The leaner (tgla) mouse mutation occurs in the gene encoding the voltage-activated Ca2+channel α1A subunit, the pore-forming subunit of P/Q-type Ca2+ channels. This mutation results in dramatic reductions in P-type Ca2+ channel function in cerebellar Purkinje neurons of tgla/tgla mice that could affect intracellular Ca2+ signaling. We combined whole cell patch-clamp electrophysiology with fura-2 microfluorimetry to examine aspects of Ca2+ homeostasis in acutely dissociated tgla/tgla Purkinje cells. There was no difference between resting somatic Ca2+ concentrations in tgla/tgla cells and in wild-type (+/+) cells. However, by quantifying the relationship between intracellular Ca2+ elevations and depolarization-induced Ca2+influx, we detected marked alterations in rapid calcium buffering between the two genotypes. Calcium buffering values (ratio of bound/free ions) were significantly reduced in tgla/tgla (584 ± 52) Purkinje cells relative to +/+ (1,221 ± 80) cells. By blocking the endoplasmic reticulum (ER) Ca2+-ATPases with thapsigargin, we observed that the ER had a profound impact on rapid Ca2+ buffering that was also differential between tgla/tglaand +/+ Purkinje cells. Diminished Ca2+ uptake by the ER apparently contributes to the reduced buffering ability of mutant cells. This report constitutes one of the few instances in which the ER has been implicated in rapid Ca2+ buffering. Concomitant with this reduced buffering, in situ hybridization with calbindin D28k and parvalbumin antisense oligonucleotides revealed significant reductions in mRNA levels for these Ca2+-binding proteins (CaBPs) in tgla/tgla Purkinje cells. All of these results suggest that alterations of Ca2+ homeostasis in tgla/tgla mouse Purkinje cells may serve as a mechanism whereby reduced P-type Ca2+ channel function contributes to the mutant phenotype.
The neurological mutant mouse leaner is a useful model of cerebellar dysfunction and pathogenesis. The leaner (tgla) mutation lies in a splice donor consensus sequence on the gene encoding the Ca2+channel α1A subunit (Fletcher et al. 1996), the pore-forming subunit of P- and Q-type voltage-activated Ca2+ channels (Gillard et al. 1997; Sather et al. 1993; Stea et al. 1994). The tgla mutation results in a dramatic reduction in P-type Ca2+ channel function in cerebellar Purkinje cells (Dove et al. 1998;Lorenzon et al. 1998), where P-type channels contribute approximately 90% to the whole cell Ca2+ current (Dove et al. 1998; Mintz et al. 1992a,b). The greatly diminished whole cell Ca2+ current density is apparently mediated by a reduction in the single-channel open probability of the mutant P-type channel (Dove et al. 1998), rather than by a reduction in the expression of the protein (Lau et al. 1998). This reduction in P-type channel function may have profound effects on intracellular Ca2+ elevations and the mechanisms of Ca2+ homeostasis in leaner Purkinje cells.
Prominent calcium signaling is a well-established aspect of cerebellar Purkinje cell physiology. The control of intracellular calcium concentrations ([Ca2+]i) in Purkinje cells is a dynamic process involving influx through voltage-activated channels, buffering and sequestration by Ca2+-binding proteins (CaBPs) and intracellular organelles, and release from intracellular inositol 1,4,5-trisphosphate (IP3) and ryanodine-sensitive stores (Eilers et al. 1996). The synchronous action of these mechanisms can be observed following excitatory transmission onto Purkinje cells, when transient increases in [Ca2+]i occur at both the dendritic and somatic levels (Eilers et al. 1995a,b). Elevations in [Ca2+]idirect many Purkinje cell functions including the induction of plasticity at both excitatory (Konnerth et al. 1992;Sakurai 1990) and inhibitory (Kano et al. 1992; Llano et al. 1991) synapses. Calcium-mediated long-term synaptic depression (LTD) is believed to be induced by the convergence of parallel and climbing fiber inputs to the Purkinje synapses that combine to activate both Ca2+ influx through voltage-activated Ca2+ channels and Ca2+release from IP3-sensitive stores (Svoboda and Mainen 1999). It is recognized that a modest, spatially restricted portion of the dendritic Ca2+ signal arises from IP3 induced Ca2+ release (Finch and Augustine 1998; Takechi et al. 1998) following parallel fiber activation. The remainder of the postsynaptic Ca2+ elevations are thought to be mediated by voltage-activated Ca2+ channels (Eilers et al. 1996), which is consistent with observations that depolarization-induced Ca2+ channel activation leads to robust increases in Purkinje cell [Ca2+]i (Kano et al. 1995b; Lev-Ram et al. 1992; Tank et al. 1988).
The magnitude and duration of [Ca2+]i elevations following Ca2+ influx or release is tightly regulated by efficient Ca2+ buffering mechanisms. These mechanisms can be functionally categorized into two types: rapid buffers that immediately reduce the free Ca2+ to a fraction of that which entered the cytoplasm, thus limiting the peak free [Ca2+]i, and slow buffers that are responsible for the decay of the Ca2+ transient and the restoration of baseline [Ca2+]i. The activities of the various Ca2+ homeostatic mechanisms overlap and interact to produce rapid and slow buffering. Purkinje cells are proposed to possess a high capacity to rapidly buffer Ca2+ (Fierro and Llano 1996). Much of this Ca2+ buffering may be attributable to the high levels of CaBPs, such as calbindin and parvalbumin (Iacopino et al. 1990; Kosaka et al. 1993; Winsky and Kuznicki 1995), present in Purkinje cells, but also may involve significant Ca2+ uptake into intracellular organelles, such as the endoplasmic reticulum (ER) and mitochondria (Berridge 1998). In this report, we compare Ca2+buffering in homozygous leaner (tgla/tgla) Purkinje cells with that in wild-type (+/+) cells. We show that leaner Purkinje cells have a diminished Ca2+ buffering ability, which we attribute to reduced uptake by the ER and reduced CaBPs. These findings illustrate the impact that a native mutation of a Ca2+ channel gene can have on Ca2+ homeostatic mechanisms.
