinylated Fab fragment of goat anti–rabbit IgG (Jackson Immunoresearch, cat. no. 111-066-047) 1 μM Qdot 605 or 625 streptavidin conjugate (Life Technologies, cat. no. Q10101MP or A10196) 1× QD binding buffer (see recipe) Sucrose Heating block 10-cm-diameter dish (e.g., Cell Culture Dishes, 100 × 20 mm style; BD Falcon, cat. no. 353003) Vacuum pump 1. Check expression of the CFP in astrocytes. Ensure that you have flat star-shaped cells with thick processes expressing CFP. If the primary culture (Basic Protocol 1) and transfection (Basic Protocol 2) procedures were successful, at least 10 transfected cells per coverslip should be observed.
2. Make a moist chamber for staining. Put Parafilm sheet (approximately 10 × 5 cm) on a heating block set at 37°C and use moist tissue paper to form a ring 9 cm in diameter. Put a 10-cm-diameter dish atop the heating block as a lid (Fig. 2.21.4A). 3. Transfer a coverslip containing transfected cells from the culture dish to the center of the moist chamber on the Parafilm sheet. Parafilm prevents the liquid from spreading and allows staining in a small volume (100 μl for an 18-mm glass coverslip).
4. Wash cells briefly by sucking medium with a vacuum pump from one corner of the coverslip and then immediately adding 250 μl of imaging medium at the other corner (Fig. 2.21.4B).
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A
lid (10-cm dish)
moist tissue paper coverslip
Parafilm
heating block (37°C)
B
imaging medium to suction pump
Figure 2.21.4 Moist chamber used for live staining and how to wash the coverslip. (A) Moist chamber consists of a 10-cm dish placed over a coverslip located in the center. Wet tissue paper surrounds the coverslip, and it is all on a Parafilm sheet on a heating block. (B) Incubation medium is removed with a sucking pipet connected to a vacuum pump, and is immediately followed by the gentle addition of imaging medium. One wash takes 10 sec.
Make sure that the coverslip does not dry out. All washes and incubations with antibodies should be performed using fresh imaging medium.
5. Incubate cells for 5 min in mGluR5 antibody solution (1.9 μg/ml). The concentration of antibody should be optimized for each antibody and for the culture conditions. Significant labeling compared to the control should be achieved, but must be well below the saturation level (Fig. 2.21.5A).
6. Wash cells three times and place in biotinylated secondary antibody (10 μg/ml) for 5 min; Fig. 2.21.5B). 7. After three brief washes, incubate cells for 1 min in 0.5 to 2 nM Qdot 625 streptavidin conjugate in 1× QD binding buffer supplemented with 215 mM sucrose (Fig. 2.21.5C). The sucrose increases the osmolarity of the buffer to a physiological level (300 mosmol). The QD suspension should be vortexed beforehand to avoid aggregation.
8. Wash cells about eight times. Wash cells thoroughly after QD incubation to remove unbound QDs. Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
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A
B
mGluR5 antibody
biotinylated Fab fragment
C
QD
Figure 2.21.5 Labeling mGluR5s with QDs. Endogenous mGluR5 is labeled first by the mGluR5 antibody at a non-saturating concentration (A), followed by incubation with biotinylated Fab fragment (B), and is subsequently labeled with QDs (C). Cells are washed after each step to remove unbound antibodies or QDs.
RECORDING AND ANALYZING mGluR5 DYNAMICS TO CALCULATE DIFFUSION PARAMETERS Diffusion coefficients and mode of diffusion (confined or free diffusing) are important measures to understand the properties of receptor diffusion (Kusumi et al., 1993). These parameters are useful for characterizing the diffusion quantitatively, and these diffusion properties can be compared between different conditions or different molecules. To evaluate diffusion behavior of mGluR5, mGluR5-QD images should be acquired at 13 Hz or faster. The trajectory of mGluR5-QDs is reconstructed from the image sequence of a single QD identified by its fluorescence intermittency (Fig. 2.21.6A; Arizono et al., 2012). This procedure consists of two main steps. First, fluorescent spots are detected by cross-correlating the image with a Gaussian model of the point spread function. A least-squares Gaussian fit is applied (around the local maximum above a threshold) to determine the center of each spot with a spatial accuracy of 5 to 10 nm. Secondly, QD trajectories are assembled automatically or manually by linking the centers of fluorescent spots that are likely from the same QD from frame to frame.
