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The Journal of Neuroscience, January 1, 1998, 18(1):16-25
Regulation of Ca2+-Dependent K+ Channel
Expression in Rat Cerebellum during Postnatal Development
Yunhua Li
Muller1,
Raven
Reitstetter1, and
Andrea J.
Yool1, 2
1 Department of Physiology and 2 Department
of Pharmacology and the Program in Neuroscience, University of Arizona
College of Medicine, Tucson, Arizona 85724-5051
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ABSTRACT |
Potassium channels govern duration and frequency of excitable
membrane events and may regulate signals that are important in neuronal
development. This study assesses the developmental expression of the
large conductance Ca2+-dependent
K+ channel in vivo and in
vitro in rat cerebellum. In vivo, transcript levels for the Ca2+-dependent K+
channel (KCa) were shown by Northern analysis
to increase during development, whereas transcript levels for the
voltage-gated K+ channel Kv3.1, a delayed rectifier
(KD), remained relatively constant. A comparable
pattern was demonstrated by expression in Xenopus
oocytes of poly(A)-enriched RNA isolated from postnatal rat cerebella.
In cerebellar cultures, increased external K+
provided a simple manipulation of cell excitability that influenced KCa transcript levels during development. With low
external K+ (5.3 mM), the levels of
KCa channel transcript (assessed by semiquantitative PCR) remained constant throughout development. However, in culture medium that supported significant dendritic outgrowth (10 mM extracellular K+), an upregulation of
KCa transcript level was observed similar to that
seen in vivo. Tetraethylammonium (TEA; 1 mM)
similarly enhanced KCa expression, suggesting that
depolarizing stimuli increased KCa expression. The
stimulatory effects of increased K+ or TEA on
KCa expression required extracellular
Ca2+ and were abolished in low external calcium (0.1 mM, buffered with EGTA), although morphological development
and survival were not impaired. The regulation of
KCa channel expression by depolarization and
Ca2+ entry provides evidence of a logical feedback
mechanism governing Ca2+ signals that may be
significant in cerebellar development.
Key words:
calcium-dependent K+ channel; rat
cerebellum; transcription; mslo; rslo; BK
channel; calbindin; depolarization; quantitative PCR
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INTRODUCTION |
The rat cerebellum is immature at
birth and develops rapidly in the first weeks of postnatal life.
Purkinje neurons in culture undergo physiological and morphological
maturation that resembles development in vivo (Woodward et
al., 1969 ) and acquire branched dendrites (Gruol and Franklin, 1987;
Baptista et al., 1994) and a pacemaker-like firing activity (Yool and
Gruol, 1987). This transition in excitability is accompanied by
increases in both Ca2+ and
Ca2+-dependent K+ channel
(KCa) membrane conductances (Yool et al.,
1988; Gruol et al., 1992). The Purkinje neuron is the primary source of
the KCa signal in the cerebellum (Knaus et al.,
1996); the channels are present in soma and dendrites (Gruol et al.,
1991) and show properties of the big conductance (BK) type (Pallotta et
al., 1981; Blatz and Magleby, 1987), including a high unitary
conductance (100 pS in physiological saline), dependence on internal
Ca2+, and sensitivity to millimolar
tetraethylammonium (Reinhart et al., 1989).
Various classes of ion channels exhibit different patterns of
expression during neuronal development (Ribera and Spitzer, 1992). The
expression of a number of K+ channel types is
developmentally regulated in the rodent brain (Swanson et al., 1990;
Trimmer, 1993). Channel expression can be regulated by depolarization
and activation of intracellular signaling pathways (Levitan et al.,
1995; Takimoto et al., 1995; Gan et al., 1996). Data presented here
show that the expression of the rslo sequence encoding the
large conductance KCa channel is regulated at the
transcriptional level during development of the rat cerebellum; in
contrast, a subtype of delayed rectifier channel (Kv3.1) is expressed
uniformly at 2-21 d postnatal. The expression pattern seen for
KCa in vivo is mimicked in cultures treated with chronic depolarizing stimuli (10 mM
K+ or 1 mM TEA) but not seen in cultures
maintained in low external K+ medium. An advantage
of using TEA as a chronic depolarizing stimulus is that its
effectiveness on electrophysiological properties of the neuron does not
seem to decrease with time of exposure (R. Reitstetter and A. J. Yool, unpublished observations), as would be predicted for other agents
such as excitatory amino acid transmitters that undergo
desensitization. In cerebellar cultures, application of 0.5-1
mM external TEA depolarizes the resting membrane potential of the Purkinje neuron by 5-8 mV and increases the voltage-dependent Ca2+ component of the action potential by slowing
the late repolarization phase (Yool et al., 1988). Our results suggest
that depolarization and calcium entry contribute to the transcriptional
regulation of KCa. Dynamic regulation of
KCa channels during development of the rat
cerebellum could influence membrane potential and calcium signaling and
thus be significant in the process of neuronal maturation.
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MATERIALS AND METHODS |
Isolation of poly(A)-enriched RNA from postnatal rat
cerebella. Sprague Dawley rat pups were anesthetized by
CO2 and decapitated at postnatal days 2, 4, 6, 7, 8, 10, and 14. The cerebellar cortices were dissected, frozen in liquid
N2, and then ground on dry ice in separate
preparations for each age. The powdered tissue was denatured in
guanidinium thiocyanate solution. After digestion with RNase-free
DNase, the total RNA was extracted by phenol and chloroform and
precipitated with isopropanol. Polyadenylated-RNA was enriched from
total RNA by oligodeoxythymidine affinity separation (Poly ATtract kit;
Promega, Madison, WI). Gel electrophoresis of the poly(A) RNA samples
showed the expected range of RNAs from 2 to 9 kb and faint but distinct
ribosomal RNA bands.
Xenopus oocyte expression. Oocytes from anesthetized
Xenopus laevis were obtained by surgical removal of lobes of
ovary and were dissociated with collagenase type IA (1.5 mg/ml; Sigma,
St. Louis, MO) for 2-3 hr in calcium-free medium containing (in
mM): NaCl, 82; KCl, 2.5; MgCl2, 1.0; and
HEPES, 5, pH 7.6. Each oocyte was injected with 100-150 ng of rat
cerebellar poly(A) RNA in 50 nl of sterile water and incubated for at
least 2 d at 18°C in buffered-Na+ bath saline
["ND96" containing (in mM): NaCl, 96; KCl, 2.0;
CaCl2, 1.8; MgCl2, 1.0; and
HEPES, 5, pH 7.6] before recording. Two-electrode voltage clamp was
used to record currents expressed in injected and uninjected oocytes,
using electrodes filled with 3 M KCl (0.5-1.5 M ).
