Correspondence to: Kamran Khodakhah, Department of Physiology & Biophysics, University of Colorado Health Sciences Center, 4200 East 9th Ave., C-240, Denver, CO 80262. Fax:303-315-8110 E-mail:kamran.khodakhah{at}uchsc.edu.
Released online: 28 February 2000
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Abstract |
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Cerebellar Purkinje neurons demonstrate a form of synaptic plasticity that, in acutely prepared brain slices, has been shown to require calcium release from the intracellular calcium stores through inositol trisphosphate (InsP3) receptors. Similar studies performed in cultured Purkinje cells, however, find little evidence for the involvement of InsP3 receptors. To address this discrepancy, the properties of InsP3- and caffeine-evoked calcium release in cultured Purkinje cells were directly examined. Photorelease of InsP3 (up to 100 µM) from its photolabile caged analogue produced no change in calcium levels in 70% of cultured Purkinje cells. In the few cells where a calcium increase was detected, the response was very small and slow to peak. In contrast, the same concentration of InsP3 resulted in large and rapidly rising calcium responses in all acutely dissociated Purkinje cells tested. Similar to InsP3, caffeine also had little effect on calcium levels in cultured Purkinje cells, yet evoked large calcium transients in all acutely dissociated Purkinje cells tested. The results demonstrate that calcium release from intracellular calcium stores is severely impaired in Purkinje cells when they are maintained in culture. Our findings suggest that cultured Purkinje cells are an unfaithful experimental model for the study of the role of calcium release in the induction of cerebellar long term depression.
Key Words: inositol trisphosphate, ryanodine, long term depression, synaptic plasticity, calcium stores
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INTRODUCTION |
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Repeated concurrent activation of the two major excitatory inputs to cerebellar Purkinje cells, the climbing fibers and the parallel fibers (PFs),1 results in the long-term depression (LTD) of the PF synaptic response (
Cultures of dissociated neurons are frequently used as simplified systems with which to study the cellular basis of neuronal plasticity. Long term depression of currents produced by ionophoretically applied glutamate has been described in cultured Purkinje cells (culture-LTD) (
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MATERIALS AND METHODS |
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Cell Culture
Cerebelli were removed from CD1 mice anaesthetized with Nembutal (50 mg/kg, i.p.) at embryonic days 1618, dissociated by the method of
Identification of Purkinje Neurons in Culture
To identify Purkinje neurons in culture, we labeled a few cultures with a monoclonal antibody to calbindin (Sigma Chemical Co.). At 7 d in vitro, >30% of neurons were positive for calbindin. The cell bodies of the calbindin-positive neurons were larger than (>20 µm) the calbindin-negative neurons, and the cells had more than two primary dendrites. Under bright-field illumination used for electrophysiological studies, Purkinje neurons were identified by their large, high profile cell bodies and the presence of more than two primary dendrites. Our visual identification of large-size neurons was confirmed electrophysiologically from their membrane capacitance. The average membrane capacitance of the cultured Purkinje cells was 13.2 ± 1.4 pF (SEM, n = 10), and the average cell input resistance was 691 ± 207 M (SEM, n = 10). All cells included in this study exhibited large (>1.5 nA) rapidly activating inward currents upon depolarization to -40 mV.
Dissociated Purkinje Cells
Dissociated cells were prepared with the protocol developed by
Whole-Cell Voltage-Clamp Recordings
The composition of the extracellular solution was (mM): 140 NaCl, 2 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, pH 7.4. The intracellular solution contained 125 Kgluconate, 20 KCl, 10 KHEPES, and 3 MgATP, pH 7.2. The internal solution also contained 200 µM Fluo-3 (Molecular Probes) and 150 µM caged inositol trisphosphate (. The voltage error due to series resistance at the peak of responses was always <10 mV. Cells were voltage clamped at -80 mV with a homemade voltage-clamp amplifier. Data were recorded with an A/D, D/A converter (PCI-MIO-16XE-10; National Instruments) and an IBM computer using custom-written software. Reagents were obtained from Fisher Chemical unless otherwise indicated. Data from 22 neurons were analyzed for this study.
