 |
INTRODUCTION |
The
-aminobutyric acid type A
(GABAA)1 receptor
is a transmitter-gated Cl
ion channel assembled from
different subunits in a pentameric composition. In the mammalian
central nervous system a large family of subunits exist that, based on
homology, are grouped into types
1-6,
1-3,
1-3,
,
,
and
(1-3). The GABAA receptor subunits are expressed
in a region- and age-specific manner (4-9). The different subunit
combinations exhibit distinct properties that presumably underlie a
precise physiological role for each subtype (Refs. 10-18; for review,
see Ref. 19). Knowledge of the distribution of different
GABAA receptor subtypes, both anatomically and
developmentally, is therefore essential for understanding the
physiological actions of GABA and the pharmacological actions of drugs
that act on different GABAA receptor subtypes.
Cerebellar granule neurons are used for studying neuronal signaling and
development because they have a relatively simple morphology and
receive most of their inhibitory input from one cell type (for review,
see Ref. 20). When cultured in serum-based medium, cerebellar granule
neurons express a wide range of receptors and develop stimulus-coupled
glutamate release (21). In the developing cerebellum, granule neurons
express GABAA receptor
2-,
3-,
3-,
1-, and
2-subunit genes (5). These subunits are replaced in the adult
cerebellum where
1,
6,
2,
3,
2, and
predominate (4,
7, 22). The
6 subunit is expressed almost exclusively in cerebellar
granule neurons, where it marks neuronal maturation. A similar
development evidently occurs in cultures of rat cerebellar granule
neurons (e.g. Refs. 23-25). It has been suggested that
45-59% of GABAA receptors in the cerebellum of the adult
rodent contain
6 subunits (25-28). The most predominant combinations are proposed to be
1
6
2/3
2,
1
2/3
2,
6
2/3
2,
1
6
2/3
, and
6
2/3
(25, 27, 29, 30);
noteworthy is the finding that the
subunit is found exclusively in
combination with
6 subunits (26, 31).
It is well known that elevated extracellular K+
concentrations or other calcium-elevating stimuli promote long term
survival of rat cerebellar granule neurons in dissociated cultures
(21). The physiological extracellular K+ concentration
is ~ 5 mM, but cerebellar granule neuronal cultures are often maintained in 25 mM K+ to enhance
survival (21). This use of chronic depolarization is questionable,
because it affects the subunit gene expression of neurotransmitter
receptors and, hence, the receptor composition and function. More
specifically, rat granule neurons do not correctly develop their AMPA
or NMDA receptor subunit expression in 25 mM K+
(32-34). In addition, the K+ concentration affects
GABAA receptor subunit expression in rat (35, 36) and mouse
(37) cerebellar granule neurons. Kainate is sometimes used to eliminate
GABAergic neurons from cultures of cerebellar granule neurons (38). As
a glutamate receptor agonist, it causes depolarization (39), but its
effect on GABAA receptor subunit expression is not as well
described as that of K+.
The aim of this work was to investigate the effects of extracellular
K+ and kainate on cell viability and on the expression of
13 different GABAA receptor subunit mRNAs in cultures
of mouse cerebellar granule neurons. The role of membrane potential in
mediating the effects of K+ and kainate was assessed by
correlating the effects on membrane potential and on mRNA
expression. Finally, the relative contribution of the
6 subunit to
receptor function was estimated from the sensitivity of the receptors
to GABA and to the
6 selective antagonist furosemide (11).
 |
MATERIALS AND METHODS |
Primary Cultures of Mouse Cerebellar Granule
Cells--
Cerebellar granule neurons were prepared from 6-8-day-old
NMRI mice (Taconic M&B) according to a procedure modified from
Courtney et al. (40) and Schousboe et al. (38).