Oocytes were held at  80 mV, uniformly stepped to 0 mV to activate
endogenous voltage-gated Ca2+ channels (and allow
Ca2+ influx if present in the bath saline), and then
stepped to various test potentials to evaluate current amplitudes
before and after perfusion of 1 mM TEA. The
KCa component was determined as the TEA-sensitive
current recorded in Na+ bath saline containing
external Ca2+. The KD component was
determined as the TEA-insensitive component in Na+
bath saline with CaCl2 omitted. Capacitance and leak were
subtracted on-line using five subthreshold pulses at one-fifth the
amplitude of the test voltage (P/5 technique). Data were collected by
GeneClamp (Axon Instruments), filtered at 2 kHz, digitized at 200 µsec, and stored on a Dell 486 computer for subsequent analyses with pClamp software. The expression of cerebellar poly(A) RNA in
Xenopus oocytes (Dascal, 1987) provided a functional assay
for the presence of K+ channel transcripts at
different developmental stages.
Northern blot analysis. Rat cerebellar poly(A) RNA from
postnatal days 2, 4, 6, 7, 8, 10, 14, and 21 was prepared from 15 to 50 rat pups per time point (more were used at the younger ages). Totals of
10 µg of each preparation of rat cerebellar mRNA were loaded on a
denaturing formaldehyde gel, separated by size via electrophoresis, and
then transferred to a nylon filter (GeneScreen) by standard techniques
(Sambrook et al., 1989). The filter was screened by hybridization with
32P-labeled antisense RNA probes prepared from the
linearized plasmids for two types of cloned cDNAs, (1) the
KCa channel mslo (Butler et al., 1993)
provided by Drs. Ganetzky and Robertson, University of Wisconsin, and
linearized with HindIII to include bases 2208-4610 (putative membrane spanning regions S9 and S10 and the C terminal) and
(2) the delayed rectifier Kv3.1 (Shaw subfamily; Swanson et al., 1990) provided by Dr. Swanson, Merck Research Laboratories, and
linearized with EcoRI to include the full-length sequence (regions S1-S6 and termini). The Kv3.1 and mslo probes were
hybridized overnight at 50°C with 50% formamide and were washed 15 min at room temperature in 1× SSC and 45 min at 62°C in 0.5× SSC,
using solutions and methods described by Sambrook et al. (1989). The constitutively expressed gene for cyclophilin (Danielson et al., 1988)
served as a control for the amount of RNA loaded on each lane. The
randomly primed cDNA probe for cyclophilin was hybridized overnight at
37°C. Semiquantitative analysis of the Northern blot was performed by
measuring the signal intensity of the bands on the scanned images of
the exposed x-ray films (Ambis 4000 laser densitometer). Samples of
equal areas were scanned with a rectangular sampling tool, set to
enclose the largest band. Net signals were obtained by subtracting an
equal area of background adjacent to the band in the same lane and were
standardized by comparison with the intensity of the net signal for
cyclophilin in the same lane.
Primary cerebellar cell culture methods. The primary
cerebellar cell cultures were established using techniques described previously (Gruol and Franklin, 1987). Purkinje cells undergo final
mitosis at approximately embryonic day 16 (Altman, 1972). The
cerebellar cortices were dissected from rat embryos at day 20 of
gestation (1 d before birth), gently minced and triturated in
Ca2+-free saline without enzymes, and then placed on
poly-D-lysine-coated dishes containing minimum essential
medium (MEM; GIBCO BRL) with 10% each heat-inactivated horse and fetal
bovine sera. The cultures were incubated at 37°C in a humidified 5%
CO2 incubator. After 3 d, the cultures were treated
with 0.02 mg/ml 5-fluorodeoxyuridine (FUDR) and then maintained in MEM
with 10% horse serum. The medium supplied from GIBCO BRL contained 5.3 mM K+ and was used as the "low K"
condition; the medium was supplemented with sterile KCl to a final
concentration of 10 mM for the "high K" condition.
Culture medium containing 1.8 mM Ca2+
provided the control level; in "low calcium" experiments, the external Ca2+ was reduced to a calculated free
concentration of 0.1 mM by 1.7 mM EGTA or to
0.1 µM by 3.5 mM EGTA (Fabiato, 1988).
Semiquantitative PCR. To assess relative levels of
KCa channel transcripts during development in
culture, we used a modified technique for PCR (the semiquantitative,
noncompetitive method) (Madsen et al., 1995; Zhao et al., 1995). This
method allows comparison of the amount of a target sequence with
respect to the amount of a constitutively expressed reference sequence
such as cyclophilin. The subsaturating level of cDNA template that is
needed to produce a dose-dependent amount of reaction product is
defined empirically in initial experiments by testing a range of
template concentrations. The relative intensity of the target sequence
product visualized with ethidium bromide can then be interpreted as
reflecting the relative abundance of the target mRNA in the original
total RNA pool (Zhao et al., 1995).
From a batch of sibling cultures, three to four culture plates were
combined to generate each RNA sample. Three sets of RNA samples were
prepared from different batches of sibling cultures. Cultures were
washed several times with PBS, and cells were scraped from the plates
and lysed in Nonidet P-40 (Sigma). Proteins were digested with
Proteinase K, and cytoplasmic RNA was extracted by phenol and
chloroform and precipitated by isopropanol. Possibly contaminating DNA
was removed by incubating with RNase-free DNase at a final
concentration of 2 µg/ml at 37°C for 60 min. The concentration of
the total RNA for each sample was determined by UV spectrophotometry, and 2 µg was used to synthesize single-stranded cDNA by reverse transcription with 1 µg of random primer, 0.2 µg of
oligodeoxythymidine (dT) primer, and 50 U of reverse transcriptase
M-MuLV (Moloney Murine Leukemia Virus; Boehringer Mannheim,
Indianapolis, IN) in 25 µl of total volume at 37°C for 1 hr by
standard techniques (Sambrook et al., 1989).