Male and female wild-type (+/+) and heterozygous leaner (tgla/+) mice on the C57BL/6J background were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and bred to obtain either wild-type (+/+) or homozygous leaner (tgla/tgla) mice. Mice were maintained on a 12-h light/dark cycle with constant temperature (23–24°C), constant humidity (45–50%), and free access to food (Wayne rodent chow) and water. As tgla/tgla mice become extremely ataxic, they were supplemented with hand feeding frompostnatal day 15–16 through postnatal day 50. Handling and care of the animals was in accordance with policies of Texas A&M University and the National Institute of Health.
Individual cerebellar Purkinje cells were obtained from 18- to 30-day-old mice using methods described previously (Dove et al. 1998). Briefly, mice were decapitated under isoflurane anesthesia and their cerebella removed. Parasagittal cerebellar slices (450 μm) were cut on a McIlwain tissue chopper and held in an oxygenated sucrose solution containing (in mM) 248 sucrose, 26 NaHCO3, 10 glucose, 5 KCl, 2 MgCl2, 1 CaCl2, and 1 Na-pyruvate (pH 7.4). Slices were enzymatically treated in sucrose solution containing 1.0 mg/ml Protease type XXIII (Sigma, St. Louis, MO) for 20 min at 35–36°C.
To isolate individual Purkinje cells, cerebellar slices were transferred to Dulbecco's modified Eagles medium (GIBCO, Grand Island, NY) and mechanically triturated through a series of fire-polished Pasteur pipettes. Isolated cells were dispersed onto the glass floor of a recording chamber pretreated with 0.1% Alcian blue solution to facilitate cell adhesion. The recording chamber was placed on the headstage of an inverted microscope (Axiovert 100, Zeiss) and the cells continuously perfused. Purkinje cells were identified morphologically by their large somata and single stump of apical dendrite.
Whole cell patch-clamp recordings were performed with an Axopatch 200A amplifier using pCLAMP software (Axon Instruments, Foster City, CA). Patch electrodes were pulled from borosilicate glass (No. 7052, Garner Glass, Claremont, CA) on a Flaming/Brown micropipette puller (Sutter Instruments, Novato, CA). Electrodes were coated with wax to reduce stray capacitance and fire-polished to final resistances of 4–5 MΩ. Cell capacitance was read directly from the potentiometer after the capacitance transients were nullified. Series resistance was compensated >75% and was adjusted as necessary throughout the course of recordings. Cells were voltage clamped at a holding potential of −60 mV, and Ca2+ currents were elicited by depolarizing voltage steps to −10 mV. Different levels of Ca2+ influx were generated by varying the duration of the voltage steps. Intervals of 1–2 min between voltage steps allowed the resulting Ca2+ transients to return to baseline. Data were low-pass filtered at 1 kHz and were acquired at a sampling rate of 0.2–4 kHz.
Intracellular [Ca2+] measurements
A dual excitation wavelength ratiometric microfluorimetry system was used to determine the spatially averaged [Ca2+]i in the somata of selected Purkinje cells loaded with fura-2 K+ 5. The excitation field (approximately 10 μm diam) was smaller than the soma of all Purkinje cells and was centered to maximally occupy cells. Illumination was provided by a xenon arc lamp (Zeiss), and the excitation wavelength was alternated between 340 and 380 nm by means of a rotating (40 Hz) filter-wheel. The fluorescence signal was collected by a photomultiplier tube (Hamamatsu) with a 510- to 560-nm band-pass filter. The output of the photomultiplier tube (340 and 380 nm wavelength samples) was directed to an analog divider circuit where the ratio of f340 to f380 signals was calculated following subtraction of background and cellular autofluorescence at each wavelength. Background fluorescence was canceled by zeroing the fluorescent signals from the 340- and 380-nm channels in a cell-free field, and autofluorescence was subtracted by reducing the f340 and f380 signals by the average amount of fluorescence recorded from cells not loaded with fura-2. Autofluorescence of patch-clamped cells was <1% of the average value of the f380 signal with the 6.0% filter at baseline [Ca2+]i and was not different between the two genotypes. A neutral density filter (1.0 or 6.0%) was placed in the excitation pathway to prevent dye bleaching and saturation of the photomultiplier.
Procedures for the estimation of [Ca2+]i and estimation of Δ[Ca2+]i have been described in detail previously (Murchison and Griffith 1998). Briefly, experimental fluorescent ratios were converted to [Ca2+] using the equation where K d is the dissociation constant for fura-2, B equals f380min/f380max,R min equals f340/f380 at zero Ca2+,R max equals f340/f380 at high Ca2+, and R equals f340/f380 measured experimentally (Grynkiewicz et al. 1985). For this report, [Ca2+]i was estimated using in vivo calibrations withK d = 230 nM,R min = 0.10,R max = 6.24, and B = 11.5.
Calculation of buffering capacity
Calcium buffering capacity was calculated by employing a modification of the method of Hille and colleagues (Tse et al. 1994). The buffering value β, was determined as the ratio of buffer-bound to free ion using the equation By this method Caint 2+ is the integral of Ca2+ influx (charge from measuredI Ca), υ is cell volume, Δ[Ca2+]i is the measured change in intracellular free Ca2+, Δ[SCa2+] is the change in concentration of Ca2+ bound to endogenous buffers, and Δ[BCa2+] is the change of Ca2+ bound to exogenous buffers (fura-2). Rearranging the above equation gives where β is the sum of the endogenous buffering strength (βS) and exogenous buffering strength (βB). The slope of the Δ[Ca2+]i versus Ca2+ entry plot is therefore the quantity The cellular Ca2+ buffering value (β) is therefore the reciprocal of the slope minus one.