Optical Measurements
Fluorescence measurements were made using the Ca2+ indicator Fluo-3, which was introduced into the cells via the whole-cell patch pipette. Light from a tungsten halogen lamp passed through a monochromator (Cairn Instruments), which restricted the excitation wavelength to 485 ± 20 nm. The excitation light was transmitted to the epifluorescence port of the microscope via a liquid light guide. The end of the light guide was focused on the specimen plane. Emitted light was collected through a 530 ± 15-nm bandpass filter and quantitatively measured with a photon counting photomultiplier (Electron Tubes) using the PCI-MIO-16XE-10 counter board and custom-written software. A pinhole in the emitted light path limited the size of the field sampled by the photomultiplier to an area just slightly larger than that of the soma of the cell.
Flash Photolysis
A xenon arc lamp (Cairn Research) was used to produce UV pulses of ~1 ms in duration. The energy stored in the flash lamp power supply could be adjusted to vary the intensity of light and the amount of InsP3 uncaged. UV light was transmitted to the microscope via a 3-mm-diameter liquid light guide, and with the aid of a dichroic mirror shared the same light path as that employed by the fluorescence excitation light. A liquid crystal shutter (Display Tech, Inc.), positioned in front of the photomultiplier was activated for 8 ms during the flash to prevent saturation of the photomultiplier. The extent of photolysis was calibrated using a fluorescent pH indicator taking advantage of the stoichiometric release of a proton with ATP during photolysis of caged MgATP, which has the same photolytic efficiency as caged InsP3 (
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RESULTS |
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We characterized the properties of InsP3-evoked calcium transients in Purkinje cells in primary cultures prepared and maintained with the same protocol as for LTD experiments (we thank Dr. David Linden, Johns Hopkins University School of Medicine, Baltimore, MD) for providing the detailed culture protocol). Purkinje cells maintained 1016 d in culture were whole-cell voltage clamped with patch pipettes containing both caged InsP3 and the fluorescent calcium indicator Fluo-3. The contents of the patch pipette solution were allowed to equilibrate with the cell, and known quantities of InsP3 were photolytically released in the cytosol. Photorelease of ~70 µM InsP3 was ineffective in producing a detectable calcium transient in the soma and proximal dendrites of five of seven cells tested. In the remaining two cells, a small and slowly rising calcium transient was observed after photorelease of InsP3. The response of one of these neurons is shown in Fig 1 A. Depolarization of the same neuron to 0 mV for 400 ms evoked a 10-fold larger Fluo-3 F/F signal. The age of the cells, 1016 d in vitro, was chosen because studies of long-term depression in cultured Purkinje neurons have typically used this range. We also examined a few neurons at 57 d in vitro since it has been shown that the percentage of Purkinje neurons with glutamate metabotropic receptorevoked calcium release peaks at this time (
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An upper estimate of the rate of release of calcium with InsP3 can be made in the few cultured Purkinje cells that demonstrated an InsP3-evoked calcium transient. Since the affinity of Fluo-3 for calcium is ~450 nM, the dye will be saturated at calcium concentrations greater than several micromolar. The amplitudes of the F/F Fluo-3 signals after photorelease of InsP3 in cultured Purkinje cells were much smaller than the amplitudes of the calcium transients produced by a 200- or 400-ms depolarization of the cell to 0 mV so it can be safely assumed that the indicator was not saturated during the InsP3-evoked calcium transients. Therefore, the concentration of calcium reached during the peak of the InsP3-evoked calcium responses is at the very most a few micromolar. Considering the slow rate of rise of the InsP3-evoked transients in cultured cells, the maximum rate of calcium release (in the few cells which did have a response) is estimated to be 0.15 µM · s-1.