Aliquots of the reverse-transcribed cDNA preparations were used for PCR
amplification with sequence-specific synthetic oligodeoxyribonucleotide primers. Primers for the KCa-related sequences were
sense 5 -GGCTGGAAGTGAATTCTGTAG-3 and antisense
5-TGAGTAAGTAGACACATTCCC-3. These primer
sequences are based on the rat brain KCa sequence
rslo in regions that correspond in mslo to
1063-1083 bases (between S3 and S4) and to 1354-1374 bases (near H5),
respectively, and produced a product of the expected size at 312 base
pairs (bp). The PCR-amplified product for KCa was
98% identical in nucleotide sequence between mouse and rat, reflecting
the high overall homology (96% at the amino acid level) that exists
between the rslo and mslo coding regions (based
on a 2.9 kb partial sequence for rslo; H. Y. Mi and T. Schwarz, personal communication). Cyclophilin served as an internal
standard for the relative quantitative comparisons; primers for the
cyclophilin sequence (Danielson et al., 1988) were sense
5-GGGGAGAAAGGATTTGGCTA-3 (from 166 to 185 bases) and antisense
5-ACATGCTTGCCATCCAGCC-3 (from 404 to 422 bp) and produced a product
at the expected size of 259 bp. Calbindin is a specific marker for
Purkinje neurons in the cerebellum; primers for the rat calbindin
sequence (Hunziker and Schrickel, 1988) were sense
5-CTGCACCATGGCAGAATCCC-3 and antisense
5-GCCACTGTGGTCAGTGTCATAC-3. These primer sequences were
located spanning the initiation methionine at 279-298 and midway
through the coding region at 611-633, respectively, and generated a
product of 355 bp. PCR reactions for KCa,
cyclophilin, and calbindin were performed in parallel in separate
reaction tubes. Each PCR amplification (50 µl of final volume)
contained 25 pmole of each primer, 0.2 mM each of
deoxynucleotides (dNTP), 10 mM Tris-HCl, 1.5 mM
MgCl2, 50 mM KCl, and 2.5 U Taq DNA
polymerase (Boehringer Mannheim) aliquoted from maximally diluted
stocks to enhance reproducibility of the pipetted volumes. The
reactions (Perkin-Elmer 9600 thermocycler) used the following steps:
denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, and
polymerization at 72°C for 1 min for 25 cycles, ending with 5 min at
72°C and storage at 4°C. Initial PCR reactions first surveyed the
different preparations to determine which seemed to have high intensity products and then tested a dilution series of template cDNA from that
same preparation to determine the concentration of template that
yielded an amount of product near the midpoint of the dose-dependent range. The empirically determined subsaturation levels of template were
50 ng of cDNA for the rslo and calbindin reactions and 5 ng
of cDNA for cyclophilin reactions; these values were held constant for
the subsequent comparisons of relative abundance across cDNA preparations from all stages. For gel electrophoresis, one-fifth of the
PCR product volumes were combined for sample-matched rslo and cyclophilin reactions into the same lanes of a 3% MetaPhor high-resolution agarose (FMC Bioproducts, Rockland, ME) gel and run at
60 V for 4 hr. Gels were stained with freshly prepared ethidium bromide
and photographed. Regions sampled by National Institutes of Health
software (NIH Image) from an imaged gel were determined from total
intensities of equal areas defined with a rectangular sampling tool
that was set to enclose the largest band; this tool was held constant
for all measurements (both signal and background) made from the same
image. The background area subtracted for each band was determined from
the adjacent region immediately above the band within the same lane.
Signal intensity data for the rslo KCa or
the calbindin bands (multiplied by 0.1 to account for the 10-fold
higher proportion of cDNA template used in the reactions) were
standardized to the intensity of the signal for cyclophilin in the same
lane. Results were obtained from three sets of total RNA preparations
from three different batches of cultures, based on two to three
different PCR reactions at each time point, with data analyzed from two
gels per PCR reaction. These multiple comparisons demonstrated that the
PCR results were comparable within a given preparation as well as
across preparations from different batches of cultures.
To ensure that the amplification of contaminating genomic DNA did not
contribute to the signals of interest, we performed a negative control
reaction in which RNA samples without reverse transcription were used
as the templates; no PCR products were found. To verify that the PCR
product was indeed the rslo sequence and not a nonspecific
product, we subjected the PCR product for rslo to
restriction enzyme digestions with NcoI (10 U) and
SacI (10 U) in final volumes of 20 µl at 37°C for 2-3
hr. Restriction digests were analyzed by gel electrophoresis (3%
MetaPhor high-resolution agarose); fragments were observed at the
predicted sizes of 99 and 213 bp for SacI and 61 and 251 bp
for NcoI, in addition to a small amount of uncut DNA at the
predicted size of 312 bp. In addition, the PCR products for
cyclophilin, calbindin, and rslo were subcloned into pGEM-T
vector (Promega) and cycle-sequenced with Taq FS DNA
polymerase and fluorescent dideoxy chain termination (Arizona Research
Laboratories, Division of Biotechnology). The DNA sequencing results
showed that the amplified products had the exact sequences that were
predicted from the published full-length sequences (100% identity;
data not shown) and confirmed the identities of the products analyzed
by semiquantitative PCR.
Immunocytochemistry and morphometric analysis. Cell cultures
were rinsed three times in MEM with 10 mM HEPES and 4%
sucrose, pH 7.3, at 37°C and were fixed in 4% paraformaldehyde (in
MEM with 10 mM HEPES and 4% sucrose) for 30 min at room
temperature. Cells were permeabilized with stop/permeabilizing buffer
(0.05% Triton X-100 in PBS, containing 0.1% bovine serum albumin, pH 7.3; repeated twice for 5 min each). Permeabilized cultures were incubated with 1 ml of primary antibody (2 hr at room temperature), rinsed with stop/permeabilizing buffer (three times for 5 min each),
and then incubated with 1 ml of secondary antibody solution (1 hr at
room temperature). The primary antibody used in this study was
anti-calbindin 28k (monoclonal raised in mouse from Sigma and used at
1:400 dilution or polyclonal raised in rabbit from Swant, Bellinzona,
Switzerland, and used at 1:500 dilution). Antibodies were diluted to
the indicated concentrations in stop/permeabilizing buffer. Secondary
antibodies were fluorescein-labeled anti-mouse IgG (made in horse, 4 ml/ml; Vector Laboratories, Burlingame, CA) and Texas Red-labeled
anti-rabbit IgG (made in goat, 6 ml/ml; Vector Laboratories). After
incubation with secondary antibody, cultures were rinsed three times
with PBS, pH 8.0, for 5 min each and visualized with fluorescein (B-2A
filter set) or Texas Red (G-2B filter set) using a Nikon Diaphot 200 inverted microscope with 40× objective (plan 40; NA 0.55; equipped
with a correction ring for culture dish thickness compensation).