A plot of Δ[Ca2+]iversus Ca2+ entry was constructed for each cell assayed by delivering depolarizing steps of varying duration to yield several levels of Ca2+ entry while measuring the resulting Δ[Ca2+]i. Calcium entry was determined by integrating the Ca2+ current over time and normalizing for cell volume (υ). Cell volume was approximated from the cellular capacitance, assuming the capacitance of a biological membrane to be 1 μF/cm2, realizing this to be an overestimate of accessible cell volume (Neher 1995). There was no difference in the capacitance of Purkinje cells from mutant or wild-type mice (Dove et al. 1998). Cells were included in our analysis only if they contained at least three data points in the linear portion of the Δ[Ca2+]i versus Ca2+ entry plot. The linear portions of the individual and composite buffering curves (the slopes of which are approximately the reciprocals of the buffering values) were fit by linear regressions, while the supralinear portions not used in any calculations were fit visually.
The rate of rise of the Ca2+ transients was determined for each cell by dividing the peak Δ[Ca2+]i by the time-to-peak and taking the average of the rates for the 100- and 200-ms voltage steps with Δ[Ca2+]i > 40 nM (Fig.3 A). Slow buffering was assessed by calculating recovery values (Murchison and Griffith 1998) that are normalized for the amplitudes of the Ca2+ transients and that take into account all processes tending to remove free Ca2+ from the cytosol without implying a linear rate. The recovery values are determined by dividing the time to recover (measured from the peak of the Ca2+transient to the point where the fluorescent ratio record first crosses the prestimulus baseline) by the peak Δ[Ca2+]i (Fig.3 B).
Solutions and drugs
Cells in the recording chamber were continuously perfused with a solution containing (in mM) 140 NaCl, 3 KCl, 2 CaCl2, 1.2 MgCl2, 10N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate (HEPES) and 33 d-glucose (pH 7.4 with NaOH, 310–330 mOsm). Prior to whole cell recordings, the external solution was exchanged for a modified recording solution containing (in mM) 132 NaCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 33 d-glucose, 10 tetraethylammonium chloride (TEA-Cl), and 0.0005 tetrodotoxin (Calbiochem, La Jolla, CA; pH 7.4 with NaOH, 310–330 mOsm). Thapsigargin (Alomone Labs, Jerusalem, Israel) was dissolved in ethanol (0.08% final concentration) and added directly to the bath. The internal pipette solution contained (in mM) 110 Cs-acetate, 15 CsCl, 10 TEA-Cl, 20 HEPES, 4 ATP, and 0.1 GTP (pH 7.2 with CsOH, 290–310 mOsm) with 50 μM fura-2 K+ 5 (Molecular Probes, Eugene, OR). Salts and other chemicals were obtained from Sigma, except as indicated.
In situ hybridization
In situ hybridization was performed as previously described (Lau et al. 1998). Coronal sections (12 μm) of cerebella from eight +/+ and eight tgla/tgla mice (postnatal day 30) were cut using a cryostat and thaw-mounted onto gelatin-coated slides. Calbindin D28k and parvalbumin single-stranded oligonucleotide probes were complementary to mouse cerebellum calbindin D28k mRNA bases 231–263 (Nordquist et al. 1988) and mouse parvalbumin mRNA bases 68–112 (Zuhlke et al. 1989). The oligonucleotide probes were radiolabeled with 35S-dATP (Dupont NEN, Boston, MA) using terminal deoxynucleotidyl transferase (GIBCO). Standard sense-strand controls confirmed the probe specificity.
The sections were fixed with 4% formaldehyde in phosphate-buffered saline, acetylated in saline (0.9% wt/vol NaCl) containing 0.25% acetic anhydride and 0.1 M triethanolamine, dehydrated in graded ethanol and delipidated using chloroform. The hybridization buffer contained 10 × 106 counts per minute (CPM)/ml oligonucleotide, 50% formamide, 10% dextran sulfate, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl-sulfate, 0.6 M NaCl, 80 mM Tris-HCl (pH 7.4), 4 mM ethylenediamine tetraacetic acid (EDTA), 0.1 M dithiothreitol, and 0.2 mg/ml heparin sulfate. Sections were hybridized overnight with oligonucleotide probe (0.25 × 106 CPM/section) in a 37°C humid chamber, then the sections were washed four times with 2 × sodium chloride plus sodium citrate (SSC) (1 × SSC; 150 mM sodium chloride, 15 mM sodium citrate) and 50% formamide and twice in 1 × SSC. The sections were rinsed first in distilled water, then 70% ethanol and air dried.
Sections were exposed for 5–7 days at room temperature to BioMax MR film (Kodak, Rochester, NY) to reveal the radiographic signal, then the sections were dipped in NTB-2 emulsion (Kodak), diluted 1:1 with deionized water. After 3–6 wk of exposure in the dark at room temperature, the sections were developed in Kodak D-19 developer, fixed in Kodak fixer, counterstained lightly with thionin, and then cover slipped.
A standardized perimeter for Purkinje cells was determined from captured images of thionin stained anterior and posterior cerebellar sections from four animals of each genotype. Using the public domain NIH Image 1.61 program (available athttp://rsb.info.nih.gov/nih-image/), Purkinje cell somata were manually outlined to determine soma size. For each animal used to determine the soma size, a total of 40 Purkinje cells were measured from the vermis and hemispheres. No significant difference in Purkinje cell soma size between +/+ and tgla/tgla mice was observed, and the modal soma size was used as the standardized cell margin.
