 |
||||
|
||||
|
||||
|
||||
The Journal of Neuroscience, July 16, 2003, 23(15):6373-6384
Previous Article | Next Article
Calcium Dependence of Retrograde Inhibition by Endocannabinoids at Synapses onto Purkinje Cells
Stephan D. Brenowitz andWade G. Regehr Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
Abstract
Top
Abstract
Introduction
Many types of neurons release endocannabinoids from their dendrites in response to elevation of intracellular calcium levels. Endocannabinoids then activate presynaptic cannabinoid receptors, thereby inhibiting neurotransmitter release for tens of seconds. A crucial step in understanding the physiological role of this retrograde signaling is to determine its sensitivity to elevations of postsynaptic calcium. Here we determine and compare the calcium dependence of endocannabinoid-mediated retrograde inhibition at three types of synapses onto cerebellar Purkinje cells. Previous studies have shown that Purkinje cell depolarization results in endocannabinoid-mediated retrograde inhibition of synapses received from climbing fibers, granule cell parallel fibers, and inhibitory interneurons. Using several calcium indicators with a range of affinities, we performed a series of in situ and in vitro calibrations to quantify calcium levels in Purkinje cells. We found that postsynaptic calcium levels of15 µM are required for half-maximal retrograde inhibition at all of these synapses. In contrast, previous studies had suggested that endocannabinoid release could occur with slight elevations of calcium above resting levels, which implies that inhibition should be widespread and continuously modulated by subtle changes in intracellular calcium levels. However, our results indicate that such small changes in intracellular calcium are not sufficient to evoke endocannabinoid release. Instead, because of its high requirement for calcium, retrograde inhibition mediated by calcium-dependent endocannabinoid release from Purkinje cells will occur under more restricted conditions and with greater spatial localization than previously appreciated.
Key words: endocannabinoids; Purkinje cell; cerebellum; parallel fiber; climbing fiber; DSE; DSI
Introduction
Endocannabinoids act as retrograde signaling molecules in many brain regions, allowing postsynaptic cells to modulate their synaptic inputs (Kreitzer and Regehr, 2002; Wilson and Nicoll, 2002
Retrograde signaling by endocannabinoids occurs throughout the brain but has been studied most extensively in the hippocampus and cerebellum. Depolarization of either CA1 pyramidal cells or Purkinje cells releases endocannabinoids, which activate presynaptic CB1 receptors and transiently reduce the strength of inhibitory synapses for tens of seconds (Kreitzer and Regehr, 2001a
The calcium dependence of endocannabinoid signaling has important implications for the magnitude and spatial extent of endocannabinoid actions. Previous studies of DSI suggest that endocannabinoid release requires only small increases in Capost. In the cerebellum, an estimated increase of 40–200 nM in a Purkinje cell was sufficient to trigger retrograde inhibition (Glitsch et al., 2000
Here we measure the calcium sensitivity of endocannabinoid-mediated retrograde inhibition for climbing fiber, parallel fiber, and inhibitory synapses onto Purkinje cells. After taking a series of steps to determine absolute levels of Capost, we found that for all three types of synapses, retrograde inhibition produced by Purkinje cell depolarization required Capost of
Materials and Methods
Electrophysiology. Parasagittal slices (300 µm thick) were cut from the cerebellar vermis of 10- to 12-d-old Sprague Dawley rats. Dissections were performed in ice-cold sucrose solution containing (in mM): 78 NaCl, 26 NaHCO3, 68 sucrose, 25 glucose, 2.5 KCl, 1.25 NaH2PO4-H2O, 7 MgCl2, 0.5 CaCl2. Slices were incubated for 30 min at 34°C in sucrose solution and then transferred to saline solution containing (in mM): 125 NaCl, 26 NaHCO3, 1.25 NaH2PO4-H2O, 2.5 KCl, 1 MgCl2, 2 CaCl2, 25 glucose, and allowed to cool to room temperature. All solutions were bubbled with 95% O2/5% CO2.Whole-cell voltage-clamp recordings of Purkinje cells were obtained using glass electrodes (1.5–2.5 M
) filled with an intracellular solution containing (in mM): 135 CsMeSO4, 15 CsCl, 15 HEPES, 0.2 EGTA, 1 MgCl2, 2 MgATP, 0.3 NaGTP, 10 phosphocreatine, 2 QX-314, and 20 TEA. The holding potential was -60 mV. This standard internal solution was supplemented with calcium indicators as described in Results. Series resistance was compensated by 75–90% during recordings, which were made using an Axon Multiclamp 700A amplifier (Axon Instruments, Union City, CA). Data were filtered at 2–5 kHz and sampled at 10 kHz. Access resistance and leak currents were monitored continuously, and experiments were rejected if these parameters changed significantly during recording. Flow rates were 1–2 ml/min. Experiments were performed at room temperature.
