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Presynaptic Calcium Measurements Using Bulk Loading of Acetoxymethyl Indicators Stephan D. Brenowitz and Wade G. Regehr Cold Spring Harb Protoc; doi: 10.1101/pdb.prot081828 Email Alerting Service Subject Categories
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Protocol
Presynaptic Calcium Measurements Using Bulk Loading of Acetoxymethyl Indicators Stephan D. Brenowitz and Wade G. Regehr
We describe a method for labeling presynaptic terminals in mammalian brain slices by focal application of calcium indicators conjugated with acetoxymethyl (AM) esters. A solution of membranepermeant, AM-conjugated calcium indicator is focally applied to the transverse cerebellar brain slice and allowed to equilibrate throughout the parallel fiber tract. Fibers are then stimulated with an extracellular electrode and fluorescence transients are measured from a location several hundred micrometers from the loading site using a photodiode or photomultiplier tube. Considerations for selecting an appropriate indicator and determining the optimum loading conditions are discussed.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
ACSF for calcium imaging Artificial cerebral spinal fluid (ACSF) is used to prepare the labeling solution and to perfuse the brain tissue under study during the procedure.
AM-conjugated calcium indicator (e.g., Fura-2 from Invitrogen) AM-conjugated calcium indicators are available from Invitrogen in 50-μg ampoules, a convenient size for preparing a loading solution for short-term use. See Discussion for considerations regarding the selection of calcium indicators.
Fast Green (1% stock solution) Dissolve 100 mg of Fast Green in 10 mL of H2O, vortex for 30 sec, and sonicate for 2 min.
Pluronic F-127/dimethylsulfoxide (DMSO; anhydrous) (25:75) Equipment
Aquarium pump or picospritzer (see Step 7) Fluorescence excitation light source An aperture in a conjugate specimen plane should be present to enable adjustment of the field of illumination.
Adapted from Imaging in Neuroscience (ed. Helmchen and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081828
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Bulk Loading of AM Indicators
Microcentrifuge tube filters (e.g., Costar Spin-X; pore size 0.22 µm) Micromanipulators (two; positioned on opposite sides of the specimen) Microscope (upright), equipped with epifluorescence optics See Imaging Setup in the Discussion for a full description of the system required.
Objectives 5× air-immersion (numerical aperture [NA] 0.15) 60× water-immersion (NA 0.90) Photodiode or photomultiplier tube An aperture in a conjugate image plane should be present to control the exposure area.
Pipettes (loading and suction) Tubing and valves These parts are used to connect loading and suction pipettes to the aquarium pump and vacuum lines, respectively. See Step 7.
METHOD Preparation of AM-Conjugated Calcium Indicator Loading Solution
1. Dissolve 50 µg of an AM-conjugated calcium indicator (e.g., Fura-2) in 20 µL of a solution of 25% pluronic F-127 and 75% DMSO. 2. Mix the solution by vortexing for 30 sec and sonicate in an ice-filled bath for 2 min. 3. Add 400 µL of ACSF and 20 µL of 1% Fast Green to the vial. Fast Green is included in the labeling solution to allow visualization during loading.
4. Mix the solution by vortexing for 30 sec, sonicate for 2 min, and filter using a microcentrifuge tube filter with 0.22-μm pore size. Keep the freshly prepared labeling solution on ice to prevent premature deesterification, and use it directly in Step 7. Loading Granule Cell Presynaptic Terminals in Rodent Transverse Cerebellar Brain Slices
5. Place a brain slice in the recording chamber of the microscope and perfuse the slice with oxygenated ACSF. For further details, see Swanson and Contractor (2004).
6. Position a suction pipette with a 20–30 µm tip diameter above the surface of the slice. 7. Fill a loading pipette (tip diameter of 20–30 µm) with labeling solution (from Step 4) and position near the suction pipette. An aquarium pump or picospritzer can be used to supply pressure for the loading pipette. To allow regulation of pressure in a very low range, we use branched tubing to connect the pressure source and pipette, with a valve at the end of one arm of the tubing to allow pressure to escape. When the valve is fully open, little or no pressure reaches the loading pipette, and partially closing the valve provides gentle pressure for loading.
8. Apply positive pressure to the loading pipette and suction to the other pipette. Using transmitted light to visualize the Fast Green present in the loading pipette, adjust the flow rate to maintain a rapidly flowing, well-confined stream of dye that is collected entirely by the suction pipette (Fig. 1). See Troubleshooting.
9. Lower the pipettes together to a position near the surface of the slice. 10. Gently lower the loading pipette to the slice surface and place it into the slice until the flow of solution from the pipette is reduced or stops entirely. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081828
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FIGURE 1. Procedure for loading granule cells in cerebellar brain slices with AM-conjugated calcium indicator. (A) Lowpower (4×), transmitted-light image of the loading pipette (right) and the suction pipette (left) as they approach the brain slice. The plume of Fast Green expelled from the loading pipette is captured by the suction pipette. The transverse cerebellar slice (out of focus) is visible below. Scale bar, 200 µm. (B) Configuration of pipettes during loading with AM-conjugated calcium indicator. In this transmitted-light image, the tip of the loading pipette (on right) is placed 15 µm below the surface of the slice and a small cloud of Fast Green is visible. To obtain focal labeling with the AM dye, the loading solution is collected by the suction pipette (on left, out of focus), which is located 50 µm above the slice. Scale bar, 25 µm. (C ) Labeled parallel fiber band. This epifluorescence image was taken shortly after loading 200 µm from the loading site. Scale bar, 25 µm.