Photographs for documentation were taken using Kodak Tri-X or T-Max 35 mm black and white film, 400 ASA; 35 mm negatives were digitized using a Nikon Coolscan slide scanner and analyzed with NIH Image software using the macro Neurite Labeling (C. Thomas, Integrated Microscopy, University of Wisconsin, 1994). Neurite lengths, numbers, and branch
orders were measured by computer-assisted linking of individual neurite
segments with a polygon tool; bifurcations defined the branchpoints and
the positions for +1 increments in branch order; and primary (1°)
branches were defined as those arising from the soma. Statistically
significant differences were determined by the Mann-Whitney rank test
with p < 0.05.
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RESULTS |
Development of K+ channels in vivo
assessed by RNA expression in oocytes
Figure 1 illustrates the current
responses evoked by voltage clamp in oocytes injected with poly(A) RNA
isolated from postnatal rat cerebella. Outward currents generated in
the presence of external Ca2+ consisted of
two components, TEA-sensitive and TEA-insensitive. In RNA from older
pups, a TEA-sensitive current was observed that was not present in the
RNA from younger pups, suggesting a developmental difference in the
level of expression of the KCa channel. At 1 mM, external TEA serves as a useful tool for selectively
identifying the KCa channels in Purkinje neurons
(Yool et al., 1988). The TEA-sensitive current was present in oocytes
injected with RNA from postnatal cerebella but not in oocytes with RNA
from young postnatal cerebella or in uninjected controls (Fig.
1A). The TEA-sensitive component showed properties of
the KCa conductance; activation of the current
required external Ca2+ and a prepulse step to 0 mV
that allowed Ca2+ entry through endogenous
voltage-gated channels, and the outward current persisted at negative
potentials (50 to 30 mV) indicating K+
selectivity. Uninjected oocytes recorded with external
Ca2+ showed currents averaging 150 nA (146 ± 48, mean ± SE; n = 9); a reversal potential of
approximately 20 mV (data not shown) suggested that this
TEA-insensitive current expressed in both injected and uninjected
oocytes was the Ca2+-dependent
Cl current native to Xenopus
oocytes.
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Figure 1.
Expression of KCa and
KD currents in Xenopus oocytes injected with
cerebellar mRNA isolated from postnatal rats. Poly(A)-enriched RNA
isolated from rat pups at postnatal day 2 (P2), P4, P6, P8, P10, and
P14 was expressed in Xenopus oocytes to assess the
presence of transcripts encoding KCa and
KD currents in vivo. A,
B, Currents were recorded by two-electrode voltage clamp
with external Ca2+ at 1.8 mM
(A) and in medium with external
Ca2+ omitted (B). A prepulse
step to 0 mV was followed by test steps to various potentials. For
clarity, only the currents evoked by test steps to +60 mV are shown for
examples of an uninjected oocyte (Uninj.) and oocytes
injected with RNA from postnatal days 4 and 10 (P4,
P10). External TEA (1 mM) was applied by
perfusion. Capacitance and leak were subtracted on-line with
subthreshold steps. C, Peak current amplitudes
(mean ± SE) of the expressed current are plotted as a function of
the developmental age (days postnatal) of the rat cerebellar source of
RNA. The net TEA-sensitive Ca2+-dependent component
identified as the KCa current (open
triangles) was significantly different at P8, P10, and P14
(*p < 0.01, unpaired t test) with
respect to P2, whereas the TEA-insensitive
Ca2+-independent current attributed to
KD channels (open circles) showed no
significant difference at any age with respect to P2. D,
The current-voltage plot shows the relationship between steady-state current amplitudes (mean ± SE) and the test voltage steps (from 80 to +60 mV; in 20 mV increments) recorded from oocytes injected with cerebellar mRNA isolated at postnatal day 10 (n = 4 for KCa; n = 6 for KD). Both
KCa and KD currents were activated at
test steps of 40 mV and above and exhibited voltage-dependent outward rectification. The outward currents seen at negative potentials (50
to 30 mV) are consistent with the identification of the currents as
K+-selective.
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Uninjected oocytes showed little current (49 ± 16 nA,
mean ± SE; n = 5) in the absence of external
Ca2+ (Fig. 1B), indicating that
the KD current endogenous to oocytes was small in
comparison with exogenously expressed currents. Oocytes injected with
cerebellar RNA recorded in nominally Ca2+-free bath
saline showed outward currents (230-320 nA) that were insensitive to 1 mM TEA and were expressed from RNA isolated at all
postnatal stages. The endogenous KD current was not
subtracted, because it could not be independently determined for the
same oocytes, but should account only for 15-20% of the total
Ca2+-independent current measured for cerebellar
RNA, assuming that the amplitude of the endogenous current remains
constant.
Figure 1C shows the net TEA-sensitive component
(KCa) calculated from pairwise comparisons in
each oocyte; data are presented as the mean difference in current
amplitude before and after application of 1 mM TEA. In this
analysis, the endogenous oocyte K+ and
Cl currents and the cerebellar KD
currents were effectively subtracted because of their insensitivity to
TEA. The mean amplitude of the KCa component
expressed in oocytes was significantly increased at postnatal days 8, 10, and 14 when compared with day 2 (p < 0.01, unpaired Student's t test), whereas KD currents
showed no statistically significant difference at any age with respect
to day 2 (p > 0.05, unpaired Student's
t test). Although oocyte expression would not necessarily be
expected to reflect the presence of mRNA in a quantitative manner, the
mean current amplitude did show a rising trend with age that matched
results of Northern analysis (see below) and suggested that the average
current amplitude could reflect relative RNA abundance in the
cerebellar preparation. The observation of a trend may have been
facilitated by standardizing the concentrations of poly(A) RNA
(determined by UV spectrophotometry) in the injection solutions.
Current-voltage plots (Fig. 1D) show the
relationship between steady state current amplitude (mean ± SE)
and voltage in oocytes injected with mRNA from postnatal day 10 (n = 4 for KCa;
n = 6 for KD). Both
KCa and KD currents exhibited outward
rectification and outward currents at negative potentials (50 to 30
mV), consistent with their identification as K+
selective conductances.
K+ channel expression in vivo
assessed by Northern blot analysis
Figure 2A shows a
scanned radiographic image of a Northern blot used to assess the levels
of transcripts homologous to cloned KCa and
KD channels in RNA isolated from postnatal rat cerebella. The delayed rectifier channels are characterized by a core region with
six putative transmembrane domains (S1-S6); the fslo,
mslo, and rslo channels cloned from
Drosophila, mouse, and rat have four additional hydrophobic
segments (S7-S10) that are unique to the KCa
channels (Atkinson et al., 1991; Adelman et al., 1992; Butler et al.,
1993) (Mi and Schwarz, personal communication on rslo). The
probe used for homology screening of the rslo-related sequence was derived from the S9 to S10 region of mslo. The
major band detected at 4.4 kb increased in intensity with age after postnatal day 7, yielding a pattern generally consistent with the
expression of KCa current in injected oocytes (Fig.