Parallel fibers were stimulated with brief pulses (100–300 µsec, 50–500 µA) using a glass theta electrode (World Precision Instruments, Sarasota, FL) filled with saline and positioned in the molecular layer. Bicuculline (20 µM) was used to block GABAA-mediated synaptic currents. Climbing fibers were activated by placing a stimulus electrode in the granule cell layer. Individual climbing fiber responses were identified by a large all-or-none response with a sharp stimulus threshold. In addition to bicuculline (20 µM), 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (250–500 nM) was added to reduce climbing fiber EPSCs by 25–75% (Foster and Regehr, 2002) and to reduce voltage-clamp errors. For experiments measuring DSI, interneurons were stimulated by placing the stimulus electrode in the molecular layer in the presence of 10 µM NBQX.
Calibration of calcium indicators. To convert fluorescence ratios to calcium concentrations, we used the relationship:
where KD is the experimentally measured dissociation constant of the indicator for calcium, Rmin and Rmax are the fluorescence ratios at 0 and saturating calcium concentrations, respectively, and
(1) is defined as the ratio of calcium-free versus calcium-bound fluorescence at the wavelength used as the denominator in R (Grynkiewicz et al., 1985
Measurement of indicator dissociation constants (KD). Previous studies have shown that the properties of indicators can change both as a result of buffer composition and from cytoplasmic interactions within cells. For example, the KD of calcium indicators can increase three- to fourfold when loaded into some types of cells (Baylor and Hollingworth, 1988
The KD of calcium indicators was measured in a spectrofluorimeter (Spex Fluorolog, Edison, NJ). Calibration solutions were prepared according to the technique of Tsien and Pozzan (1989
Addition of BSA to the calibration buffer increased the KD of several of the calcium indicators. As shown for fura-2FF in Figure 1 Aa, BSA increased the KD by fourfold, from 8 to 32 µM. Similarly, BSA increased the KD of fura-2 and mag-fura-5 by 3.3-fold and 4.0-fold, respectively (see Table 1). Although there is uncertainty regarding the cytoplasmic protein concentration in Purkinje cells, the shift that we observe in KD suggests that the affinity of these indicators may also change when loaded in a Purkinje cell. Because of the possibility of such a shift in affinity, using cuvette KD values to estimate calcium concentrations with these indicators could cause underestimation of calcium levels by a factor of 3–4.
View larger version (28K):
[in this window]
[in a new window]
Figure 1. Calibration of calcium indicators. A, Calcium dependence of fura-2FF (Aa) and fluo-5N(Ab)fluorescence in the CsMeSO4 calibration buffer in the absence (circles) and presence (triangles) of BSA (20 mg/ml). BSA increased the KD offura-2FF from 8 to 32 µM but did not alter the KD of fluo-5N. B, Fluorescence excitation spectra for a combination of fura-2FF (500 µM) and fluo-5N (250 µM) measured in a cuvette, measured at 0 mM Ca (6 mM EGTA) and 10 mM Ca. This dye combination has an isosbestic point of 355 nm ( 1). Fura-2FF and fluo-5N have peaks at 380 nm (
View this table:
[in this window]
[in a new window]
Table 1. Summary of indicator properties
Fluo-5N, however, is not prone to such large uncertainties. We found that BSA did not significantly alter the affinity of fluo-5N (Fig. 1 Ab), which suggests that the dissociation constant determined in a cuvette is appropriate for quantifying calcium levels within a cell, regardless of the extent to which fluo-5N is bound to protein within the cell. Our measurements of KD and the effects of BSA are summarized in Table 1.
Magnesium sensitivity of the indicators. For some indicators, sensitivity to magnesium can complicate the quantification of calcium levels. KD values for magnesium are 5.6 mM for fura-2 (Grynkiewicz et al., 1985
Indicator pairs. The insensitivity of the KD of fluo-5N to proteins suggested a strategy to determine the appropriate calibration parameters for fura-2, fura-2FF, and mag-fura-5. It is possible to estimate calcium levels simultaneously with fluo-5N and one of these UV-excited indicators as illustrated in Figure 1 B, in which excitation spectra of a combination of fura-2FF and fluo-5N are shown. Our goal was to test the accuracy of calibration parameters by comparing simultaneous calcium measurements from two indicators. Using the wavelengths indicated in Figure 1 B, we calculated the fluorescence ratio of fura-2FF as
For calibration and imaging experiments using indicator pairs, a filter cube containing a 505 nm dichroic and 515 long-pass emission filter was used. For experiments with either fura-2FF or mag-fura-5 alone, a 455 nm dichroic and 455 nm long-pass emission filter were used.