11. Gradually increase the pressure on the loading pipette until the flow resumes. Typical loading times range from 2 min to 20 min depending on the preparation, the indicator used, and the desired degree of loading (see Discussion). The brain slice should be continuously superfused with oxygenated ACSF during loading to minimize undesirable background labeling. The total volume of loading solution applied to the brain slice varies depending on the loading time, pressure, and pipette tip diameter, but it is typically 1–5 µL.
12. After loading the labeling solution into the brain slice, allow the indicator to equilibrate within the cell for 1 h–2 h. Imaging Calcium Transients in Parallel Fiber Presynaptic Terminals
13. Position the stimulus electrode in the center of the labeled parallel fiber tract near the loading site. 14. Image parallel fibers 600–800 µm from the loading site in a 150-μm-diameter region. Near the loading site, both neurons and glia are indiscriminately labeled, and dye may enter intracellular organelles that do not respond to intracellular calcium changes. However, more distant sites are not exposed to the AM indicator and the only dye reaching these sites is the membrane impermeant acid form. See Troubleshooting.
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Bulk Loading of AM Indicators
TROUBLESHOOTING Problem (Step 8): The loading pipette becomes clogged. Solution: This can be corrected by further sonication and filtration of the loading solution. If the
problem persists, a loading pipette with a larger tip can help. Problem (Step 14): No fluorescence change is observed on stimulation. Solution: It is often necessary to reposition the stimulus electrode and adjust the position of the labeled
fibers by moving the slice to optimize the fluorescence transient. Stimulation of a large proportion of labeled fibers will maximize the relative change in fluorescence on stimulation. Problem (Step 14): The stimulus-evoked calcium transient is small or decays slowly. Solution: This is a symptom of excessive loading. To reduce the level of calcium indicator in the
labeled fibers, shorten the loading time or increase the dilution of calcium indicator in the loading solution. Problem (Step 14): Indicators are not retained in presynaptic boutons at physiological temperatures. Solution: For experiments conducted at room temperature (20 C–24 C), the acid form of the indi-
˚
˚
cator is stable inside cells for hours. At elevated temperatures, the indicator is removed from neurons, limiting the time available for fluorescence measurements (Beierlein et al. 2004). It may therefore be necessary to use calcium indicators conjugated to high-molecular-weight dextrans.
DISCUSSION
Calcium ions entering presynaptic terminals regulate neurotransmitter release as well as multiple types of both short- and long-term synaptic plasticity (Zucker and Regehr 2002). To directly examine the role of calcium in synaptic transmission in the mammalian brain, it is necessary to measure presynaptic calcium levels and determine the time course of presynaptic calcium entry. However, the small size of presynaptic boutons in the mammalian brain (1 µm) makes measurement of presynaptic calcium technically challenging. Classic studies measuring presynaptic calcium used the squid giant synapse and neuromuscular junction, large presynaptic structures that enabled direct calcium measurements with microspectrophotonic absorption and charge-coupled device imaging, respectively. Because these structures are many orders of magnitudes larger than typical mammalian presynaptic boutons, different approaches have been used to measure presynaptic calcium in the mammalian central nervous system. One approach that has proven useful in measuring presynaptic calcium signals in small presynaptic structures is the use of membrane-permeant indicators to label populations of presynaptic boutons (Regehr and Tank 1991). Rather than attempting to measure calcium levels in individual terminals, the strategy is to make an aggregate measurement from many terminals. These methods have been used for studies of presynaptic calcium in presynaptic terminals of cerebellar granule cells (Regehr and Atluri 1995) and hippocampal mossy fibers (Regehr et al. 1994). Many other synapses are also well suited to this method. The loading technique described in this protocol is not difficult and can provide calcium measurements with signal-to-noise ratios much greater than can be obtained with single-bouton measurements using two-photon microscopy. Also, problems associated with presynaptic rundown that can occur with whole-cell recordings of presynaptic neurons can be avoided with bulk loading, because the cell contents are not dialyzed with pipette solution. Membrane-permeant calcium indicators have been developed by conjugating the negatively charged dye molecule to acetoxymethyl (AM) esters (Tsien 1981). The AM-conjugated dye is uncharged and passes across the cell membrane. Once inside cells, the AM group is removed by endogenous esterases, releasing the ion-sensitive acid form of the indicator. AM-conjugated calcium indicators are available with a range of optical properties and calcium affinities, providing Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081828
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S.D. Brenowitz and W.G. Regehr
flexibility for measuring calcium in different concentration ranges and in combination with other fluorophores (Zhao et al. 1997).