The numbers of silver grains located over Purkinje cells were counted as previously described (Nakagawa et al. 1996) with minor modifications. Bright-field color images were captured with uniform brightness through a Hamamatsu video camera attached to an upright microscope (Axioplan 2, Zeiss). The images were converted to grayscale and the brightness readjusted to achieve a consistent threshold level. The standardized perimeter was applied to captured images, and only Purkinje cells that fit this template were counted. A total of 80 cells per animal were measured equally from the vermis and hemispheres of cerebellar folia IV + V and VIII + IX (20 cells from each region). Adobe Photoshop 5.0 was used to count the number of silver grains within the template by identifying pixels above the threshold level. The number of silver grains per cell was obtained by dividing the total number of pixels occupied by silver grains on a Purkinje cell by the average size a single silver grain. The mRNA expression level was described by the number of silver grains per cell. Nonspecific binding was corrected by subtracting the average number of silver grains observed in an identical area of the adjacent granule cell layer. All measurements were carried out with the investigator blind to genotype. Accuracy of this counting method was confirmed by several duplicate counts in which the grain count was performed manually. The use of the photoshop software reduces the subjective problems of manual grain counting techniques.
In situ hybridization data were analyzed using an ANOVA for a two-factor experiment (SAS, SAS Institute) followed by Scheffé'sF-test for post hoc analysis (α = 0.05). All other data were analyzed using two-way ANOVA or t-tests where appropriate. Statistical significance was based on P < 0.05, with all values reported as means ± SE.
Basal [Ca2+]i was assessed in acutely dissociated +/+ and tgla/tgla cerebellar Purkinje cells loaded with 50 μM fura-2 K+ 5 via the patch pipette and in cells loaded with sufficient fura-2 AM to provide approximately the same fluorescent intensity as in patched cells. This concentration of fura-2 does not contribute much to the endogenous buffering capacity of Purkinje neurons (Fierro et al. 1998) and therefore should operate effectively as a [Ca2+] indicator (Murchison and Griffith 1998). Data were collected 5–10 min after establishment of the whole cell patch-clamp configuration or 40–50 min after wash out of fura-2 AM from the bath (to allow for intracellular deesterification). Loading of the fluorescent indicator was assessed by monitoring the increase in 380 nm fluorescence. Despite ample evidence for reduced Ca2+ influx in tgla/tgla Purkinje cells due to reduced P-type Ca2+ current (Dove et al. 1998; Lorenzon et al. 1998), we found no differences in resting somatic [Ca2+]i between +/+ and tgla/tgla Purkinje cells. In patched cells, the values were 70.3 ± 3.4 nM (mean ± SE; n = 19) and 71.8 ± 5.1 nM (n = 13); and in unpatched cells they were 119.5 ± 8.3 nM (n = 10) and 113.9 ± 4.4 nM (n = 20) for +/+ and tgla/tgla cells, respectively. These levels of resting Ca2+ are slightly higher than the 25–40 nM previously estimated for Purkinje cells in slice preparations (Fierro et al. 1998;Kano et al. 1995a; Llano et al. 1994), but are consistent with levels observed in other neurons (Miller 1991; Murchison and Griffith 1998). Thus tgla/tgla Purkinje cells are able to maintain a normal basal [Ca2+]i despite reduced Ca2+ entry.
Calcium buffering capacity
Cerebellar Purkinje cells are thought to have a high endogenous Ca2+ binding ratio (Fierro and Llano 1996) which is a measure of the ability to buffer elevations in [Ca2+]i. We investigated whether the reduced function of P-type Ca2+channels in tgla/tglaPurkinje cells could affect the changes in intracellular calcium concentration (Δ[Ca2+]i) induced by the activation of these channels. We addressed this question with combined whole cell voltage-clamp and fura-2 microfluorimetric recordings, as previously described (Murchison and Griffith 1998). Five to 10 min after establishment of the whole cell recording configuration, voltage-gated Ca2+channels were activated by depolarizing pulses of varying duration, and the accompanying Ca2+ influx and Δ[Ca2+]i were measured, as detailed in methods.
Figure 1 A shows Ca2+ currents elicited by voltage steps to −10 mV from a holding potential of −60 mV in acutely dissociated +/+ and tgla/tgla Purkinje cells. As previously observed, there was a marked reduction in peak current amplitude in the tgla/tglacells (note the calibration scales). Figure 1 B shows the corresponding change in fluorescence ratio (f340/f380) associated with the Ca2+ currents in Fig. 1 A. The smaller of these same fluorescence ratios are shown separated and expanded in Fig. 1 C. These are the same traces used to measure the linear portion of the buffering curves below. In Fig.1 D, the calculated Ca2+ entry is plotted against the peak Δ[Ca2+]i. For both the +/+ and tgla/tgla cell, the plot contains an initial linear portion, followed by a clear break from linearity at larger levels of Ca2+ influx. This supralinearity is interpreted as an indication of Ca2+-induced Ca2+ release (CICR), a process that amplifies Ca2+ signals by the release of Ca2+ sequestered in ryanodine-sensitive intracellular stores (Llano et al. 1994; Verkhratsky and Shmigol 1996). Supralinearity was observed in plots from all +/+ and tgla/tglacells where Δ[Ca2+]ireached threshold levels. Also, caffeine application caused Δ[Ca2+]i in all mutant cells examined (n = 6, not shown), presumably by the activation of caffeine-sensitive ryanodine receptors, as described previously for these cells (Kano et al. 1995a). This suggests that ER signaling functions are maintained in tgla/tgla cells.
The linear portion of the plot was used to calculate the rapid Ca2+ buffering capability, with the reciprocal of the slope yielding the Ca2+ buffering value of the cell. For the +/+ cell depicted in Fig. 1 D, the plot yielded a buffering value of 1,227, a large value consistent with previous characterization of Purkinje cells (Fierro and Llano 1996). In contrast, the tgla/tgla cell in Fig.1 D possessed a much lower buffering value of 697. These values are typical of others in our analysis.