In situ measurement of Rmax. We first used this approach of measuring calcium with a pair of indicators to make in situ measurements of the calibration parameter Rmax for fura-2, fura-2FF, and mag-fura-5 directly in Purkinje cell dendrites. This parameter is subject to change when indicators are introduced into a cell. As calcium levels become sufficiently high to cause the ratio to approach Rmax (the fluorescence ratio at saturating Capost), measurements of Capost become extremely sensitive to Rmax. Hence, errors in measuring Rmax greatly distort measurements of calcium at concentrations above the KD of the indicator. To measure Rmax in situ, we elevated calcium in the cell by bath applying the calcium ionophore ionomycin (Erdahl et al., 1995
It was not possible to elevate calcium levels within cells sufficiently to saturate the fluo-5N response and directly measure Rmax for this indicator. However, fluo-5N has such a low affinity (Table 1) that calcium levels in our experiments do not lead to ratio changes that approach Rmax for fluo-5N, and as a result slight changes in Rmax have minor effects on Capost measurements with fluo-5N. We therefore used cuvette values of Rmax to convert fluo-5N fluorescence to calcium concentrations.
We also estimated the calibration parameter Rmin within cells by bath applying ionomycin in the presence of
Cuvette measurements of Rmin and Rmax were made using the experimental imaging system with a 30 µM path length cuvette placed on the stage of the microscope. Our standard cesium intracellular solution was supplemented with indicators. Rmin was measured in the presence of 6 mM EGTA and no added calcium. Rmax was measured with 10 mM CaCl2.
For measuring cellular calcium transients with fura-2, fura-2FF, and mag-fura-5, we used the Rmin value obtained in a cuvette and the Rmax value obtained in situ. Because we could not experimentally verify that fluo-5N was saturating during ionomycin application, cuvette values of Rmin and Rmax were used.
Cellular imaging. Imaging was conducted using a Cooke Sensicam QE mounted on an Olympus BX51 upright microscope equipped with a 60x, 0.9 numerical aperture objective. The imaging protocols for measurement of calcium transients evoked by voltage steps consisted of sets of three images (20 msec exposure) acquired in succession at the three appropriate excitation wavelengths (
Calcium indicators were obtained from Molecular Probes (Eugene, OR) with the exception of fura-2FF, which was from Teflabs (Austin, TX). Ionomycin was from Molecular Probes. NBQX was from Tocris (Ellisville, MO), and bicuculline was from Sigma (St. Louis, MO).
Results
Our strategy for determining the calcium sensitivity of retrograde inhibition was to combine calcium measurements with voltage-clamp recordings of synaptic responses to quantify the relationship between postsynaptic calcium levels and retrograde endocannabinoid inhibition. This approach required two preliminary steps. First we needed to establish recording conditions for controlled elevation of calcium. This is difficult because Purkinje cell dendrites tend to fire calcium action potentials, which result in large calcium transients that do not depend on the length of the depolarization. Second, we needed to quantify postsynaptic calcium levels. Although there have been many studies of calcium signaling in Purkinje cells (Ross and Werman, 1987Using voltage steps to produce graded calcium transients
Our first goal was to control calcium influx. We took several steps to achieve voltage control sufficient to prevent spiking and permit regulation of the duration of calcium currents. First, we used a cesium-based internal solution with added TEA and QX-314 to block potassium and sodium channels. Second, we recorded from Purkinje cells from postnatal day (P) 10–P12 rats, which have electrically compact dendritic trees. The size of the dendritic arbor of the Purkinje cells used in this study is indicated by the fluorescence image of a cell loaded with the calcium-indicator fura-2FF (Fig. 2A). It was difficult to control calcium entry into Purkinje cells from animals older than P12, because brief depolarizations evoked dendritic calcium spikes. Finally, delivery of a 200 msec voltage ramp from -90 to -60 mV after the voltage step helped to suppress spiking (Fig. 2B, top). Under these conditions, voltage steps generally did not produce dendritic spikes and uncontrolled calcium entry (Fig. 2B, bottom). Recordings were terminated if cells spiked during voltage steps. Calcium transients were measured in dendritic regions selected from Figure 2A (right) and are shown here asF/F (Fig. 2C). The amplitudes of calcium transients were graded, progressively increasing with the duration of the voltage step. These recording methods were used in subsequent experiments for indicator calibration and measurements of retrograde inhibition.
View larger version (83K):
[in this window]
[in a new window]
Figure 2. Graded calcium transients in cerebellar Purkinje cells. A, Purkinje cell from a P10 rat in a sagittal slice. The cell was loaded with fura-2FF and excited at 380 nm. B, Top, Voltage steps from -60 to 0 mV were delivered for a duration of 100–1000 msec, followed by a 200 msec ramp from -90 to -60 mV. Bottom, Calcium currents recorded in the presence of intracellular Cs, TEA, and QX-314. C, Graded fluorescence transients were produced by voltage steps from B, delivered at the time indicated by the arrow, with the largest transients corresponding to the longest depolarizations. A region selected from the image in A (right) was used for analysis. Exposures were obtained at 250 msec intervals.