Briefly, trypsin (0.25 mg/ml, Sigma)-dissociated and DNase (50 units/ml, Sigma)-treated cells were plated at ~300,000
cells/cm2 in 35-mm Petri dishes coated with
poly-D-lysine (Sigma). Cells were cultured in Dulbecco's
minimum essential medium (Invitrogen) supplemented with 10% (v/v)
fetal bovine serum (Invitrogen), 31 mM glucose, 0.2 mM glutamine (Sigma), 4 µg/liter insulin (Sigma), 7.3 µM p-aminobenzoic acid (Sigma), and 50 units/ml penicillin (LEO Pharma). As appropriate, the medium was
further supplemented with 20 mM KCl (to a final
K+ concentration of 25 mM) and/or 50 µM kainate (Sigma). Culture medium was replaced after
~24 h with the inclusion of 10 µM cytosine arabinoside
(Sigma) to reduce non-neuronal proliferation; after this treatment the
medium was not changed. The cells were cultured in a humidified 5%
CO2 atmosphere at 37 °C. The cultures were used in
experiments within 12 days in vitro (DIV).
Measurement of Cell Viability--
The amount of viable cells in
the cerebellar granule neuron cultures was quantified using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as
described (41). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is reduced to formazan by cells that have functional
mitochondria; this process has been shown to correlate well with cell
viability (42). The cultures were tested at 7 and 12 DIV.
Semiquantitative Reverse Transcription-PCR--
Reverse
transcription-PCR was performed as previously described (43). The
cultures were tested at 7 and 12 DIV. Primers (Table
I) for the GABAA receptor
subunits
1,
2,
3,
4,
5,
6,
1,
2,
3,
1,
2,
3, and
were used. Briefly, RNA was isolated with RNeasy
Mini Kit (Qiagen) after lysis of the cells, and the concentration and
purification of RNA was measured on a spectrophotometer (Ultrospec4000,
Pharmacia Biosciences). cDNA was synthesized using 0.25 µg of
RNA, and 1 µl of the resulting mixture was used for PCR for each
subunit. cDNA amplifications were performed in 42 cycles, and
aliquots (15 of 100 µl in total) of the PCR products were taken from
cycles 27, 30, 33, 36, 39, and 42. Relative intensity of the ethidium
bromide-stained bands on gels were measured using computer-assisted
image analysis and compared with the 400-bp band of the molecular
weight marker (100 Base-Pair-Ladder, Amersham Biosciences) added in
fixed amount for all gels. Because the efficiency of the PCR
amplification is primer-dependent, the relative intensities
could not be compared between different subunit mRNAs.
Electrophysiology--
Before recordings, the culture medium was
exchanged for an extracellular recording solution (artificial balanced
salt solution (ABSS)), and the Petri dish with cells was transferred to
an inverted phase-contrast Zeiss Axiovert 10 microscope stage. The
cells were constantly perfused with ABSS (0.5 ml/min) at room
temperature from a gravity-fed 7-barrel perfusion pipette (List) ~100
µm from the recorded neuron. By switching application from one barrel to another, the extracellular solution surrounding the neuron was
exchanged with a time constant of ~50 ms. Individual cerebellar granule neurons were approached with micropipettes of
3-5-megaohm resistance manufactured from 1.5-mm-outer-diameter glass
(World Precision Instruments). Standard patch clamp technique (44) in
current or voltage clamp mode was used to record from neurons in the
whole-cell configuration using an EPC-9 amplifier (HEKA Elektronik).
Whole-cell membrane currents and potentials were plotted on a low
fidelity chart recorder during the experiment and stored on computer
hard disk and video tape using a VR-100 digital data recorder (Instrutech).
For recordings of the membrane potentials with extracellular
K+ concentration = 5 mM (physiological
K+), the cells were perfused with ABSS composed of 138.5 mM NaCl, 5 mM KCl, 1.25 mM
Na2HPO4, 2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.35. For recordings with extracellular
K+ concentration = 25 mM (depolarizing
K+) the concentration of NaCl was reduced to 118.5 mM, and the concentration of KCl was increased to 25 mM. Kainate was dissolved in ABSS to a final concentration
of 50 µM, where indicated. The intrapipette solution
contained 10 mM NaCl, 130 mM potassium
gluconate, 1 mM MgCl2, 1 mM
CaCl2, 10 mM EGTA, 2 mM MgATP, and
10 mM HEPES, pH 7.3. Initially the cells were perfused with
"physiological" ABSS containing 5 mM K+
without kainate. The perfusion was then switched to ABSS with either 25 mM K+ or 50 µM kainate or both.