Most experiments studying the properties of InsP3-evoked calcium transients in Purkinje cells have used rat cerebellar slices. We ascertained that the properties of InsP3-evoked calcium transients in acutely prepared mouse Purkinje cells are like those described in rat Purkinje cells by performing similar experiments in freshly dissociated Purkinje cells from young mice. We chose dissociated neurons over Purkinje cells in slices to improve the space clamp and reduce the time taken to equilibrate the patch pipette contents with the cytosol. As in rat Purkinje cells, intracellular photorelease of >10 µM InsP3 increased calcium in mouse-dissociated Purkinje cells (Fig 1 B). The calcium transient shown in Fig 1 B, evoked by release of ~70 µM InsP3, peaked within 80 ms. This response is typical of all those observed in mouse-dissociated Purkinje neurons (n = 7), and demonstrates that properties of InsP3-evoked calcium transients in mouse Purkinje cells are similar to those in rat Purkinje cells (
Ryanodine receptors are present throughout Purkinje cells (
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The average InsP3-evoked Fluo-3 F/F was 3.3 ± 0.7 (SEM, n = 7), 40-fold larger than the same in cultured Purkinje cells (0.08 ± 0.07, SEM, n = 10). We also calculated the mean of the InsP3- or caffeine-evoked responses, normalized to peak calcium transients induced by 200-ms depolarizations to 0 mV for all cultured and dissociated Purkinje cells (Fig 3). The average normalized InsP3-evoked transient in the dissociated neurons was over 40-fold larger than in cultured neurons, and that of the caffeine-evoked response was 15-fold larger.
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We postulated that the lack of prominent InsP3- and caffeine-evoked calcium transients in cultured Purkinje cells was a result of depleted calcium stores. Rapid depletion of calcium stores has been shown to occur in cells maintained in primary cultures (
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DISCUSSION |
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We directly examined the properties of calcium stores in cultured and acutely prepared mouse Purkinje cells by intracellular photolytic release of InsP3, and by bath application of caffeine. We find that both InsP3 and ryanodine receptormediated calcium release are severely impaired in cultured Purkinje cells. 70% of the cultured Purkinje cells tested showed no InsP3-evoked calcium transient with as much as 100 µM InsP3. This is in marked contrast to acutely prepared Purkinje cells where all cells challenged with >10 µM InsP3 responded with a large and rapidly rising transient. The maximal rate of calcium release in cultured Purkinje cells is estimated to be 0.15 µM · s-1, three to four orders of magnitude less than that found in acutely prepared Purkinje cells (as much as 1500 µM · s-1) (
There are several reports of intracellular calcium release in cultured Purkinje cells in response to activation of metabotropic glutamate receptors. In these studies, where calcium increases are observed, they are small (
In these studies we examined InsP3-evoked calcium release in the soma and proximal dendrites of cultured Purkinje neurons, and compared them with responses in the soma of acutely dissociated cells. It is possible that cultured Purkinje cells have prominent InsP3-evoked responses in their distal dendrites that we missed. However, in cultured Purkinje neurons, labeling with InsP3 receptor-specific antibodies suggests that InsP3 receptors are evenly distributed throughout the Purkinje cell, including the somata and fine dendrites (
While caffeine mobilized calcium in all the acutely prepared Purkinje cells tested in this study, we find that it is less potent in cultured Purkinje cells. Our results are in agreement with the finding that caffeine-evoked responses in cultured Purkinje neurons are quite labile (
The reason for the impaired calcium release in Purkinje cells maintained in culture is not clear. Although InsP3 and ryanodine receptors are present in both the cell bodies and dendrites of cultured Purkinje cells (
This study was prompted by the discrepancy in the data obtained regarding the role of InsP3-evoked calcium release in the induction of LTD in Purkinje cells in acutely prepared slices, and those maintained in culture (
Despite impaired calcium release, it is interesting that a form of plasticity is observed in cultured Purkinje cells. Work from many laboratories has demonstrated that several intracellular second messengers contribute to culture-LTD. These include nitric oxide and cyclic GMP (
The use of culture-LTD as a model for cerebellar long-term depression is based on the assumption that it shares the same fundamental mechanisms as cerebellar LTD. While minor differences between acutely prepared and cultured Purkinje cells have been described previously (summarized in
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Footnotes |
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1 Abbreviations used in this paper: InsP3, inositol trisphosphate; LTD, long-term depression; PF, parallel fiber.
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Acknowledgements |
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We thank Dr. David Linden for providing the Purkinje cell culture protocol, and Drs. Brian Salzberg, A.R. Martin, and David Ogden for comments on the manuscript.
This study was supported, in part, by the National Ataxia Foundation.
Submitted: 28 October 1999
Revised: 6 December 1999
Accepted: 31 January 2000
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