Quantification of dendritic calcium transients in Purkinje cells
We addressed two issues to quantify calcium levels with fluoro-metric indicators. First, we needed to select an indicator with appropriate calcium sensitivity. If the affinity were too high, the indicator would become saturated during calcium transients and it would not provide accurate estimates of Capost. If the affinity were too low, the indicator would have poor sensitivity. A second issue is that the properties of indicators can change both as a result of buffer composition and from cytoplasmic interactions within cells (Konishi et al., 1988As described in Materials and Methods, several steps were taken to address these concerns. Indicators were chosen for calibration on the basis of their affinities and spectral properties (Table 1). We selected three ratiometric indicators (fura-2, fura-2FF, and mag-fura-5) that can be calibrated using Equation 1 (Grynkiewicz et al., 1985
View larger version (29K):
[in this window]
[in a new window]
Figure 3. Quantification of dendritic calcium transients. Purkinje cells were loaded with different indicator pairs, fura-2/fluo-5N (A), fura-2FF/fluo-5N (B), and mag-fura-5/fluo-5N (C). Fluorescence transients produced by a series of voltage steps were then measured simultaneously for each pair of indicators. Fluorescence ratios are plotted for fura-2, fura-2FF, or mag-fura-5 in a, and Rmin and Rmax are indicated in with dotted line. Fluorescence ratios for fluo-5N are plotted in b. Ratios determined with indicator pairs are compared in c, where ratios were normalized so that Rmin = 0 and Rmax = 1. Ind and e, calciumvalues were then calculated from the traces in a and b, respectively, using the using calibration parameters described in Materials and Methods. In f the values of calcium determined for each of a pair of indicators are plotted for the purpose of comparison. A line with a slope of 1 is indicated with dotted lines, and regressions of the data points are indicated with solid lines with slopes of 1.06 for Af, 1.02 for Bf, and 1.04 for Cf. In A and B, voltage steps (50, 100, 250, 500, 1000 msec) were delivered, and in C, the 50 msec voltage step was omitted. In Aa, Ab, Ad, and Ae, gray traces correspond to 50 and 100 msec depolarizations. In Ae and Af, calcium concentrations calculated from fura-2 fluorescence ratios that exceeded 2 µM were not displayed. Points corresponding to calculated values of Capost >77 µM (10 times the KD of fura-2FF) or 380 µM (10 times the KD of mag-fura-5) were not displayed in Bf and Cf, respectively.
Experiments in which Purkinje cells were loaded with fura-2 and fluo-5N revealed that the affinity of fura-2 is too high for our purposes. Fluorescence ratios for both indicators were measured simultaneously during a series of voltage steps of 50–1000 msec (Fig. 3Aa,Ab). Fura-2 exhibited nonsaturating ratio changes during voltage steps of 50 or 100 msec duration (Fig. 3Aa, gray traces), but because of its high affinity for calcium (KD = 131 nM), fura-2 saturated completely during voltage-step durations of 250–1000 msec (Fig. 3Aa). Fluo-5N, because of its low affinity for calcium (KD = 410 µM), did not exhibit a detectable fluorescence change during voltage steps of 50–100 msec (Fig. 3Ab, gray traces). Comparison of the normalized ratio changes indicates that fura-2 saturates completely with calcium signals that cause a <1% change in the fluorescence ratio of fluo-5N (Fig. 3Ac). Fluorescence ratios from fura-2 and fluo-5N were converted to calcium concentrations (Fig. 3Ad,Ae). Calcium measurements from these two indicators are difficult to compare because their dissociation constants differ by a factor of several thousand (Fig. 3Af). It is apparent, however, that fura-2 readily saturates and is not suitable for measuring dendritic calcium transients during voltage steps >100 msec.
Similar experiments revealed that fura-2FF is well suited to measuring the Capost transients that produce retrograde inhibition. Fluorescence ratios obtained during voltage steps of 50–1000 msec are shown for fura-2FF (Fig. 3Ba) and fluo-5N (Fig. 3Bb). The plot of the fluorescence ratio of fura-2FF versus the ratio of fluo-5N shows that there is some saturation of the fura-2FF response during calcium transients that cause large fluorescence signals in fluo-5N (Fig. 3Bc). We then compared the calcium concentrations estimated with fura-2FF (Fig. 3Bd) and fluo-5N (Fig. 3Be). Because peak calcium levels during long voltage steps saturated fura-2FF (Fig. 3Ba,Bc), we discarded calcium measurements that exceeded 77 µM (10-fold above the KD of fura-2FF). In this concentration range, calcium measurements made with fura-2FF and fluo-5N were in close agreement (Fig. 3Bf), as indicated by the linear regression of fura-2FF calcium values versus fluo-5N calcium values, which had a slope of 1.03. In a group of eight cells that were loaded with fura-2FF and fluo-5N, the slope of this regression was 0.99 ± 0.03. We conclude that fura-2FF and fluo-5N report calcium concentrations that are in agreement up to
Similar experiments suggested that mag-fura-5 is also suitable for quantifying calcium levels arising from Purkinje cell depolarization (Fig. 3C). After conversion of fluorescence ratios (Fig. 3Ca,Cb) to calcium concentrations (Fig. 3Cd,Ce), we compared calcium estimates obtained with mag-fura-5 and fluo-5N, considering values up to 10-fold greater than the KD of mag-fura-5, or 380 µM. Because of its low affinity (KD = 38 µM), mag-fura-5 does not saturate during calcium transients that cause large fluorescence changes in fluo-5N (Fig. 3Cc). For the cell shown in Figure 3C, the slope of the linear regression of calcium concentrations measured with fluo-5N versus mag-fura-5 had a slope of 1.04 (Fig. 3Cf). For a group of 10 cells examined in this manner, the slope of a regression of calcium measurements from magfura-5 versus fluo-5N was 1.02 ± 0.03. We conclude that measurements of calcium with mag-fura-5 and fluo-5N are in agreement at concentrations up to 380 µM. Moreover, despite its sensitivity to magnesium (see Materials and Methods), magfura-5 appears to be a suitable indicator for measuring calcium transients in Purkinje cells.