IMAGING SETUP
The methods described here require an upright microscope equipped with epifluorescence optics and appropriate filter sets for the selected indicator. Illumination is provided by either a 150-W xenon bulb or a 100-W halogen bulb powered by a low-noise power supply. Fluorescence is measured with either a photodiode or a photomultiplier tube. The collected light is focused onto the surface of the detector by a lens placed in the image plane. To optimize the signal-to-noise ratio, it is important to control both the area of fluorescence excitation and the region in which fluorescence emission is collected from the specimen. This can be performed with apertures placed in the excitation and emission light paths. Movement artifacts caused by opening the shutter can be minimized by using a remote light source coupled to the microscope by a light guide. To monitor the placement of loading and suction pipettes, it is convenient to use a low-power air objective with a long working distance. For fluorescence measurements, it is desirable to use a highpower water-immersion objective with a high numerical aperture. Selecting the Appropriate Indicator
The same basic procedure is used for all experiments of this type; however, the choice of the indicator and the degree of loading depend on the specifics of the experiment. To illustrate this we consider the use of high- and low-affinity indicators for (1) measurement of the time course of presynaptic calcium transients and (2) detection of changes in the amplitude of presynaptic calcium influx. Measurement of the Time Course of Presynaptic Calcium Transients
Calcium transients on the tens-of-milliseconds to tens-of-seconds timescale have been implicated in several use-dependent forms of plasticity that include facilitation, posttetanic potentiation, and calcium-dependent recovery from depression. Accurate determination of the time course of calcium transients on these timescales relies on the correct choice of indicator (Regehr and Atluri 1995; Feller et al. 1996). A comparison of the time course of fluorescence transients measured with two different indicators is shown in Figure 2A. Magnesium Green, which has a dissociation constant (Kd) for calcium of 7 µM, is effective at detecting large calcium transients (Zhao et al. 1996) and provides an accurate measure of the time course of the calcium transient. In contrast, the high-affinity indicator Fura-2 (Kd 200 nM) is sufficiently sensitive to slow the transient as a consequence of saturation of the indicator (Regehr and Atluri 1995). Detection of Changes in the Amplitude of Presynaptic Calcium Influx
Such measurements are required in determining the relationship between calcium influx and transmitter release, as well as in studies of the contribution of presynaptic calcium-channel modulation to alterations in synaptic strength (Sabatini and Regehr 1995). Consider the measurement of fluorescence changes (ΔF/F ) for stimulus trains in which fibers are activated twice in rapid succession (Fig. 2B). With a low-affinity indicator, such as Magnesium Green, the second stimulus produces the same increment in fluorescence, but with Fura-2 the fluorescence transient produced by the second stimulus is smaller. The difference between the responses of these two indicators is a reflection of their different affinities. For low-affinity calcium-sensitive indicators, such as benzothiazole coumarin, Magnesium Green, MagFura-5, Furaptra, Fura-FF, and others, there is a roughly linear relationship between calcium entry and the resulting ΔF/F signals, and it is a simple matter to detect changes in calcium entry. Although some low-affinity indicators such as Furaptra and Mag754
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Bulk Loading of AM Indicators
FIGURE 2. Detection of presynaptic calcium transients and changes in calcium influx in cerebellar parallel fibers. Magnesium Green (Kd for calcium = 7 µM) and Fura-2 (Kd for calcium = 200 nM) were used to measure the time course of ΔF/F signals. Fluorescence changes were evoked by one pulse (A) and by two pulses separated by 10 msec (B). ΔF/F signals for Fura-2 are inverted for clarity. Experiments were conducted in cerebellar brain slices from young rats at room temperature.
nesium Green are sensitive to magnesium as well as calcium, other low-affinity indicators are insensitive to magnesium (e.g., Fura-FF and Fluo-4FF). For high-affinity calcium indicators, such as Fura2, Calcium Orange, and Calcium Crimson, there is a distortion of the calcium transient owing to saturation of the indicator. Despite the saturation of high-affinity indicators such as Fura-2, these indicators are useful for the studies of presynaptic calcium. Their sensitivity makes them well suited to detecting small elevations of calcium, and they also have the advantage that they are relatively insensitive to magnesium. Furthermore, their degree of saturation provides a way of quantifying calcium levels reached in presynaptic terminals and, with care, can be used to quantify changes in calcium entry (for a detailed description, see Regehr and Atluri 1995; Sabatini and Regehr 1995). Determining the Appropriate Loading Conditions
The introduction of an indicator into a cell always alters the calcium dynamics to some extent. Therefore, with a loading method such as that described here, it is important to determine how much indicator has been introduced into the presynaptic terminal and what effect this might have on the parameter being measured. With high-affinity indicators, such as Fura-2, it is straightforward to use the degree of saturation as a measure of the extent of calcium buffering. For example, compare the measurements of fluorescence transients produced by one and two stimuli in Figure 3A. For a fiber tract that has a low concentration of Fura-2 (Fig. 3A, left), there is a smaller incremental increase in fluorescence for the second stimulus than for the first, indicating that the calcium transients are sufficiently large to begin to saturate the response of the indicator. In a fiber tract that has a higher
FIGURE 3. Assessing the degree of indicator loading. The effects of loading time are shown for Fura-2 (A) and MagFura-5 (B) fluorescence transients. Presynaptic terminals were loaded for 5 min (left) and 30 min (right). Experiments were conducted at room temperature in cerebellar parallel fibers from young rats. Peak changes in ΔF/ F produced by the first stimulus are in A, 4.85% (left) and 5.9% (right), and the single stimulus in B, 0.96% (left) and 0.97% (right).