There was a significant reduction (P < 0.001) in average Ca2+ buffering values for tgla/tgla Purkinje cells (584 ± 52, n = 10) relative to +/+ (1,221 ± 80, n = 11) as shown in Fig.2 B. Figure 2 A plots cumulative data for all +/+ and tgla/tgla cells included in our analysis. Despite the reduction in rapid buffering detected in the mutant cells, the mean rate of rise of the tgla/tglaCa2+ transients was significantly (P < 0.001) slower (196 ± 21 nM/s,n = 10) than that of the +/+ cells (543 ± 124 nM/s n = 11). An example of this difference is shown in Fig. 3 A. In the face of constant Ca2+ influx, a reduction in rapid buffering would be expected to increase the rate of rise of the intracellular Ca2+ signal. However, the reduced function of the tgla/tglaP-type Ca2+ channel not only limits the peak amplitude of the Ca2+ current, but also suppresses the rate of Ca2+ influx. Thus it appears that the rate of rise of an intracellular Ca2+ transient is critically influenced by the rate of Ca2+ influx. It is of interest to note in this regard, that the maximum Ca2+ current density, the P-type channel open probability (Dove et al. 1998) and the rate of rise of the Ca2+transient in the tgla/tglaneurons are each about one-third that of the +/+ cells, while the buffering values are only reduced about 50%. That the diminished influx rate in mutant Purkinje neurons does not appear to functionally limit the peak Δ[Ca2+]iemphasizes the decrement in the ability of the mutant cells to buffer Ca2+.
Although we were specifically interested in obtaining data regarding the peak Δ[Ca2+]i to assess rapid buffering, we also acquired information on the slow buffering Ca2+ clearance (return of Ca2+ transient to baseline) from some cells. In agreement with Fierro et al. (1998), we observed both fast and slow components of Ca2+ clearance, with the fast component becoming more prominent with increasing Ca2+ load. Because of this, the net rate of recovery increases with increasing amplitude of the Ca2+ transient. We therefore calculated recovery values according to our procedures in Murchison and Griffith (1998) for transients of <350 nM amplitude (small load) and for transients of >350 nM amplitude (large load). Examples of recovery from a small load are presented in Fig. 3 B. For each cell for which recovery time information was available, we averaged the recovery values of all transients under 350 nM to obtain a single value and likewise for transients over 350 nM. The values for +/+ neurons were as follows: small load, 0.47 ± 0.04 s/nM (n= 9); large load, 0.22 ± 0.04 s/nM (n = 7). The values for large and small loads were significantly different (P < 0.001). These +/+ values were not significantly different from those of the mutant neurons: small load, 0.40 ± 0.06 s/nM (n = 4); large load, 0.20 ± 0.06 s/nM (n = 5).
Endoplasmic reticulum in rapid calcium buffering
To address the differences in endogenous buffering capacity between the two genotypes, we first examined the contribution of the ER using thapsigargin, an irreversible inhibitor of the ER Ca2+ pump (Thastrup et al. 1990). Thapsigargin (400 nM) was bath-applied for 5–7 min and subsequently washed out, a treatment previously shown to effectively block ER calcium uptake (Murchison and Griffith 1998) without affecting voltage-gated Ca2+ channel function (Shmigol et al. 1995). Following application of thapsigargin, cells were stimulated with one to three depolarizing pulses to deplete from the ER any previously sequestered Ca2+ that might remain available for CICR, although in the absence of reloading, the ER stores are thought to spontaneously deplete within a few minutes (Brorson et al. 1991). Thereafter, several levels of Ca2+entry were generated as described above, and the accompanying Δ[Ca2+]i was measured. Figure 4 A shows the plots of Δ[Ca2+]iversus Ca2+ entry for a +/+ and a tgla/tgla Purkinje cell pretreated with thapsigargin. For the +/+ cell, a buffering value of 187 was determined, while the tgla/tgla exhibited a buffering value of 182. Interestingly, buffering values following thapsigargin pretreatment were not significantly different for tgla/tgla Purkinje cells (301 ± 66, n = 9) relative to +/+ cells (377 ± 60, n = 11). However, for both genotypes thapsigargin pretreatment significantly reduced (P < 0.001) buffering values relative to untreated controls (Fig.4 B), suggesting a major role for the ER in rapid Ca2+ buffering in Purkinje neurons. These data also imply that the ER is differentially involved in rapid buffering between the two genotypes, with +/+ Purkinje cells using this organelle more prominently. A decreased contribution of the ER to rapid buffering may account for the reduced Ca2+ buffering capacity of tgla/tglaPurkinje cells.
In addition to reducing buffering values, thapsigargin pretreatment linearized the plot of Δ[Ca2+]i versus Ca2+ entry for both genotypes. This is consistent with depletion of ER Ca2+ stores normally available for CICR, although the supralinearity of the control plots also may be partially explained by saturation of the ER buffering ability (see discussion). An alternative explanation of the dual effects of thapsigargin might be that blocking of the ER calcium pump raises the [Ca2+]isuch that the concentration threshold for CICR is reached at lower levels of Ca2+ entry. In this scenario, the buffering values obtained from the Δ[Ca2+]i versus Ca2+ entry plots would be skewed by the appearance of CICR from residual Ca2+ remaining in the ER. However, this is unlikely because thapsigargin had no effect on resting [Ca2+]i in either +/+ (68.2 ± 4.5 nM, n = 12) cells or tgla/tgla (78.7 ± 6.6 nM, n = 10) cells, and previously sequestered Ca2+ was depleted prior to collecting data. Because thapsigargin treatment is well known to delay the restoration of basal [Ca2+]i(Fierro et al. 1998; Murchison and Griffith 1998) and there was no difference between the recovery values of the +/+ and tgla/tglacells, recovery in thapsigargin was not assessed.
Removal of ER buffering by thapsigargin also significantly increased the rate of rise of the Ca2+ transients in tgla/tgla neurons from 196 ± 21 nM/s (n = 10) to 300 ± 41 nM/s (n = 9, P = 0.03), but not in +/+ neurons (control: 543 ± 124 nM/s; thapsigargin: 720 ± 74 nM/s,n = 11 for each). These data suggest that, in addition to reduced participation of the ER, some further buffering decrement may exist in mutant neurons. Because CaBPs are often considered to be mediators of rapid Ca2+ buffering, and CaBPs have been shown to control the rate of rise of Ca2+transients (Chard et al. 1993), we determined the different level of mRNA expression of CaBPs between the two genotypes.