On the basis of these experiments, we conclude that fura-2FF and mag-fura-5 are suitable for measuring absolute calcium concentrations during depolarization of Purkinje cells. This conclusion rests on the close agreement of these two indicators with fluo-5N when the indicators were simultaneously loaded and imaged. On the basis of its fivefold lower KD, fura-2FF is better suited to the smaller calcium levels produced by brief depolarizations. Fura-2, however, readily saturates during brief depolarizations, and because of its high affinity it could not resolve calcium transients during depolarizations >100 msec. The agreement between the simultaneously loaded indicators suggests that despite the shift in KD seen for fura-2FF and mag-fura-5 caused by addition of BSA (Fig. 1, Table 1), such a shift does not occur in the cell. If we had been unable to determine the KD values of these indicators within Purkinje cells, our estimates of Capost on the basis of in vitro KD values would be lower bounds subject to the uncertainty of the extent to which the KD shifted to higher values within cells. Also, the linear relation between calcium measurements obtained with the simultaneously loaded dyes indicates that the experiments with ionomycin provide an accurate measurement of Rmax. For subsequent experiments that measured the calcium dependence of retrograde inhibition, we used a single calibrated indicator, either fura-2FF or mag-fura-5, to minimize the duration of fluorescence excitation and bleaching.
Capost dependence of DSE
We next combined synaptic recordings with calcium imaging to determine the relationship between peak dendritic Capost and the extent of retrograde inhibition by endocannabinoids at synapses onto Purkinje cells. Our experimental approach was to deliver a series of voltage steps to the Purkinje cell to elevate Capost to different levels and then determine the relationship between Capost and the resulting synaptic inhibition. Recent studies have suggested that metabotropic receptor activation may modulate the calcium dependence of cannabinoid release or even evoke calcium-independent cannabinoid release (Maejima et al., 2001Parallel fiber DSE
We first examined DSE at the parallel fiber to Purkinje cell synapse (Fig. 4). Each Purkinje cell receives inputs from thousands of parallel fibers that form synapses onto distal portions of its dendrites (Palay and Chan-Palay, 1974
View larger version (20K):
[in this window]
[in a new window]
Figure 4. Calcium dependence of DSE at the parallel fiber to Purkinje cell synapse. A, DSE of parallel fiber EPSCs was measured after voltage steps of 100–1000 msec delivered to a Purkinje cell loaded with fura-2FF. Voltage steps (top) and resulting calcium transients (bottom) are imaged in the dendritic region of parallel fiber stimulation. B, DSE was evoked by a voltage step delivered after the 10th parallel fiber stimulus (top). Traces are averages of three EPSCs at the indicated time (pre, post, rec). C, Peak DSE was calculated as 1 - (EPSCpost /EPSCpre) and plotted versus Capost. DSE was half-maximal at Capost = 14 µM, and the Hill coefficient was 2.5. DSEmax was 0.89. DSEmax and Ca0.5 are indicated by horizontal and vertical dashed lines, respectively. Data in A–C were obtained from one cell.
To determine the calcium dependence of PF DSE, this experimental approach was repeated on five neurons loaded with fura-2FF. The relation between peak Capost and peak DSE is shown in Figure 5A. Maximum DSE (DSEmax) after long voltage steps was 0.89 ± 0.02. Fits of the Hill equation have been made to data from each cell. For clarity, DSE values were normalized to the maximum DSE obtained in each cell so that data range between 0 and 1. For these experiments, Ca0.5 = 15.3 ± 1.1 µM, and the Hill coefficient was 2.3 ± 0.2.