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concentration of Fura-2 (Fig. 3A, right), the size of the calcium transient is reduced by the increased buffer capacity, and there is less saturation of the indicator. Thus, for a high-affinity indicator, the degree of saturation is sensitive to the amount of indicator introduced into the presynaptic terminal. Changes in the degree of saturation can be used to quantify the amount of buffer introduced into the presynaptic terminal relative to the endogenous buffer capacity, using an approach similar to that described previously (Sabatini and Regehr 1995). For low-affinity indicators, the extent of loading and the degree to which calcium signaling is distorted can be examined by measuring the time course of the calcium transient, as in Figure 3B. Lightly labeled fiber tracts provide an accurate measure of the calcium transient, whereas transients in heavily labeled tracts are significantly slowed. The degree of slowing reflects the extent to which the buffer capacity of the presynaptic terminal has been increased by the indicator (Neher and Augustine 1992; Tank et al. 1995). Because the loading technique described here can be used to measure fluorescence in a population of presynaptic terminals, an advantage of this approach is that low intracellular concentrations of indicator can provide a fluorescence signal with minimal perturbation of the endogenous calcium transient and yet retain excellent signal-to-noise characteristics. CONCLUSION
The loading procedure described here has proven to be a useful tool in the study of calcium’s role in synaptic transmission in the mammalian brain (Regehr et al. 1994; Wu and Saggau 1994; Mintz et al. 1995; Atluri and Regehr 1996; Dittman and Regehr 1996; Sabatini and Regehr 1996, 1997, 1998; Chen and Regehr 1997). Provided that care is taken in the choice of indicator and loading conditions, it is possible to measure the time course of presynaptic calcium transients, to quantify changes in calcium influx, and to measure the time course of presynaptic calcium entry. RECIPE ACSF for Calcium Imaging
125 mM NaCl 26 mM NaHCO3 1.25 mM NaH2PO4 2.5 mM KCl 1.0 mM MgCl2 2.0 mM CaCl2 25 mM glucose Bubble 95% O2 and 5% CO2 through the solution.
ACKNOWLEDGMENTS
This work was supported by the Intramural Program of the National Institute on Deafness and Other Communication Disorders (SDB) and by National Institutes of Health R01DA024090 and R37NS032405 (WGR). REFERENCES Atluri PP, Regehr WG. 1996. Determinants of the time course of facilitation at the granule cell to Purkinje cell synapse. J Neurosci 16: 5661–5671. Beierlein M, Gee KR, Martin VV, Regehr WG. 2004. Presynaptic calcium measurements at physiological temperatures using a new class of dextran-conjugated indicators. J Neurophysiol 92: 591–599.
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Chen C, Regehr WG. 1997. The mechanism of cAMP-mediated enhancement at a cerebellar synapse. J Neurosci 17: 8687–8694. Dittman JS, Regehr WG. 1996. Contributions of calcium-dependent and calcium-independent mechanisms to presynaptic inhibition at a cerebellar synapse. J Neurosci 16: 1623–1633.
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Bulk Loading of AM Indicators
Feller MB, Delaney KR, Tank DW. 1996. Presynaptic calcium dynamics at the frog retinotectal synapse. J Neurophysiol 76: 381–400. Mintz IM, Sabatini BL, Regehr WG. 1995. Calcium control of transmitter release at a cerebellar synapse. Neuron 15: 675–688. Neher E, Augustine GJ. 1992. Calcium gradients and buffers in bovine chromaffin cells. J Physiol 450: 273–301. Regehr WG, Atluri PP. 1995. Calcium transients in cerebellar granule cell presynaptic terminals. Biophys J 68: 2156–2170. Regehr WG, Tank DW. 1991. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J Neurosci Methods 37: 111–119. Regehr WG, Delaney KR, Tank DW. 1994. The role of presynaptic calcium in short-term enhancement at the hippocampal mossy fiber synapse. J Neurosci 14: 523–537. Sabatini BL, Regehr WG. 1995. Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice. Neuropharmacology 34: 1453–1467. Sabatini BL, Regehr WG. 1996. Timing of neurotransmission at fast synapses in the mammalian brain. Nature 384: 170–172. Sabatini BL, Regehr WG. 1997. Control of neurotransmitter release by presynaptic waveform at the granule cell to Purkinje cell synapse. J Neurosci 17: 3425–3435.
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Sabatini BL, Regehr WG. 1998. Optical measurement of presynaptic calcium currents. Biophys J 74: 1549–1563. Swanson GT, Contractor A. 2004. Recording in the cerebellar slice. Curr Protoc Neurosci 2004: 6.18.1–6.18.14. Tank DW, Regehr WG, Delaney KR. 1995. A quantitative analysis of presynaptic calcium dynamics that contribute to short-term enhancement. J Neurosci 15: 7940–7952. Tsien RY. 1981. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290: 527–528. Wu LG, Saggau P. 1994. Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus. Neuron 12: 1139–1148. Zhao M, Hollingworth S, Baylor SM. 1996. Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers. Biophys J 70: 896–916. Zhao M, Hollingworth S, Baylor SM. 1997. AM-loading of fluorescent Ca2+ indicators into intact single fibers of frog muscle. Biophys J 72: 2736–2747. Zucker RS, Regehr WG. 2002. Short-term synaptic plasticity. Annu Rev Physiol 64: 355–405.