1). A probe for another type of K+ channel that is
expressed at high levels in the cerebellum (Perney et al., 1992) was
generated from the full-length sequence of the delayed rectifier Kv3.1
(Shaw family). The Kv3.1 probe showed continuous expression
of a major band at 5.5 kb, also in a pattern generally consistent with
the expression of KD current in injected oocytes. Faint
secondary bands were seen on the Northern blots; a band at 1.8 kb
detected by the mslo probe and a band at 2.4 kb detected by
the Kv3.1 probe were present in RNA at postnatal day 8 and older (data
not shown). KCa and Kv3.1 genes are known to
generate alternatively spliced transcripts (Luneau et al., 1991;
Adelman et al., 1992), but an extensive diversity was not evident in
the relatively simple pattern observed in the Northern analysis of
cerebellar RNA. Cyclophilin (0.6 kb) served as a control for the
amounts of mRNA loaded.
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Figure 2.
Northern blot analysis of K+
channel transcripts in postnatal rat cerebellum in vivo.
A, Poly(A)-enriched RNA was isolated from rat pup
cerebella at P2, P4, P6, P7, P8, P10, P14, and P21 and screened by
Northern blot analysis with 32P-labeled antisense probes
for rslo (mslo probe, region S9-S10) and
Kv3.1 (full-length probe) sequences. A 4.4 kb band detected by the
mslo probe was increased in signal intensity at day 7 and thereafter. A 5.5 kb band detected by the Kv3.1 probe was present at all stages. Constitutively expressed cyclophilin
(cyclo; 0.6 kb) served as a standard for the amount of
RNA loaded in each lane. All bands in
lanes P2 and P4 appeared to run slightly faster than
those in other lanes; ladders run on both sides of the
sample lanes showed a comparable difference. The shifted
positions were taken into account when analyzing signals at P2 and P4.
B, Densitometric analysis of the Northern blot provided
an estimate of relative intensities of the rslo and
Kv3.1 signals after subtraction of adjacent background and
standardization to the signal for cyclophilin. Signal intensities are
calculated as a percentage of the signal measured at postnatal day 2 (the first time point assayed) and plotted as a function of
developmental age of the rat cerebellum.
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The intensities of the bands detected by Northern analysis were
measured by densitometer to evaluate relative levels of expression in
postnatal cerebellum (Fig. 2B). Band intensities were
adjusted by subtraction of an adjacent background region within the
same lane to determine the signal values. Signals for
K+ channel sequences were standardized to the signal
for cyclophilin in the same lanes and are plotted as the percentage of
the signal detected on postnatal day 2 (Fig. 2B). The
results suggested an upregulation of the levels of
rslo-related transcripts at postnatal day 7 and older and a
fairly constant level of expression of Kv3.1 during postnatal
development.
Semiquantitative analysis of rslo transcription
in vitro assessed by PCR
Semiquantitative PCR (Zhao et al., 1995) allowed the evaluation of
relative amounts of rslo transcripts at different stages in
culture. Figure 3A shows the
products resulting from PCR reactions done with a dilution series of
reverse-transcribed cDNA templates. Intensities of ethidium
bromide-stained bands showed a dose-dependent relationship with
template concentration (Fig. 3B). Products were obtained
from separate amplifications with primers for rslo,
calbindin, or cyclophilin and were combined on gels for the same
cerebellar template. The products ran at the expected sizes of 312 bp
for rslo, 259 bp for cyclophilin, and 355 bp for calbindin.
Saturation of the amount of reaction product was evident at cDNA
template levels of >10 ng for cyclophilin and >200 ng for
rslo and calbindin (Fig. 3B). Subsaturating
amounts of cDNA template at 50 ng for rslo and calbindin and
at 5 ng for cyclophilin were used for developmental analyses.
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Figure 3.
PCR analysis of the dose-dependent amplification
of rslo, calbindin, and cyclophilin sequences from
reverse-transcribed RNA isolated from cerebellar cultures.
A, Example of a gel showing PCR products generated from
a dilution series of template, using sequence-specific primers for
rslo and cyclophilin (cyc), is shown. Products stained with ethidium bromide were seen at the expected sizes
(rslo at 312 bp; cyclophilin at 259 bp) and showed a
dose-dependent relationship to the amount of template. Template for the
analysis was prepared from cytoplasmic RNA of rat cerebellar cell
cultures at 4 d in vitro for cyclophilin and 7 d in vitro for rslo. Comparable results
were obtained from replicate analyses of rslo and
calbindin, amplified in parallel with cyclophilin. B,
Relative signal intensities of the PCR products were summed for uniform
areas by NIH Image software, adjusted by subtraction of adjacent
background values, and plotted as a proportion of the maximum signal as
a function of template concentration. The maximum signal
(Imax) was set as the intensity of
the product at saturating amounts of template. Lines were fit using:
I = Imax *
[C]/(K0.5 + [C]), where I is the measured intensity
and C is the concentration of template (nanograms per 50 µl). Data are mean ± SE from triplicate experiments.
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Figure 4 shows that the levels of
rslo transcript (assessed at 14 d in vitro)
were smallest in the low K+ culture condition (5.3 mM K+) and were enhanced by treatment
either with high K+ (10 mM
K+) or with 1 mM TEA. All cultures were
initially established in low K+ medium and then were
transferred to experimental media at either day 3 or day 13 in
vitro. Some cultures were maintained continuously in low
K+ medium for comparison. All cultures were
harvested in these experiments on day 14 in vitro.
Significant increases in rslo transcript levels were
observed after the long-term (11 d) treatment with the high K+ or TEA media. Although not significantly
different in the short-term (1 d) treatment, similar trends indicated
that the effects of the stimulatory agents could be observed after only
24 hr of treatment preceding the cell harvest and suggested that the
cells remained capable of responding to depolarizing cues with an
increase in rslo transcription despite their long incubation
in low K+ medium. The stimulation of rslo
expression by depolarizing stimuli depended on the presence of external
Ca2+. When external free Ca2+ was
buffered with EGTA from 1.8 to 0.1 mM, the levels of
rslo transcripts remained low (at a level comparable with
that seen in the low K+ treatment) regardless of the
treatment with 10 mM K+ or 1 mM TEA. Low Ca2+ medium did not seem to
affect adversely the overall development of cerebellar neurons or the
survival of Purkinje neurons (see Figs. 6, 7). In essence, the lack of
available external Ca2+ seemed to prevent the
expected change in levels of rslo transcripts and suggested
that the developmental change in rslo expression may depend
on environmental cues that promote voltage-gated
Ca2+ signaling.