View larger version (22K):
[in this window]
[in a new window]
Figure 5. Summary of calcium dependence of parallel fiber DSE. A, Data from five cells loaded with fura-2FF. For clarity, peak DSE values from each cell were normalized. Each cell is represented by a different plot symbol. Fits of the Hill equation to the data points from each cell are superimposed. For five cells, half-maximal DSE occurred at peak Capost = 15.3 ± 1.1 µM, cooperativity was 2.3 ± 0.2, and peak DSE was 0.89 ± 0.02. B, Data from five cells loaded with mag-fura-5. Half-maximal DSE occurred at peak Capost = 17.4 ± 1.0 µM, cooperativity was 1.2 ± 0.10, and DSEmax was 0.91 ± 0.02.
To confirm these measurements of the calcium dependence and cooperativity of PF DSE, we performed further experiments using a different calcium indicator, mag-fura-5. Using the same experimental technique as illustrated in Figure 4, we measured the relationship between Capost and PF DSE in five neurons using mag-fura-5 to measure Capost (Fig. 5B). In these cells, Ca0.5 = 17.4 ± 1.0 µM, and the Hill coefficient was 1.2 ± 0.1. DSEmax was 0.91 ± 0.02. Measurements of Ca0.5 obtained with fura-2FF and mag-fura-5 for DSE at the parallel fiber synapse are statistically indistinguishable (p = 0.36; t test); however, estimates of the Hill coefficient obtained with fura-2FF and mag-fura-5 differed (p < 0.01, t test). The lower estimate of cooperativity obtained with mag-fura-5 may result from the difficulty of resolving small calcium transients, less than one-tenth of the KD of mag-fura-5 (<
Climbing fiber DSE
We next determined whether the calcium dependence of DSE was similar at the other excitatory synapse onto Purkinje cells, the climbing fiber synapse (Fig. 6). Purkinje cells receive a single powerful climbing fiber synapse that consists of hundreds of release sites located in proximal regions of Purkinje cell dendrites (Palay and Chan-Palay, 1974
View larger version (16K):
[in this window]
[in a new window]
Figure 6. Calcium dependence of DSE at the climbing fiber to Purkinje cell synapse. A, DSE was evoked with Purkinje cell depolarizations of 200–2000 msec (top), which evoked calcium transients measured in proximal dendrites (bottom). B, EPSCs were evoked at 30 sec intervals. EPSCs marked post and rec were evoked at 5 and 35 sec after thet ermination of the voltage step. C, Peak DSE was calculated for each trial as 1 - (EPSCpost /EPSCpre) and plotted against peak Capost. DSEmax and Ca0.5 are indicated by horizontal and vertical dashed lines, respectively. Data fromA and B were obtained from four consecutive trials. Data from A–C were obtained from one cell. In this cell, half-maximal DSE occurred at Capost = 16 µM, cooperativity was 2.6, and DSEmax was 0.68. D, Data from six cells, as obtained in C. Each cell is indicated by a different plot symbol, and fits of the Hill equation for each cell are superimposed. For six cells, half-maximal DSE occurred at peak Capost = 18.5 ± 0.7 µM, cooperativity was 2.0 ± 0.2, and DSEmax was 0.47 ± 0.08.
To determine the relationship between peak Capost and DSE for climbing fiber synapses, we repeated this protocol of delivering voltage steps and measuring Capost and DSE (Fig. 6C). For the neuron shown in Figure 6A–C, half-maximal DSE occurred at Capost = 16.0 µM, and the Hill coefficient was 2.6. This procedure was repeated on six neurons (Fig. 6D). Each cell is indicated with a different plot symbol. For these cells, DSE at the climbing fiber synapse was half-maximal at Capost = 18.5 ± 0.7 and had a Hill coefficient of 2.0 ± 0.2. The values of Ca0.5 and the Hill coefficient at CF synapses are very similar to those described above for PF synapses. DSEmax at the climbing fiber synapse was 0.47 ± 0.08. This value is roughly half of the maximum DSE observed at parallel fiber synapses. Thus DSE at CF and PF synapses shows a similar low sensitivity to Capost and high cooperativity, although the maximal extent of endocannabinoid inhibition is less at CF compared with PF synapses.