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Presynaptic Calcium Measurements Using Bulk Loading of Acetoxymethyl Indicators Stephan D. Brenowitz and Wade G. Regehr Cold Spring Harb Protoc; doi: 10.1101/pdb.prot081828 Email Alerting Service Subject Categories
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Protocol
Presynaptic Calcium Measurements Using Bulk Loading of Acetoxymethyl Indicators Stephan D. Brenowitz and Wade G. Regehr
We describe a method for labeling presynaptic terminals in mammalian brain slices by focal application of calcium indicators conjugated with acetoxymethyl (AM) esters. A solution of membranepermeant, AM-conjugated calcium indicator is focally applied to the transverse cerebellar brain slice and allowed to equilibrate throughout the parallel fiber tract. Fibers are then stimulated with an extracellular electrode and fluorescence transients are measured from a location several hundred micrometers from the loading site using a photodiode or photomultiplier tube. Considerations for selecting an appropriate indicator and determining the optimum loading conditions are discussed.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
ACSF for calcium imaging Artificial cerebral spinal fluid (ACSF) is used to prepare the labeling solution and to perfuse the brain tissue under study during the procedure.
AM-conjugated calcium indicator (e.g., Fura-2 from Invitrogen) AM-conjugated calcium indicators are available from Invitrogen in 50-μg ampoules, a convenient size for preparing a loading solution for short-term use. See Discussion for considerations regarding the selection of calcium indicators.
Fast Green (1% stock solution) Dissolve 100 mg of Fast Green in 10 mL of H2O, vortex for 30 sec, and sonicate for 2 min.
Pluronic F-127/dimethylsulfoxide (DMSO; anhydrous) (25:75) Equipment
Aquarium pump or picospritzer (see Step 7) Fluorescence excitation light source An aperture in a conjugate specimen plane should be present to enable adjustment of the field of illumination.
Adapted from Imaging in Neuroscience (ed. Helmchen and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081828
750
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Bulk Loading of AM Indicators
Microcentrifuge tube filters (e.g., Costar Spin-X; pore size 0.22 µm) Micromanipulators (two; positioned on opposite sides of the specimen) Microscope (upright), equipped with epifluorescence optics See Imaging Setup in the Discussion for a full description of the system required.
Objectives 5× air-immersion (numerical aperture [NA] 0.15) 60× water-immersion (NA 0.90) Photodiode or photomultiplier tube An aperture in a conjugate image plane should be present to control the exposure area.
Pipettes (loading and suction) Tubing and valves These parts are used to connect loading and suction pipettes to the aquarium pump and vacuum lines, respectively. See Step 7.
METHOD Preparation of AM-Conjugated Calcium Indicator Loading Solution
1. Dissolve 50 µg of an AM-conjugated calcium indicator (e.g., Fura-2) in 20 µL of a solution of 25% pluronic F-127 and 75% DMSO. 2. Mix the solution by vortexing for 30 sec and sonicate in an ice-filled bath for 2 min. 3. Add 400 µL of ACSF and 20 µL of 1% Fast Green to the vial. Fast Green is included in the labeling solution to allow visualization during loading.
4. Mix the solution by vortexing for 30 sec, sonicate for 2 min, and filter using a microcentrifuge tube filter with 0.22-μm pore size. Keep the freshly prepared labeling solution on ice to prevent premature deesterification, and use it directly in Step 7. Loading Granule Cell Presynaptic Terminals in Rodent Transverse Cerebellar Brain Slices
5. Place a brain slice in the recording chamber of the microscope and perfuse the slice with oxygenated ACSF. For further details, see Swanson and Contractor (2004).
6. Position a suction pipette with a 20–30 µm tip diameter above the surface of the slice. 7. Fill a loading pipette (tip diameter of 20–30 µm) with labeling solution (from Step 4) and position near the suction pipette. An aquarium pump or picospritzer can be used to supply pressure for the loading pipette. To allow regulation of pressure in a very low range, we use branched tubing to connect the pressure source and pipette, with a valve at the end of one arm of the tubing to allow pressure to escape. When the valve is fully open, little or no pressure reaches the loading pipette, and partially closing the valve provides gentle pressure for loading.
8. Apply positive pressure to the loading pipette and suction to the other pipette. Using transmitted light to visualize the Fast Green present in the loading pipette, adjust the flow rate to maintain a rapidly flowing, well-confined stream of dye that is collected entirely by the suction pipette (Fig. 1). See Troubleshooting.
9. Lower the pipettes together to a position near the surface of the slice. 10. Gently lower the loading pipette to the slice surface and place it into the slice until the flow of solution from the pipette is reduced or stops entirely. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081828
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FIGURE 1. Procedure for loading granule cells in cerebellar brain slices with AM-conjugated calcium indicator. (A) Lowpower (4×), transmitted-light image of the loading pipette (right) and the suction pipette (left) as they approach the brain slice. The plume of Fast Green expelled from the loading pipette is captured by the suction pipette. The transverse cerebellar slice (out of focus) is visible below. Scale bar, 200 µm. (B) Configuration of pipettes during loading with AM-conjugated calcium indicator. In this transmitted-light image, the tip of the loading pipette (on right) is placed 15 µm below the surface of the slice and a small cloud of Fast Green is visible. To obtain focal labeling with the AM dye, the loading solution is collected by the suction pipette (on left, out of focus), which is located 50 µm above the slice. Scale bar, 25 µm. (C ) Labeled parallel fiber band. This epifluorescence image was taken shortly after loading 200 µm from the loading site. Scale bar, 25 µm.
