1Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center, College Station 77843-1114; and 2Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4458
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dove, Leonard S.,
Sang-Soep Nahm,
David Murchison,
Louise C. Abbott, and
William H. Griffith.
Altered Calcium Homeostasis in Cerebellar Purkinje Cells of
Leaner Mutant Mice.
J. Neurophysiol. 84: 513-524, 2000.
The leaner (tgla) mouse mutation
occurs in the gene encoding the voltage-activated Ca2+
channel 1A 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/tgla
and +/+ 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.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1A 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+]i
direct 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.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
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 from postnatal 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.
Isolated cells
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.
Electrical recording
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 M. 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
[Ca2+]i have been
described in detail previously (Murchison and Griffith 1998
). Briefly, experimental fluorescent ratios were converted to [Ca2+] using the equation
![]() |
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
, was determined as the ratio of
buffer-bound to free ion using the equation
![]() |
![]() |
![]() |
A plot of [Ca2+]i
versus 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
[Ca2+]i.
Calcium entry was determined by integrating the
Ca2+ current over time and normalizing for cell
volume (
). 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
[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
[Ca2+]i by the
time-to-peak and taking the average of the rates for the 100- and
200-ms voltage steps with
[Ca2+]i > 40 nM (Fig.
3A). 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
[Ca2+]i (Fig.
3B).
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, 10 N-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 at http://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.
Statistical analysis
In situ hybridization data were analyzed using an ANOVA for a
two-factor experiment (SAS, SAS Institute) followed by Scheffé's F-test for post hoc analysis ( = 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Resting [Ca2+]i
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/tgla
Purkinje cells could affect the changes in intracellular calcium concentration
(
[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
[Ca2+]i were measured,
as detailed in METHODS.
Figure 1A 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/tgla
cells (note the calibration scales). Figure 1B shows the
corresponding change in fluorescence ratio (f340/f380) associated with
the Ca2+ currents in Fig. 1A. The
smaller of these same fluorescence ratios are shown separated and
expanded in Fig. 1C. These are the same traces used to
measure the linear portion of the buffering curves below. In Fig.
1D, the calculated Ca2+ entry is
plotted against the peak
[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/tgla
cells where
[Ca2+]i
reached threshold levels. Also, caffeine application caused
[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. 1D, 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.
1D 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.
2B. Figure 2A 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/tgla
Ca2+ 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. 3A. 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/tgla
P-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/tgla
neurons 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
[Ca2+]i
emphasizes the decrement in the ability of the mutant cells to buffer
Ca2+.
|
|
Although we were specifically interested in obtaining data regarding
the peak [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. 3B. 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
[Ca2+]i was measured.
Figure 4A shows the plots of
[Ca2+]i
versus 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.
4B), 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/tgla
Purkinje cells.
|
In addition to reducing buffering values, thapsigargin pretreatment
linearized the plot of
[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+]i
such that the concentration threshold for CICR is reached at lower
levels of Ca2+ entry. In this scenario, the
buffering values obtained from the
[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/tgla
cells, 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.
Calcium-binding proteins
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. Figure
5A 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 5B 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.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The leaner (tgla) mouse mutation occurs in
the gene encoding the voltage-activated Ca2+
channel 1A 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
[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
[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/tgla
cells. While mRNA levels may not directly reflect the expression of
functional protein, our previous investigation of
1A 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+]i
transients 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/tgla
cells 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 of
Chard 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
[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
[Ca2+]i
for 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
[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
[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/tgla
Purkinje cells, we observed supralinearity in plots of
[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.
![]() |
ACKNOWLEDGMENTS |
---|
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.
![]() |
FOOTNOTES |
---|
Address for reprint requests: W. H. Griffith.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 October 1999; accepted in final form 6 March 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|