BASIC PROTOCOL 4
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B
C
process
** * soma 0
2
4
% Confined
100
6⫻10⫺2
D (m2/sec)
75
***
50 25 0
process
soma
Figure 2.21.6 Analysis of diffusion parameters. (A) Representative trajectories of mGluR5-QDs in the process (top) and soma (bottom) recorded for 15.2 sec. Scale bar: 1 μm. (B) Median diffusion coefficients of mGluR5QDs (arrowhead) plus (hatched bar) and minus (filled bar) interquartile range (IQR) at the process and soma. Leftward shift of the diffusion coefficient in the soma indicates slower diffusion of mGluR5-QDs. ***: p < 0.005, Mann–Whitney’s U-test. (C) Percentage of mGluR5-QDs showing confined lateral diffusion (average ± SEM, n = 27 cells). ***: p < 0.005, (paired t-test). 1268 QDs were analyzed for processes. 253 QDs were analyzed for soma.
Materials Cells on coverslips labeled with QDs (Basic Protocol 3) Imaging medium (see recipe) Recording chamber that allows recording in imaging medium (e.g., Ludin chamber; Life Imaging Service, http://www.lis.ch/) Inverted fluorescent microscope (e.g., IX70, Olympus) equipped with a Plan Apo 60× objective lens (NA 1.42) (e.g., Olympus) Cooled charge-coupled device (CCD) camera (e.g., ORCA II-ER, Hamamatsu Photonics) or EM-CCD camera (e.g., ImagEM, Hamamatsu Photonics) Appropriate filter sets (e.g., excitation: 470 to 490 nm, emission: 515 to 550 nm for CFP or GFP signal and excitation: 420 to 490 nm, emission: 595 to 615 nm for QD signal) Light-emitting diode (LED) illumination system (e.g., precisExcite, CoolLED 490 nm for CFP or GFP signal and 440 nm for QD signal) or mercury/xenon lamp (e.g., Olympus) PC and software for image acquisition (e.g., MetaMorph, Molecular Devices) 1. Mount coverslip in recording chamber, add imaging medium (>200 μl) to chamber, and place on microscope stage. 2. Set light source and filters for CFP, look for a CFP transfected astrocyte, take a snapshot, and save image. 3. Change filters for QD and carry out real-time recording. Obtain QD images with an integration of 75 msec for at least 200 consecutive frames. 4. Save recorded images on computer hard drive. 5. Merge CFP image and the sequential images of QD. Throughout analysis, analyze only signals that overlap with the CFP signal (Fig. 2.21.7). 6. Reconstruct trajectory from image sequence of a single QD identified by its fluorescence intermittency using analysis software for SPT. Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots
We use custom-made analysis software for SPT “TI Workbench” written by Dr. Takafumi Inoue, Waseda University, Japan (Bannai et al., 2009). For TI Workbench, contact the following e-mail address: [email protected].
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mGluR5-QD
CFP
mGluR5-QD/CFP
Figure 2.21.7 mGluR5-s on CFP-positive astrocyte. An appropriate density of mGluR5-QDs on the astrocytic membrane. Only the mGluR5-QDs that overlap with CFP signal are analyzed. Scale bar, 10 μm.
Another option for SPT analysis is G-track from G-Angstrom Inc. (http://www.gangstrom.com/eng/products/gtrack.php).
7. Calculate diffusion parameters from trajectories reconstructed from sequential recordings of mGluR5-QDs as described in the following steps.
Calculating the diffusion coefficient 8. Calculate mean square displacement (MSD) as the function of time for each trajectory of mGluR5-QD by applying the relation:
MSD (nτ ) =
N −n 1 (x ((i + n) τ ) − x (iτ ))2 + (y ((i + n) τ ) − y (iτ ))2 N −n i=1
Equation 2.21.1
where τ is the acquisition time, N is the total number of frames, and n and i are positive integers, with n determining the time increment (Saxton and Jacobson, 1997). 9. Calculate the diffusion coefficients (D) by fitting first four points of MSD versus time curves with the equation: MSD (nτ ) = 4Dnτ + b Equation 2.21.2
An mGluR5-QD with diffusion coefficient (D)