11. Gradually increase the pressure on the loading pipette until the flow resumes. Typical loading times range from 2 min to 20 min depending on the preparation, the indicator used, and the desired degree of loading (see Discussion). The brain slice should be continuously superfused with oxygenated ACSF during loading to minimize undesirable background labeling. The total volume of loading solution applied to the brain slice varies depending on the loading time, pressure, and pipette tip diameter, but it is typically 1–5 µL.
12. After loading the labeling solution into the brain slice, allow the indicator to equilibrate within the cell for 1 h–2 h. Imaging Calcium Transients in Parallel Fiber Presynaptic Terminals
13. Position the stimulus electrode in the center of the labeled parallel fiber tract near the loading site. 14. Image parallel fibers 600–800 µm from the loading site in a 150-μm-diameter region. Near the loading site, both neurons and glia are indiscriminately labeled, and dye may enter intracellular organelles that do not respond to intracellular calcium changes. However, more distant sites are not exposed to the AM indicator and the only dye reaching these sites is the membrane impermeant acid form. See Troubleshooting.
752
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Bulk Loading of AM Indicators
TROUBLESHOOTING Problem (Step 8): The loading pipette becomes clogged. Solution: This can be corrected by further sonication and filtration of the loading solution. If the
problem persists, a loading pipette with a larger tip can help. Problem (Step 14): No fluorescence change is observed on stimulation. Solution: It is often necessary to reposition the stimulus electrode and adjust the position of the labeled
fibers by moving the slice to optimize the fluorescence transient. Stimulation of a large proportion of labeled fibers will maximize the relative change in fluorescence on stimulation. Problem (Step 14): The stimulus-evoked calcium transient is small or decays slowly. Solution: This is a symptom of excessive loading. To reduce the level of calcium indicator in the
labeled fibers, shorten the loading time or increase the dilution of calcium indicator in the loading solution. Problem (Step 14): Indicators are not retained in presynaptic boutons at physiological temperatures. Solution: For experiments conducted at room temperature (20 C–24 C), the acid form of the indi-
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cator is stable inside cells for hours. At elevated temperatures, the indicator is removed from neurons, limiting the time available for fluorescence measurements (Beierlein et al. 2004). It may therefore be necessary to use calcium indicators conjugated to high-molecular-weight dextrans.
DISCUSSION
Calcium ions entering presynaptic terminals regulate neurotransmitter release as well as multiple types of both short- and long-term synaptic plasticity (Zucker and Regehr 2002). To directly examine the role of calcium in synaptic transmission in the mammalian brain, it is necessary to measure presynaptic calcium levels and determine the time course of presynaptic calcium entry. However, the small size of presynaptic boutons in the mammalian brain (1 µm) makes measurement of presynaptic calcium technically challenging. Classic studies measuring presynaptic calcium used the squid giant synapse and neuromuscular junction, large presynaptic structures that enabled direct calcium measurements with microspectrophotonic absorption and charge-coupled device imaging, respectively. Because these structures are many orders of magnitudes larger than typical mammalian presynaptic boutons, different approaches have been used to measure presynaptic calcium in the mammalian central nervous system. One approach that has proven useful in measuring presynaptic calcium signals in small presynaptic structures is the use of membrane-permeant indicators to label populations of presynaptic boutons (Regehr and Tank 1991). Rather than attempting to measure calcium levels in individual terminals, the strategy is to make an aggregate measurement from many terminals. These methods have been used for studies of presynaptic calcium in presynaptic terminals of cerebellar granule cells (Regehr and Atluri 1995) and hippocampal mossy fibers (Regehr et al. 1994). Many other synapses are also well suited to this method. The loading technique described in this protocol is not difficult and can provide calcium measurements with signal-to-noise ratios much greater than can be obtained with single-bouton measurements using two-photon microscopy. Also, problems associated with presynaptic rundown that can occur with whole-cell recordings of presynaptic neurons can be avoided with bulk loading, because the cell contents are not dialyzed with pipette solution. Membrane-permeant calcium indicators have been developed by conjugating the negatively charged dye molecule to acetoxymethyl (AM) esters (Tsien 1981). The AM-conjugated dye is uncharged and passes across the cell membrane. Once inside cells, the AM group is removed by endogenous esterases, releasing the ion-sensitive acid form of the indicator. AM-conjugated calcium indicators are available with a range of optical properties and calcium affinities, providing Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081828
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flexibility for measuring calcium in different concentration ranges and in combination with other fluorophores (Zhao et al. 1997).