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Figure 4.
Quantitative comparison of the relative abundance
of rslo transcripts in cerebellar cultures after
short-term (1 d) and long-term (11 d) treatments with different
K+, TEA, and Ca2+ conditions.
A, PCR products were generated with template synthesized from cytoplasmic RNA that was isolated from cerebellar cultures at
14 d in vitro. Products stained with ethidium
bromide were seen at the expected sizes (rslo at 312 bp;
cyclophilin at 259 bp). Treatments are summarized at the
bottom of B. B, The
box plot summarizes the intensities of
rslo signals resulting from different treatments
in vitro, standardized to the corresponding intensity of
the signal for cyclophilin. Box boundaries indicate the
full range of data values; internal horizontal bars
represent median values. Long-term treatments with TEA and with 10 mM extracellular K+ significantly
increased the abundance of rslo transcripts in comparison with the low K+ treatment
(*p < 0.05, nonparametric Mann-Whitney test). In
low calcium medium (0.1 mM), the effects of TEA and high
extracellular K+ were negated. In short-term
treatments, similar trends were evident but not statistically
significant.
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Intensities of signals, standardized as the ratio of rslo to
cyclophilin signals, are summarized from duplicate analyses of gels
from two complete sets of PCR reactions per RNA preparation for two
different RNA preparations, producing eight measurements per sample
point (Fig. 4B). In long-term treatments (from day 3 to day 14 in culture) with high K+ or TEA, the
intensity of signal for rslo was increased significantly when compared with the level observed in cultures maintained
continuously in low K+ (p < 0.05, nonparametric Mann-Whitney test). Short-term treatments (24 hr)
with high K+ or TEA showed similar trends in the
intensities of signals for rslo. In low calcium (0.1 mM) culture medium, the levels of rslo transcripts were not significantly different from those in the low
K+ condition alone, indicating that the
expression-promoting effects of chronic TEA or 10 mM
external K+ were inhibited by the reduction of
external Ca2+. Restriction digests and DNA
sequencing confirmed that the PCR product analyzed in our studies
accurately represented the rslo sequence encoding the rat
KCa channel.
Figure 5 shows the developmental profile
of rslo expression. The pattern of expression observed for
RNA in vivo (above, as assessed by oocyte expression and
Northern analysis) also was observed in vitro when the
cerebellar cultures were grown in the presence of a chronic
depolarizing stimulus but not when grown in its absence. Cytoplasmic
RNA was isolated from cerebellar cultures at 4, 8, 10, 14, and 21 d in vitro and analyzed by semiquantitative PCR (Fig.
5A). Relative intensities of the PCR product bands were calculated from the ratio of rslo to cyclophilin after
background subtraction, in replicate experiments from four PCR analyses
of two separate batches of culture preparations (Fig. 5B).
For cultures grown in the low K+ culture medium, the
intensity of signals for rslo remained low and exhibited no
significant difference at any age (p > 0.05, nonparametric Mann-Whitney test) when compared with day 4. However, in
cultures maintained in low external K+ in
combination with 1 mM TEA, the rslo signal
intensity was increased significantly with development at 10, 14, and
21 d when compared with the age-matched low K+
cultures (p < 0.05, nonparametric Mann-Whitney
test). A comparison of the patterns of rslo expression
suggests that the cerebellar culture system is more representative of
the developing cerebellum in vivo, at least with respect to
KCa channel expression, in a background of increased
neuronal excitability.
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Figure 5.
Quantitative comparison of the relative abundance
of rslo transcripts during development of cerebellar
cultures in low K+ medium with and without 1 mM TEA. A, PCR products were generated from
reverse-transcribed cytoplasmic RNA that was harvested from cerebellar
cultures at different ages (days in vitro). All cultures were maintained in low K+ medium, either without TEA
(left) or with 1 mM TEA treatment initiated
at 3 d in vitro (right).
B, The box plot summarizes the
rslo signals standardized to the corresponding signals
for cyclophilin as a function of developmental age, for quadruplicate experiments. Box boundaries indicate the full range of
data values; internal horizontal bars represent median
values. Significant differences between age-matched cultures with and
without TEA are indicated by asterisks
(*p < 0.05, unpaired Mann-Whitney test).
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Morphological development in culture
A quantitative morphological analysis of Purkinje neurons at
different ages in culture allowed a comparison of the effects of low
K+ (5.3 mM), high K+
(10 mM), and reduced external Ca2+ (0.1 µM) treatments on the overall development of the neurons. Purkinje neurons were identified by fluorescence immunohistochemistry with antibody to calbindin, a specific marker within the cerebellum for
Purkinje neurons (Fig. 6). Greater
neurite outgrowth was seen for Purkinje neurons maintained in the high
K+ culture medium (Fig. 6B) when
compared with those in low K+ medium (Fig.
6A). The stimulatory effect of high
K+ was not blocked by low Ca2+
(Fig. 6C); these neurons showed an enhanced neurite
outgrowth when compared with neurons grown in the low
K+ condition.
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Figure 6.
Morphological development of identified Purkinje
neurons in cerebellar cultures after long-term (12 d) treatments with
different K+ and Ca2+ conditions.
Rat cerebellar Purkinje neurons in cultures at 15 d in
vitro were identified by anti-calbindin fluorescence imaging. Digital images show (A) Purkinje neurons grown in
low K+ (5.3 mM) medium,
(B) a Purkinje neuron grown in high
K+ (10 mM) medium, and
(C) a Purkinje neuron grown in high
K+ medium in which the external free
Ca2+ was buffered to 100 nM with EGTA.
High K+ medium stimulates neurite development; low
external Ca2+ does not seem to prevent this
development (see Table 1).
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Table 1 summarizes the average neurite
lengths and numbers of branchpoints for cerebellar Purkinje neurons
grown in the various treatment conditions. Neurons grown in high
K+ had significantly longer neurites and a tendency
toward more neurite branching than did those grown in the low
K+ condition. Reduced external
Ca2+ was not overtly detrimental to overall
development; growth in high K+ was not significantly
different when compared between the normal Ca2+ (1.8 mM) and reduced Ca2+ (0.1 µM) conditions. In low K+ medium, the
presence of 1 mM external TEA increased neurite outgrowth in a manner similar to that seen in the high K+
treatment (data not shown).
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Table 1.