It has been suggested that CaBPs of the “EF-hand” family, such as calbindin D28k and parvalbumin, may act as endogenous Ca2+ buffers in neuronal cells (Chard et al. 1993; Fierro and Llano 1996). We utilized in situ hybridization histochemistry to assess the levels of mRNA for these two CaBPs in coronal cerebellar sections from +/+ and tgla/tgla mice. Figure5 A shows high-power bright-field images of calbindin D28k mRNA hybridization for representative sections of +/+ and tgla/tgla cerebellum. In both genotypes, silver grains representing positive hybridization for calbindin D28k mRNA are present at the level of Purkinje cell somata. Figure 5 B displays silver grains representing positive hybridization for parvalbumin in +/+ and tgla/tgla sections. Silver grains showing strong hybridization for parvalbumin mRNA were principally observed over Purkinje cell somata and to a lesser degree over somata in the molecular layer. Levels of mRNA for the two CaBPs were compared for individual +/+ and tgla/tgla Purkinje cells by quantifying silver grain density within the area of Purkinje cell somata as described in methods. Quantitative analyses of grain density were performed on high magnification images of cerebellar sections from +/+ (n = 8) and tgla/tgla(n = 8) mice. For both parvalbumin and calbindin D28k, mRNA levels were significantly reduced in Purkinje cells of tgla/tgla mice, as shown in Fig. 6. For calbindin D28k mRNA, average grains per cell equaled 120 ± 2 for +/+ mice and 98 ± 2 for tgla/tgla mice (P < 0.001). The reduction in parvalbumin mRNA level was even more pronounced, with average silver grains per cell 274 ± 7 for +/+ and 150 ± 4 for tgla/tgla(P < 0.001). While corresponding protein levels were not determined, decreased mRNA levels for calbindin D28k and parvalbumin are consistent with the altered rate of rise of Ca2+ transients and reductions in Ca2+ buffering detected in tgla/tgla Purkinje cells.
The leaner (tgla) mouse mutation occurs in the gene encoding the voltage-activated Ca2+channel α1A subunit (Fletcher et al. 1996). Recent work by this laboratory (Dove et al. 1998) and others (Lorenzon et al. 1998) has demonstrated that this mutation leads to dramatic reductions in the function of P-type voltage-activated Ca2+channels in cerebellar Purkinje cells of homozygous leaner (tgla/tgla) mice. P-type channels mediate roughly 90% of all voltage-activated Ca2+ current in Purkinje cells (Dove et al. 1998; Mintz et al. 1992a,b). In this report, we have described the consequences of reduced P-type voltage-activated Ca2+ channel function on the regulation of [Ca2+]i in tgla/tgla Purkinje cells.
There was no difference in resting [Ca2+]i between tgla/tgla and +/+ Purkinje cells. Apparently normal resting [Ca2+]i in tgla/tgla cells, despite reduced influx through P-type channels suggested that Ca2+ signaling might be modified in these mutant cells. By quantifying the relationship between Δ[Ca2+]i and Ca2+ influx, we detected a marked reduction in rapid Ca2+ buffering in tgla/tgla Purkinje cells relative to +/+ cells. For any given level of Ca2+ influx through voltage-gated Ca2+ channels, tgla/tgla cells exhibited somatic Ca2+ elevations of greater magnitude than those displayed by +/+ cells. Likewise, a Ca2+transient of similar amplitude is attained for a given stimulation, despite the diminished Ca2+ entry in the mutant neurons. This implies that reduced P-type channel function in tgla/tgla cells does not result in reduced Δ[Ca2+]i following membrane depolarization, and that Ca2+ signaling processes may not be profoundly disrupted.
Basis of altered Ca2+ homeostasis
The regulation of [Ca2+]i is crucial to cellular physiology. Calcium ions control a variety of neuronal processes including transmitter release, cell excitability, and gene expression (Berridge 1998; Clapham 1995;Volpe et al. 1993). Modifications in Ca2+ regulation may represent important compensatory mechanisms initiated to maintain signaling function in tgla/tgla Purkinje cells. We believe that the reductions in rapid Ca2+buffering that we have observed in the mutant cells probably represent compensatory homeostatic efforts to maintain normal Ca2+ signaling functions, such as CICR, despite greatly reduced Ca2+ influx through voltage-activated Ca2+ channels. Compensatory changes in Ca2+ buffering mechanisms are believed to occur during aging in other neurons (Murchison and Griffith 1998; Tsai et al. 1998), and during chronic depolarization of cultured neurons (Fickbohm and Willard 1999). There is an intriguing possibility that this cellular attempt to provide normal Ca2+ signaling ultimately results in the death of the mutant neurons. It is well known that excessive Ca2+, particularly in mitochondria, can act as the trigger for neuronal death (Budd and Nicholls 1996; Nicotera and Orrenius 1998;Stout et al. 1998). The tgla/tgla animals that we examined were younger than 30 days, and so would not yet be expected to have suffered extensive loss of Purkinje cells (Herrup and Wilczynski 1982). However, older mutant animals suffer a dramatic loss of Purkinje neurons by a currently unknown mechanism (Heckroth and Abbott 1994). Our results suggest that reductions in both ER buffering and CaBPs may place a greater burden on mitochondrial Ca2+ buffering, which could in turn result in the induction of cell death through mitochondrial Ca2+ overload. The possibility that compensatory changes in the nervous system might eventually prove deleterious has been suggested also as a mechanism of age-related neuronal dysfunction (Cotman et al. 1995).