Calcium dependence of DSI
Thus far we have seen that extremely high levels of calcium are required for DSE, almost 100 times as large as has been reported previously for DSI. This raises the question of whether there are large differences in the calcium levels needed to produce retrograde suppression of excitatory and inhibitory synapses or whether previous studies may have underestimated the calcium levels required for DSI. We therefore determined the Capost dependence of DSI.Purkinje cells receive inhibitory synaptic inputs from stellate and basket cells that are located in the molecular layer of the cerebellum (Palay and Chan-Palay, 1974
In contrast to the stereotypical localization of excitatory PF and CF synapses on Purkinje cells, the location of inhibitory synapses is more varied. Stellate cells form synapses over a wide area of the dendritic tree, and basket cell terminals form specialized arbors in axosomatic regions. In our experiments, IPSCs were evoked at 0.5 Hz with a stimulus electrode placed in the molecular layer. DSI was quantified by measuring inhibition of evoked IPSCs (eIPSCs) evoked 3–7 sec after the depolarization in the same manner as measurements of PF DSE. We measured calcium transients (Fig. 7A) and inhibition of eIPSCs (Fig. 7B) in Purkinje cells after depolarizations of different durations. Dendritic calcium transients were measured near the location of the stimulus electrode, but the location of the activated synapses was not certain. Data from one cell are shown in Figure 7A–C. Depolarizations that ranged from 150 to 1500 msec produced increasing DSI (Fig. 7B). As in previous experiments, voltage step protocols were repeated to determine the relationship between Capost and DSI. Results from a single neuron are shown in Figure 7C. Half-maximal DSI in this cell occurred at Capost = 14.5 µM, and the Hill coefficient was 1.0. Maximal DSI was 0.80. This experiment was repeated on four neurons (Fig. 7D). For these cells, DSI was half-maximal at Capost = 16.0 ± 0.7 µM and had a Hill coefficient of 1.3 ± 0.1. The maximal extent of DSI was 0.82 ± 0.01. We conclude that the calcium sensitivity of DSI is very similar to that of both PF and CF DSE.
View larger version (16K):
[in this window]
[in a new window]
Figure 7. Calcium dependence of DSI at inhibitory synapses on Purkinje cells. A, DSI was evoked by voltage steps of 150–1500 msec (top), and resulting calcium transients are shown below (bottom). B, (top) IPSCs were evokeda t0. 5 Hz. DSI was evoked by voltage steps delivered after the 10th stimulus. C, Peak DSI is plotted against peak Capost for each trial. DSEmax and Ca0.5 areindicated by horizontal and vertical dashed lines, respectively. Data in A–C are from one cell. Half-maximal DSI occurred at Ca-post =14.5 µM, the hill coefficient was 1.0, and DSI max was 0.80. D, The relationship between DSI and peak Capost for fourcells. Each cell is represented by a different plot symbol, and fits of data from each cell to the Hill equation are shown. For four cells, half-maximal DSI occurred at peak Capost = 16.0± 0.7 µM, cooperativity was 1.3 ± 0.1, and DSImax was 0.82 ± 0.01.
Discussion
Our primary finding is that half-maximal retrograde inhibition by endocannabinoids occurs with postsynaptic calcium levels ofQuantifying calcium levels within Purkinje cells
The approach that we took to quantify dendritic calcium transients in Purkinje cells has general implications for the fluoro-metric measurements of calcium levels for cells in brain slices. The key steps were to perform both cuvette and in situ calibrations and to assess the responses of ratiometric indicators by simultaneously monitoring responses of the single-wavelength indicator fluo-5N. First, we selected the indicators with the appropriate sensitivities and found that fura-2FF and mag-fura-5 were both appropriate for measuring calcium transients evoked by depolarizations of sufficient duration to produce retrograde synaptic inhibition. In contrast, fura-2 was not appropriate because it readily saturated during brief voltage steps as a consequence of its high affinity. The dissociation constant of fura-2FF (7.7 µM) makes it well suited to measuring calcium transients <5 µM that enabled us to resolve the steep cooperativity of the relationship between Capost and DSE. Second, we determined Rmax in situ for fura-2, fura2-FF, and mag-fura-5 by using a calcium ionophore to increase Capost and used fluo-5N to ensure that Capost had been elevated sufficiently. Finally, we assessed whether binding to protein might affect the sensitivity of indicators within Purkinje cells. Although in cuvette measurements the addition of protein increased the KD of fura-2, fura-2FF, and mag-fura-5, the KD of fluo-5N was unaffected. We took advantage of this insensitivity of fluo-5N to determine the appropriate dissociation constants for fura-2 FF and mag-fura-5 in Purkinje cells. Our results suggest that neither of these indicators undergoes a shift in affinity within Purkinje cells. By eliminating a three- to fourfold uncertainty in the KD, we were better able to determine Capost.Calcium dependence of retrograde inhibition
We found similarities and differences in the Capost dependence of retrograde inhibition at the three synapses that we studied. Although Ca0.5 was similar at all of the synapses (In considering the factors underlying such differences in the extent and Capost dependence of retrograde inhibition, it is necessary to consider the many steps between the initial elevation in Capost and the reduction in synaptic strength. (1) Endocannabinoids are synthesized and released in a calcium-dependent manner (Devane et al., 1992