Maximal neurite lengths and numbers of branchpoints per
cell at 15 d in vitro for Purkinje neurons grown in
altered K+ and Ca2+ conditions
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The ability of Purkinje neurons to respond to high
K+ with enhanced outgrowth, despite a reduction in
external Ca2+, indicated that the blocking effect of
reduced Ca2+ on rslo gene expression did
not result indirectly from stunting development. This supported our
interpretation that intracellular Ca2+ signaling may
be involved in the regulation of rslo expression. However, a
possibility remained that the observed decrease in the rslo
signal after low Ca2+ treatment was caused by the
selective death of a hypothetical subpopulation of Purkinje neurons
that expressed the rslo gene. To rule out this alternative
hypothesis, we assessed the effects of altered K+
and Ca2+ on the levels of expression of a Purkinje
specific marker, calbindin, referenced to a marker for all cells in the
heterogeneous cultures, cyclophilin. If the selective deaths of
Purkinje neurons were occurring, it would be apparent as a decrease in
the ratio of calbindin to cyclophilin signals.
Figure 7 shows that low external
Ca2+ treatment (which antagonized the stimulation of
rslo transcript levels in 10 mM
K+) did not decrease the relative calbindin signal,
and thus the effect on rslo level cannot be explained simply
as a loss of Purkinje neurons. Cultures were treated beginning on day 3 in low K+ (5.3 mM) or high
K+ (10 mM) conditions without or with
EGTA to buffer free Ca2+ to 0.1 mM and
were harvested on day 14 in vitro. PCR was used to confirm
the subsaturation range of the dose-response relationship for the
reverse-transcribed templates, and the semiquantitative method was used
to assess the relative levels of transcripts (Fig. 7A). In
the high K+ condition with normal
Ca2+, the level of rslo transcript,
standardized to cyclophilin, was significantly increased with respect
to the other three treatment conditions, and this stimulatory effect
was blocked in the low Ca2+ condition (Fig.
7B). In contrast, the calbindin signal did not decrease in
the low Ca2+ conditions (Fig. 7C), as
would be expected if Purkinje neurons were dying during the treatment.
Calbindin levels were not stimulated by the high K+
treatment nor reduced by the imposition of low Ca2+
in addition to the low K+ or high
K+ treatments, indicating that the survival of
Purkinje neurons was not appreciably compromised. This interpretation
presumes that the levels of calbindin transcripts per Purkinje neuron
are similar, an idea that is supported qualitatively from our
observations that (1) all neurons with distinctive Purkinje
morphologies were positive for calbindin (there were no calbindin-free
subpopulations), (2) the numbers of Purkinje neurons per culture plate
did not appear to be different (comparable numbers of neurons were
imaged from all culture plates), and (3) the calbindin
immunofluorescent signals used to image the Purkinje cells did not show
any obvious differences in intensities. Direct counting of the numbers
of Purkinje neurons per plate was impractical because the cultures were
established as explants of small chunks of cerebellar tissue (from
which cells migrated out to cover the plate), and thus the initial
numbers of neurons per plate were variable. Assessing the ratio of
signals for a Purkinje-specific molecular marker with reference to a
general marker for all cells is a useful method for determining the
relative survival of Purkinje neurons within the mixed population of
cells that are present in primary cerebellar cultures.
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Figure 7.
Comparison of the effects of altered
K+ and Ca2+ concentrations on the
expression of rslo and calbindin in cerebellar cultures. A, PCR products were generated with template synthesized
from cytoplasmic RNA that was isolated from cerebellar cultures at 14 d in vitro. Products stained with ethidium
bromide were seen at the expected sizes (rslo at 312 bp;
calbindin at 355 bp; and cyclophilin at 259 bp). Treatments, initiated
on day 3 in culture, were low K+ (5.3 mM) and high K+ (10 mM)
conditions, without or with EGTA (low Ca2+) to
buffer the free Ca2+ concentration to 0.1 mM. RNA template prepared without reverse transcriptase
(no RT) yielded no detectable PCR product
(top) and was comparable with a blank
lane (bottom), indicating that genomic
DNA did not contribute to the amplified signals. The molecular weight
standard ladder (Lad.) shows bands at 200 and 400 bp for reference. B, C, The box
plots summarize the rslo
(B) and the calbindin (C)
signals standardized to the corresponding signals for cyclophilin, for
triplicate experiments. Box boundaries indicate the full
range of data values; internal horizontal bars represent median values. Significant differences between treatments are indicated
by asterisks (*p < 0.05, unpaired
Mann-Whitney test).
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DISCUSSION |
The pattern of rslo transcription in the rat cerebellum
showed an upregulation during the first 2 weeks of postnatal
development in vivo. A comparable pattern was seen in
cultures that were chronically exposed to mildly depolarizing stimuli.
In contrast, the levels of rslo transcript showed no change
during development in low K+ culture medium, which
may represent a chronically understimulated environment. The resting
membrane potential in the Purkinje neuron is set primarily by the
K+ equilibrium potential; a brief application of
bath saline in which the NaCl is substituted with KCl (a "symmetrical
K+" condition) results in a rapid transient
depolarization by ~60 mV (Yool et al., 1992), in agreement with the
prediction of the Nernst equation. Thus, the effects of altered
external K+ described here for the cerebellar
cultures are likely to be a direct consequence of depolarization of the
resting membrane potential. The difference in external
K+ concentration between 5.3 and 10 mM
is predicted from the Nernst equation to cause a difference in the
K+ equilibrium potential of approximately +16 mV.
Based on published studies of current-clamp recordings in cultured
Purkinje neurons, this difference in membrane potential could shift the
cell from slow firing or virtual quiescence to a steady fast
pacemaker-like firing activity (Yool et al., 1988).
The effects of TEA on the development of the cerebellar cultures may
result not only from depolarization of the resting membrane potential
but also from an increase in the voltage-dependent
Ca2+ component of the action potentials that results
from a slowing of the late repolarization phase (Yool et al., 1992).
Because the effects of 10 mM K+ and TEA
treatments on rslo transcript levels are comparable in magnitude and Ca2+ dependence and because the
effects of these treatments complement the developmental pattern of
rslo expression observed in vivo, it seems
unlikely that the upregulation of rslo in the TEA treatments here is caused simply by the block of the channels per se. Instead, the
effect of TEA on channel transcription is more easily ascribed to an
alteration in transmembrane signaling that is comparable with the
effect of 10 mM K+.