There is also evidence supporting an alternative explanation in which delayed maturation of Purkinje neurons in mouse cerebellar mutants results in the phenotype (Sotello 1990). In this scenario, the reduced rapid buffering ability of tgla/tgla cells is simply part of the continuum of delayed physiological maturation that is presumably mediated by reduced P-type Ca2+channel function in early development. Several lines of evidence support this possibility. Reduced Ca2+ buffering in the mutant Purkinje cells would be consistent with the developmental increase in buffering reported by Fierro and Llano (1996). Also, persistent multiple synaptic contacts on dendritic spines of tgla/tgla Purkinje cells and altered spinogenesis are reminiscent of the situation in immature +/+ Purkinje cells (Rhyu et al. 1999). Additionally, tyrosine hydroxylase, which is known to be transiently expressed in early development of normal Purkinje cells, is persistently expressed in the mutant cells (Austin et al. 1992; Hess and Wilson 1991).
Rapid Ca2+ buffering
In situ hybridization histochemistry analysis revealed significant reductions in the levels of mRNA for the CaBPs calbindin D28k and parvalbumin in tgla/tglacells. While mRNA levels may not directly reflect the expression of functional protein, our previous investigation of α1A Ca2+ channel mRNA in these cells revealed a relative correlation between levels of message and protein expression (Lau et al. 1998). Based on their presumed role as endogenous Ca2+ buffers, reductions in CaBPs in tgla/tgla Purkinje cells are consistent with the increased rate of rise of Ca2+ transients in the presence of thapsigargin, and with a possible role in the reduced buffering capacity of these cells. Interestingly, a Ca2+-responsive element appears to control expression of calbindin D28k in Purkinje cells at the transcriptional level (Arnold and Heintz 1997). This mechanism has been proposed to alter the Ca2+buffering capacity of these cells depending on Ca2+ loads. Likewise, expression of parvalbumin is up-regulated in neurons of the deep cerebellar nuclei in response to loss of Purkinje cell input in several mouse mutants that suffer Purkinje cell degeneration, including leaner (Baurle et al. 1998). This increase in parvalbumin could be a compensatory mechanism to enhance Ca2+ buffering in response to increased Ca2+ influx accompanying the increased excitation in cells of the cerebellar nuclei that are deprived of the tonic inhibitory input of Purkinje cells. Similar feedback mechanisms could operate to produce the compensatory changes in CaBPs proposed here. While CaBPs can profoundly alter the shape and amplitude of [Ca2+]itransients when transfected into or exogenously applied to cells (Chard et al. 1993; Lledo et al. 1992), the role these proteins play in Ca2+ buffering under physiological conditions remains largely unknown. Some insight has come from studies of calbindin D28k null mice, where postsynaptic Ca2+ transients in Purkinje cells are greater in magnitude than those observed in wild-type mice and have a larger rapidly decaying component (Airaksinen et al. 1997). This is consistent with a role for Ca2+ binding proteins as rapid buffers.
We have also observed a prominent role for the ER in Purkinje cell rapid Ca2+ buffering. Cerebellar Purkinje cells are known to express high levels of ER Ca2+ATPases (Baba-Aissa et al. 1996b), including one isoform not expressed elsewhere in the CNS (Baba-Aissa et al. 1996a; Wu et al. 1995). These pumps allow the sequestration of Ca2+ into the ER lumen. A significant contribution of the ER to rapid Ca2+buffering in Purkinje cells contradicts the presumption that the high endogenous buffering capacity of these cells is attributable solely to calcium-binding proteins (Fierro and Llano 1996). Surprisingly, the ER appears differentially involved in Ca2+ buffering between tgla/tgla and +/+ Purkinje cells, as exclusion of this organelle from rapid buffering by inhibition of the Ca2+ ATPases yielded similar buffering capacities for the two genotypes. These data suggest that a lessened contribution of the ER may be the primary basis for the reduced rapid Ca2+ buffering observed in tgla/tgla Purkinje cells, with possible reductions in CaBPs complimenting this change.
It might be anticipated that Purkinje cells with reduced CaBPs, like the tgla/tgla cells, would continue to show a decrement in rapid Ca2+buffering ability relative to +/+ cells, even in the presence of thapsigargin. However, block of ER buffering appears to account for almost the entire difference in the rapid buffering values of the two genotypes. There are several possible explanations for this observation involving the presumed interactions between the CaBPs and the ER. It should be emphasized, however, that the interactions of Ca2+ buffering mechanisms are not well understood, particularly in the case of CaBPs. The thapsigargin data suggest that even the reduced amount of CaBPs apparently present in the tgla/tgla neurons is sufficient to buffer the Ca2+ loads imposed in these experiments to a similar extent as the CaBPs present in the +/+ cells. Thus it may be that the +/+ CaBPs are present in considerable excess. The linearity of the Ca2+ buffering curves in thapsigargin shows that the non-ER rapid buffering mechanisms remain unsaturated in the presence of substantial Ca2+ loads. Alternatively, the apparent lack of additional rapid buffering deficit in the tgla/tglacells treated with thapsigargin could be explained by increased activity of another rapid buffer, such as mitochondria. The primary impact of the reduced CaBPs in the mutant cells appears to be on the rate of rise of the Ca2+ transients, which was significantly increased after thapsigargin treatment, but not changed in the wild-type cells. This is consistent with the findings ofChard et al. (1993), showing that exogenous CaBPs were able to decrease the rate of rise of Ca2+transients.