IMAGING SETUP
The methods described here require an upright microscope equipped with epifluorescence optics and appropriate filter sets for the selected indicator. Illumination is provided by either a 150-W xenon bulb or a 100-W halogen bulb powered by a low-noise power supply. Fluorescence is measured with either a photodiode or a photomultiplier tube. The collected light is focused onto the surface of the detector by a lens placed in the image plane. To optimize the signal-to-noise ratio, it is important to control both the area of fluorescence excitation and the region in which fluorescence emission is collected from the specimen. This can be performed with apertures placed in the excitation and emission light paths. Movement artifacts caused by opening the shutter can be minimized by using a remote light source coupled to the microscope by a light guide. To monitor the placement of loading and suction pipettes, it is convenient to use a low-power air objective with a long working distance. For fluorescence measurements, it is desirable to use a highpower water-immersion objective with a high numerical aperture. Selecting the Appropriate Indicator
The same basic procedure is used for all experiments of this type; however, the choice of the indicator and the degree of loading depend on the specifics of the experiment. To illustrate this we consider the use of high- and low-affinity indicators for (1) measurement of the time course of presynaptic calcium transients and (2) detection of changes in the amplitude of presynaptic calcium influx. Measurement of the Time Course of Presynaptic Calcium Transients
Calcium transients on the tens-of-milliseconds to tens-of-seconds timescale have been implicated in several use-dependent forms of plasticity that include facilitation, posttetanic potentiation, and calcium-dependent recovery from depression. Accurate determination of the time course of calcium transients on these timescales relies on the correct choice of indicator (Regehr and Atluri 1995; Feller et al. 1996). A comparison of the time course of fluorescence transients measured with two different indicators is shown in Figure 2A. Magnesium Green, which has a dissociation constant (Kd) for calcium of 7 µM, is effective at detecting large calcium transients (Zhao et al. 1996) and provides an accurate measure of the time course of the calcium transient. In contrast, the high-affinity indicator Fura-2 (Kd 200 nM) is sufficiently sensitive to slow the transient as a consequence of saturation of the indicator (Regehr and Atluri 1995). Detection of Changes in the Amplitude of Presynaptic Calcium Influx
Such measurements are required in determining the relationship between calcium influx and transmitter release, as well as in studies of the contribution of presynaptic calcium-channel modulation to alterations in synaptic strength (Sabatini and Regehr 1995). Consider the measurement of fluorescence changes (ΔF/F ) for stimulus trains in which fibers are activated twice in rapid succession (Fig. 2B). With a low-affinity indicator, such as Magnesium Green, the second stimulus produces the same increment in fluorescence, but with Fura-2 the fluorescence transient produced by the second stimulus is smaller. The difference between the responses of these two indicators is a reflection of their different affinities. For low-affinity calcium-sensitive indicators, such as benzothiazole coumarin, Magnesium Green, MagFura-5, Furaptra, Fura-FF, and others, there is a roughly linear relationship between calcium entry and the resulting ΔF/F signals, and it is a simple matter to detect changes in calcium entry. Although some low-affinity indicators such as Furaptra and Mag754
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Bulk Loading of AM Indicators
FIGURE 2. Detection of presynaptic calcium transients and changes in calcium influx in cerebellar parallel fibers. Magnesium Green (Kd for calcium = 7 µM) and Fura-2 (Kd for calcium = 200 nM) were used to measure the time course of ΔF/F signals. Fluorescence changes were evoked by one pulse (A) and by two pulses separated by 10 msec (B). ΔF/F signals for Fura-2 are inverted for clarity. Experiments were conducted in cerebellar brain slices from young rats at room temperature.
nesium Green are sensitive to magnesium as well as calcium, other low-affinity indicators are insensitive to magnesium (e.g., Fura-FF and Fluo-4FF). For high-affinity calcium indicators, such as Fura2, Calcium Orange, and Calcium Crimson, there is a distortion of the calcium transient owing to saturation of the indicator. Despite the saturation of high-affinity indicators such as Fura-2, these indicators are useful for the studies of presynaptic calcium. Their sensitivity makes them well suited to detecting small elevations of calcium, and they also have the advantage that they are relatively insensitive to magnesium. Furthermore, their degree of saturation provides a way of quantifying calcium levels reached in presynaptic terminals and, with care, can be used to quantify changes in calcium entry (for a detailed description, see Regehr and Atluri 1995; Sabatini and Regehr 1995). Determining the Appropriate Loading Conditions
The introduction of an indicator into a cell always alters the calcium dynamics to some extent. Therefore, with a loading method such as that described here, it is important to determine how much indicator has been introduced into the presynaptic terminal and what effect this might have on the parameter being measured. With high-affinity indicators, such as Fura-2, it is straightforward to use the degree of saturation as a measure of the extent of calcium buffering. For example, compare the measurements of fluorescence transients produced by one and two stimuli in Figure 3A. For a fiber tract that has a low concentration of Fura-2 (Fig. 3A, left), there is a smaller incremental increase in fluorescence for the second stimulus than for the first, indicating that the calcium transients are sufficiently large to begin to saturate the response of the indicator. In a fiber tract that has a higher
FIGURE 3. Assessing the degree of indicator loading. The effects of loading time are shown for Fura-2 (A) and MagFura-5 (B) fluorescence transients. Presynaptic terminals were loaded for 5 min (left) and 30 min (right). Experiments were conducted at room temperature in cerebellar parallel fibers from young rats. Peak changes in ΔF/ F produced by the first stimulus are in A, 4.85% (left) and 5.9% (right), and the single stimulus in B, 0.96% (left) and 0.97% (right).