View larger version (23K):
[in this window]
[in a new window]
Figure 8. Contributions of the relation ship between Capre and EPSC to the Capost dependence of retrograde inhibition. Aa, Dependence of the EPSC on Capre at parallel fiber synapses, which is described by an equation of the form EPSC = k(Capre)3. Ab, Relationship between PF–DSE and Capost, which is described by DSE = DSEmax/(1 + (Ca0.5/Capost)n). Parameters of this plot are from Figure 5A, with DSEmax = 0.89, Ca0.5 = 15.3, and n = 2.3. Ac, We next used the power law, above, to calculate the relationship between endocannabinoid-mediated inhibition of Capre and Capost. Capre was calculated using Aa and Ab. Maximum inhibition of Capre is 52%, inhibition of Capre is half-maximal with Capost = 20.8 µM, and the Hill coefficient of 2.0. Ba, Dependence of the CF EPSC on Capre, which is described by EPSC = 1.1 (Capre n/(Capre n + KD n), where n = 4.5 and KD = 0.6 mM (Foster et al., 2002
We therefore assessed how the relationship between Capre and EPSCs influences the Hill coefficient, the Capost sensitivity, and the extent of retrograde inhibition at PF and CF synapses. For PF and CF synapses, we used the known relationship between Capre and EPSC amplitude (Fig. 8Aa,Ba) and our experimentally determined measurement of the dependence of DSE on Capost (Fig. 8Ab,Bb) to express modulation of Capre as a function of Capost during retrograde inhibition (Fig. 8Ac,Bc). Although multiple mechanisms may contribute to endocannabinoid modulation, at the PF and CF synapses the effects of endocannabinoids reflect a reduction of presynaptic calcium influx (Kreitzer and Regehr, 2001b
We also found that the relationship between Capre and EPSC amplitude at PF versus CF synapses altered the apparent Capost sensitivity of retrograde inhibition. At PF synapses, Ca0.5 for inhibition of EPSCs and for inhibition of Capre was 15.3 and 20.9 µM, respectively. At CF synapses, Ca0.5 for inhibition of EPSCs and Capre was 18.5 and 12.3 µM, respectively. This comparison of postsynaptic calcium required for half-maximal inhibition of EPSCs versus Capre revealed that at a saturating synapse such as CF synapses, the Capost required for half-maximal inhibition of Capre compared with inhibition of EPSCs shifts to lower values and at a nonsaturating synapse such as PF synapses the shift is to higher values. Thus, the relationship between Capre and EPSC amplitude alters the Capost dependence of retrograde inhibition, but the effects are small.
The final issue that we addressed was the source of cooperativity in endocannabinoid-mediated retrograde inhibition. We considered the possibility that the steep relationship between Capre and the EPSC might account for steep Hill coefficients of 2–2.3 for the relationship between Capost and DSE. On the basis of the analysis in Figure 8, however, we found that the cooperativity changes only slightly when we consider the effects of Capost on Capre, shifting the Hill coefficients to 1.8–2.0, thus indicating that the cooperativity of DSE occurs at a stage before presynaptic calcium influx. This is consistent with previous studies that found varying degrees of cooperativity (Hill coefficients of 1.2–3) in channel modulation by neurotransmitters (Ito et al., 1992
The Hill coefficient that we measured for the relationship between Capost and DSI was 1.3, consistent with values of 1.3 and 1 reported previously for DSI in hippocampus and cerebellum, respectively (Glitsch et al., 2000
Functional implications
The high Capost levels required for retrograde inhibition and the Hill coefficients ofAdditional factors may also be important under physiological conditions. To accurately determine the Capost dependence of retrograde inhibition, we performed experiments at room temperature in young animals in the absence of metabotropic receptor activation. Considering the many steps between elevation of Capost and retrograde inhibition, it is likely that the Capost dependence of retrograde inhibition differs somewhat at physiological temperatures. The Capost dependence may also be developmentally regulated. Last, recent studies point to a role for metabotropic glutamate and acetylcholine receptors in potentiating Capost-dependent release in evoking release through calcium-independent mechanisms (Varma et al., 2001
To more fully appreciate the functional implications of the Capost dependence of endocannabinoid release, it is necessary to know the magnitude and spatial location of dendritic calcium signals under different physiological conditions. Numerous studies have shown that calcium signals in Purkinje cells arise from spontaneous activity and synaptic activation, both of which can open voltage-gated calcium channels and release calcium from internal stores (Tank et al., 1988
Footnotes
Received Mar. 24, 2003; revised May. 1, 2003; accepted May. 2, 2003.This work was supported by National Institutes of Health Grant RO1-NS32405-01. We thank Michael Beierlein, Dawn Blitz, Solange Brown, Kelly Foster, Anatol Kreitzer, Patrick Safo, and Matthew Xu-Friedman for comments on this manuscript and Kim Irwin for technical assistance.
Correspondence should be addressed to Wade Regehr, 220 Longwood Avenue, Harvard Medical School, Boston, MA 02115. E-mail: wade_regehr{at}hms.harvard.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/236373-12$15.00/0
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.
[Abstract/Free Full Text] Baylor SM, Hollingworth S (1988) Fura-2 calcium transients in frog skeletal muscle fibres. J Physiol (Lond) 403: 151–192.
[Abstract/Free Full Text]<