The ER has not been shown to be involved in the rapid buffering of peak Δ[Ca2+]i in other cell types. Block of the ER Ca2+ pumps by thapsigargin has generally been associated with a reduction in the slow buffering restoration of basal [Ca2+]i in neurons (Markram et al. 1995; Miller 1991;Murchison and Griffith 1998; Shmigol et al. 1994b, 1995). In the present study, thapsigargin affected rapid Ca2+ buffering as evidenced by an increased peak Δ[Ca2+]ifor any given level of Ca2+ entry. Fierro et al. (1998) accorded the ER a role in slow buffering in Purkinje neurons based on evidence of prolonged Ca2+ transients in the presence of pump blockers, thapsigargin, and cyclopiazonic acid. However, these investigators had to use shorter duration whole cell depolarizations in the presence of the blockers to attain the same Ca2+ transient amplitude as in the controls, implying that there was some effect on peak Δ[Ca2+]i, and thus on rapid Ca2+ buffering. These results support the hypothesis that the ER buffers Ca2+ differently in Purkinje cells than in other neuronal types. For instance, an investigation of rat basal forebrain neurons by this laboratory concluded that those cells had rapid buffering values of 200–400 and that the ER was involved primarily in slow buffering without obviously contributing to CICR (Murchison and Griffith 1998). In contrast, we find the Purkinje neurons to have much greater buffering values and an ER that is involved in rapid buffering and in robust CICR. There is an additional implication of the interpretation of the ER as a rapid buffer with respect to the supralinearity of buffering curves observed in Purkinje and other cell types. While this has previously been considered to be due to CICR, there also is a possibility that some of the supralinearity is actually due to “saturation” of the ER buffering ability as the store becomes maximally filled, the slope of the supralinear line then reflecting a combination of the buffering ability of the non-ER rapid buffers and the contribution of CICR. A similar explanation involving saturation of rapid buffers has been proposed recently for supralinear [Ca2+]i responses in the dendrites of Purkinje neurons where Ca2+ influx in the restricted dendritic space produces Δ[Ca2+]i of tens of micromolar (Maeda et al. 1999).
Nature of altered ER Ca2+ regulation
Alterations in Ca2+ buffering by the ER have been reported elsewhere. For example, in rat adrenergic neurons, reduced ER buffering is thought to account for an age-related increase in norepinephrine release (Tsai et al. 1998). The nature of reduced ER buffering remains to be determined. The ER is probably partially full at rest (Garaschuck et al. 1997;Murchison and Griffith 1999; Shmigol et al. 1994a). It is conceivable that the ER in tgla/tgla Purkinje cells may be more fully loaded under resting conditions, lessening its capacity to sequester Ca2+. This might be expected for cells with reduced CaBPs. Not only would this place a greater burden on other rapid buffering mechanisms, in this case ER uptake, but also Ca2+ might be expected to spread further from sites of influx, thus enhancing the opportunities to fill the ER stores. Increased rapid buffering ability has been associated with decreased loading of ER Ca2+ stores in aged rat basal forebrain neurons (Murchison and Griffith 1999), and the opposite situation may pertain to Purkinje neurons of the mutant mouse. From the perspective of the +/+ Purkinje neurons, the full compliment of CaBPs would be expected to reduce the loading of the ER relative to that of the tgla/tgla cells, giving the wild-type ER a greater capacity to buffer Ca2+. Alternatively, the ER may merely be repositioned (Subramanian and Meyer 1997) more distal to calcium entry sites in tgla/tgla Purkinje cells.
As mentioned above, the ER is known to contribute to slow buffering in Purkinje and other neurons. Although we did not directly assess the relative contributions of mutant and wild-type ER to slow buffering, there was no change in the sum process of Ca2+clearance between the two genotypes. This suggests that either the participation of the ER in slow buffering in the mutant cells is not disrupted as the involvement in rapid buffering is, or that other slow buffering mechanisms compensate by increased activity. Another explanation could involve the apparent reduction in the rapid buffering abilities of CaBPs. Because increased buffering by rapid buffers is known to slow Ca2+ clearance (Chard et al. 1993), a decrease in this buffering might enhance clearance and offset the diminished ER uptake. When clearance time is normalized to the amplitude of the Ca2+ transient, as in our lab's method of calculating recovery values (Murchison and Griffith 1998), mouse Purkinje neurons show a pattern of slow recovery from small amplitude transients and relatively more rapid recovery following larger transients. We found the relative recovery to be about twice as fast for transients >350 nM, as for those below that concentration. Fierro et al. (1998) attributed an increased rapid decay of large amplitude transients in Purkinje cells to ER uptake. An increased contribution of the ER to Ca2+ buffering of large Ca2+ loads was proposed also in basal forebrain neurons, but the relative recovery of Ca2+transients is significantly slowed with increasing transient amplitude in those cells (Murchison and Griffith 1998). The basis of this difference in the physiology of the two cell types is unknown, but it further emphasizes the contrasts between their Ca2+ homeostatic mechanisms.
In addition to contributing to Ca2+ buffering, Purkinje cell ER serves as an important reservoir of releasable Ca2+. Cultured Purkinje neurons provided some of the early evidence that the ER can function as a Ca2+ source or sink (Brorson et al. 1991); an interpretation that is now widely accepted as a general principle in neurons (Berridge 1998). The ER appears to be a continuous network (Martone et al. 1993) containing IP3 (Furuichi et al. 1993) and ryanodine receptors (Kuwajima et al. 1992), which mediate Ca2+release (Pozzan et al. 1994; Verkhratsky and Shmigol 1996). Calcium release attributable to activation of these receptors is an important aspect of cell signaling (Clapham 1995) and appears to contribute to the induction of synaptic plasticity (Inoue et al. 1998;Kohda et al. 1995; Khodakhah and Armstrong 1997). For both +/+ and tgla/tglaPurkinje cells, we observed supralinearity in plots of Δ[Ca2+]i versus Ca2+ entry, which was prevented by thapsigargin. As this is generally considered evidence of Ca2+-induced Ca2+ release (Llano et al. 1994; Verkhratsky and Shmigol 1996), it would appear that alterations in Ca2+ homeostasis in the mutants preserve Ca2+ signaling processes of the ER. These results suggest that the reduced contribution of the ER to Ca2+ buffering in tgla/tgla Purkinje cells does not result from generalized ER dysfunction, but is part of an adaptive process to conserve function in a cell where the natural influx of Ca2+ is greatly reduced.
This work was supported in part by National Institutes of Health Grants AG-07805 to W. H. Griffith and NS-01681 to L. C. Abbott and by Texas A&M University Interdisciplinary Research Initiatives to W. H. Griffith and L. C. Abbott.
W. H. Griffith.
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