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concentration of Fura-2 (Fig. 3A, right), the size of the calcium transient is reduced by the increased buffer capacity, and there is less saturation of the indicator. Thus, for a high-affinity indicator, the degree of saturation is sensitive to the amount of indicator introduced into the presynaptic terminal. Changes in the degree of saturation can be used to quantify the amount of buffer introduced into the presynaptic terminal relative to the endogenous buffer capacity, using an approach similar to that described previously (Sabatini and Regehr 1995). For low-affinity indicators, the extent of loading and the degree to which calcium signaling is distorted can be examined by measuring the time course of the calcium transient, as in Figure 3B. Lightly labeled fiber tracts provide an accurate measure of the calcium transient, whereas transients in heavily labeled tracts are significantly slowed. The degree of slowing reflects the extent to which the buffer capacity of the presynaptic terminal has been increased by the indicator (Neher and Augustine 1992; Tank et al. 1995). Because the loading technique described here can be used to measure fluorescence in a population of presynaptic terminals, an advantage of this approach is that low intracellular concentrations of indicator can provide a fluorescence signal with minimal perturbation of the endogenous calcium transient and yet retain excellent signal-to-noise characteristics. CONCLUSION
The loading procedure described here has proven to be a useful tool in the study of calcium’s role in synaptic transmission in the mammalian brain (Regehr et al. 1994; Wu and Saggau 1994; Mintz et al. 1995; Atluri and Regehr 1996; Dittman and Regehr 1996; Sabatini and Regehr 1996, 1997, 1998; Chen and Regehr 1997). Provided that care is taken in the choice of indicator and loading conditions, it is possible to measure the time course of presynaptic calcium transients, to quantify changes in calcium influx, and to measure the time course of presynaptic calcium entry. RECIPE ACSF for Calcium Imaging
125 mM NaCl 26 mM NaHCO3 1.25 mM NaH2PO4 2.5 mM KCl 1.0 mM MgCl2 2.0 mM CaCl2 25 mM glucose Bubble 95% O2 and 5% CO2 through the solution.
ACKNOWLEDGMENTS
This work was supported by the Intramural Program of the National Institute on Deafness and Other Communication Disorders (SDB) and by National Institutes of Health R01DA024090 and R37NS032405 (WGR). REFERENCES Atluri PP, Regehr WG. 1996. Determinants of the time course of facilitation at the granule cell to Purkinje cell synapse. J Neurosci 16: 5661–5671. Beierlein M, Gee KR, Martin VV, Regehr WG. 2004. Presynaptic calcium measurements at physiological temperatures using a new class of dextran-conjugated indicators. J Neurophysiol 92: 591–599.
756
Chen C, Regehr WG. 1997. The mechanism of cAMP-mediated enhancement at a cerebellar synapse. J Neurosci 17: 8687–8694. Dittman JS, Regehr WG. 1996. Contributions of calcium-dependent and calcium-independent mechanisms to presynaptic inhibition at a cerebellar synapse. J Neurosci 16: 1623–1633.
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Feller MB, Delaney KR, Tank DW. 1996. Presynaptic calcium dynamics at the frog retinotectal synapse. J Neurophysiol 76: 381–400. Mintz IM, Sabatini BL, Regehr WG. 1995. Calcium control of transmitter release at a cerebellar synapse. Neuron 15: 675–688. Neher E, Augustine GJ. 1992. Calcium gradients and buffers in bovine chromaffin cells. J Physiol 450: 273–301. Regehr WG, Atluri PP. 1995. Calcium transients in cerebellar granule cell presynaptic terminals. Biophys J 68: 2156–2170. Regehr WG, Tank DW. 1991. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J Neurosci Methods 37: 111–119. Regehr WG, Delaney KR, Tank DW. 1994. The role of presynaptic calcium in short-term enhancement at the hippocampal mossy fiber synapse. J Neurosci 14: 523–537. Sabatini BL, Regehr WG. 1995. Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice. Neuropharmacology 34: 1453–1467. Sabatini BL, Regehr WG. 1996. Timing of neurotransmission at fast synapses in the mammalian brain. Nature 384: 170–172. Sabatini BL, Regehr WG. 1997. Control of neurotransmitter release by presynaptic waveform at the granule cell to Purkinje cell synapse. J Neurosci 17: 3425–3435.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081828
Sabatini BL, Regehr WG. 1998. Optical measurement of presynaptic calcium currents. Biophys J 74: 1549–1563. Swanson GT, Contractor A. 2004. Recording in the cerebellar slice. Curr Protoc Neurosci 2004: 6.18.1–6.18.14. Tank DW, Regehr WG, Delaney KR. 1995. A quantitative analysis of presynaptic calcium dynamics that contribute to short-term enhancement. J Neurosci 15: 7940–7952. Tsien RY. 1981. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290: 527–528. Wu LG, Saggau P. 1994. Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus. Neuron 12: 1139–1148. Zhao M, Hollingworth S, Baylor SM. 1996. Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers. Biophys J 70: 896–916. Zhao M, Hollingworth S, Baylor SM. 1997. AM-loading of fluorescent Ca2+ indicators into intact single fibers of frog muscle. Biophys J 72: 2736–2747. Zucker RS, Regehr WG. 2002. Short-term synaptic plasticity. Annu Rev Physiol 64: 355–405.
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