The Northern analysis complemented the studies using oocyte expression
of RNA, matching the molecular detection of specific signals with a
broader analysis of functional expression. Although the sequences used
as probes for the Northern analysis (rslo and Kv3.1) proved
to be representative of a general pattern of KCa and
KD channel expression in developing cerebellum, it is
important to note that the results from oocyte recordings must in fact
be considered as the summed effect of a more complex state, involving the differential expression of multiple K+ channel
subtypes (Drewe et al., 1992; Perney et al., 1992; Maletic-Savatic et
al., 1995; Gurantz et al., 1996). Kv3.1 is expressed at highest levels
in cerebellum (Drewe et al., 1992; Perney et al., 1992). Other types of
delayed rectifiers, including Kv1.1, Kv1.2, Kv1.3, and Kv3.4, also are
present in cerebellum (Wang et al., 1993; Goldman-Wohl et al., 1994;
Sheng et al., 1994; Veh et al., 1995; McNamara et al., 1996). The
mslo region S9-S10 used as the probe for Northern analysis
is in a region that is alternatively spliced (Adelman et al., 1992); it
is possible other KCa gene products are expressed in
cerebellum but were not detected in our analysis of in vivo
expression.
The RNA detected by Northern analysis and the currents expressed in
oocytes represented the transcripts present at the age of cerebellar
harvest and showed that rslo and Kv3.1 channels continued to
be actively expressed during development of the cerebellum. The
correlation between the oocyte expression and Northern analysis indicated that full-length RNA sequences for K+
channels were successfully obtained in the poly(A) RNA isolation procedure. We would not expect to distinguish between different types
of delayed rectifiers when analyzing oocytes by two-electrode voltage
clamp. Thus, the overall constancy of KD current expression may reflect overlapping patterns of expression of various types of
delayed rectifiers. However, the conclusion relevant to this study is
that the combined delayed rectifier component seems to remain fairly
constant during development and supports the results of previous single
channel studies that showed an increase in KCa but
not in KD channel abundance during the development of Purkinje neurons in culture (Yool et al., 1988).
There is an interesting similarity between the developmental pattern of
rslo transcription in vivo and that seen in
vitro in the presence of TEA or increased external
K+. The ability of 1 mM TEA to stimulate
rslo expression in low K+ suggests that
the important factor is not increased external K+
per se but its likely effect in creating a mild depolarization of the
resting membrane potential that seems to provide the signal for an
enhanced rslo expression over basal levels. Presumably, this
key aspect also would be a natural part of the developmental environment in vivo. One possible mechanism in
vivo might be that depolarizing stimuli are provided from
developing excitatory synaptic inputs from climbing fibers or parallel
fibers, or both. For example, excitatory synapses from multiple
climbing fibers are clearly formed in vivo on Purkinje
neurons in rat cerebellum during the first week postnatal and are
progressively eliminated to a single fiber innervation pattern by the
end of the second week (Mariani and Changeux, 1981). Purkinje neurons
in vivo also undergo extensive synaptogenesis with parallel
fibers from granule cells and show a dramatic increase in the number of
synaptic junctions during early postnatal development (Herndon and
Oster-Granite, 1975). Additional indirect support for the idea that the
cultured neurons may be chronically understimulated (relatively
hyperpolarized) in the absence of an exogenous depolarizing stimulus
comes from the observation that the average resting membrane potential
recorded in Purkinje neurons in slice preparations is more negative
than that in vivo, suggesting that the removal (by the
slicing process) of excitatory synaptic inputs lowers the resting
membrane potential (Llinás and Sugimori, 1980). Presumably, in
cerebellar cultures, a similar lack of excitatory input could exist
because the inferior olivary nucleus is absent and the numbers of
granule cells are suppressed by the mitotic inhibitor FUDR. Exogenous
depolarizing agents such as K+ or TEA may partially
mimic aspects of the natural condition in vivo. The
rslo expression in vivo (Fig. 2) and in
vitro (Fig. 5) suggested a possible peak in transcript levels near
day 14 and a small decline by day 21. This decrease may be simply
coincidental or might reflect a limited duration of sensitivity of the
neurons to the putative signal that upregulates the expression of
KCa channels.
Intracellular Ca2+ is a potent regulatory element in
the control of gene expression (Bading et al., 1993; Ghosh et al.,
1994; Ginty, 1997; Hardingham et al., 1997), including the expression of ion channels (Huang et al., 1994; Linsdell and Moody, 1995). Membrane depolarization regulates gene expression in cerebellar granule
cells (Bessho et al., 1994; Harris et al., 1995; Resink et al., 1995).
Our results show that the stimulation of rslo expression by
chronic depolarization in culture requires the presence of external
Ca2+. This suggests a possible involvement of
voltage-gated Ca2+ channels in mediating the
response, an idea that could be tested further with
Ca2+ channel-selective antagonists. The ability of
the Purkinje neurons in culture to undergo morphological development in
an even lower external Ca2+ concentration (0.1 µM) than that used in the studies of transcript levels
(100 µM) suggests that the low external
Ca2+ treatment itself is not detrimental and that
the cells in low Ca2+ medium are able to maintain a
sufficient level of normal function to survive and grow. Interestingly,
this finding also suggests that the depolarization-stimulated
morphological outgrowth of Purkinje cell neurites is not strongly
Ca2+ dependent or relies on Ca2+
from intracellular stores. In contrast, the mechanism of
depolarization-induced increase in rslo expression would
seem to require mM amounts of external
Ca2+ to be effective.
KCa channels provide a sensitive mechanism for the
control of Ca2+ influx through voltage-gated
Ca2+ channels (Gorman and Thomas, 1978; Blatz and
Magleby, 1987). The existence of a calcium-dependent upregulation of
KCa channel expression would seem to represent a
logical feedback control in the cerebellar neurons. Therefore, it will
be of interest to determine whether the sensitivity of neurons to the
control signal is truly enhanced during early developmental stages of
the cerebellum. The regulated expression of KCa
channel abundance and subcellular localization could exert a
substantial influence on actively propagated dendritic
Ca2+ spikes (Llinás and Sugimori, 1980;
Markram and Sakmann, 1994). Regulated expression of the
KCa channel thus could be an important factor in the
development of information processing capabilities in the cerebellar
Purkinje neuron.
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FOOTNOTES |
Received Aug. 11, 1997; revised Oct. 6, 1997; accepted Oct. 9, 1997.
This work was supported by Whitehall Foundation Research Grant W95-27.
We thank Drs. B. Ganetzky, G. Robertson, R. Swanson, H. Y. Mi, and
T. Schwarz for providing cloned cDNAs.
Correspondence should be addressed to Dr. Andrea J. Yool, Department of
Physiology, University of Arizona College of Medicine, Tucson, AZ
85724-5051.
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Abstract
Introduction