After a new stable membrane potential was reached, the perfusion was
switched back to physiological ABSS. Each of the depolarizing solutions
was tested at least twice on each cell. Membrane potentials were
corrected for liquid junction potentials.
In experiments addressing the GABA concentration-response relationships
or furosemide sensitivities, the ABSS contained 140 mM
NaCl, 3.5 mM KCl, 1.25 mM
Na2HPO4, 2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.35. The intrapipette solution contained 140 mM KCl, 1mM MgCl2, 1 mM
CaCl2, 10 mM EGTA, 2mM MgATP, and
10 mM HEPES, pH 7.3. The neurons were voltage-clamped at
60 mV. The high intracellular Cl
concentration shifted
the Cl
reversal potential to ~0 mV and substantially
increased the currents recorded at
60 mV. Series resistance was 65%
compensated. GABA (Sigma) was dissolved in distilled water at a
concentration at least 100× greater than that required for perfusion
and diluted with ABSS. Furosemide (Sigma) was dissolved in
Me2SO and diluted with ABSS; the content of
Me2SO in the final solution was at most 0.1% and had no
effect of its own on membrane current. Different concentrations of GABA
were applied for 5 s at 1-min intervals. Furosemide was preapplied
for 10 s immediately before the application of a premixed solution
of GABA and furosemide. Between drug applications the cell was perfused
with ABSS.
Membrane currents and potentials were analyzed using Pulse (HEKA
Elektronik) and Igor Pro (Wavemetrics) software. Current responses were
quantified by measuring the peak current during application of GABA or
GABA plus furosemide. GABA concentration-response relationships were
fitted to the equation,
|
(Eq. 1)
|
where I is the peak membrane current induced by the
GABA concentration, [GABA], Imax is the
maximum peak current that GABA can induce, EC50 is the GABA
concentration eliciting 50% of Imax, and
n is the Hill coefficient.
Statistics--
Data were described using mean and S.E. or 95%
confidence intervals. Mean values were compared using either Student's
t test or analysis of variance (ANOVA); when relevant, the
Tukey test was used as a means of post-hoc multiple comparisons.
Two-way ANOVA was used to separate the effects of two variables and
determine their interaction. Probabilities (p) < 0.05 were considered statistically significant. The term "occlusion"
refers to situations where the effect due to simultaneous variation of
two variables was smaller than the sum of the effects due to variation
of each variable separately.
 |
RESULTS |
K+ and Kainate Are Acutely Depolarizing, but Prolonged
Exposure Induces Compensatory Hyperpolarization--
Fig.
1 shows the influence of the culture
medium as well as the extracellular recording solution (ABSS) on the
membrane potentials of the neurons. The same K+ and kainate
concentrations were used for the culture media and extracellular
recording solutions: 5 or 25 mM K+ and 0 or 50 µM kainate.

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Fig. 1.
Membrane potentials of cerebellar granule
neurons depend on K+ and kainate concentrations in the
culture media as well as in the extracellular recording solution.
Each column represents the mean ± S.E. from 6-11
neurons. The effects of K+ and kainate were analyzed using
two-way ANOVA. A, at 2-3 DIV the composition of the culture
medium was without effect. In the extracellular recording solution both
elevation of K+ (p < 0.001) and addition
of kainate (p < 0.001) depolarized the membrane
potential with total occlusion (p = 0.026).
B, after 7-8 DIV a significant effect of the culture medium
had emerged (p < 0.001) in addition to, but
independent of, the effect of the extracellular recording solution
(p < 0.001). In the culture medium both elevated
K+ (p = 0.004) and kainate
(p = 0.034) hyperpolarized the membrane potential
without interaction. In the extracellular recording solution both
elevation of K+ (p < 0.001) and kainate
(p < 0.001) depolarized the membrane potential with
partial occlusion (p < 0.001). In columns marked with
the symbol # membrane potentials were measured in the presence of the
same extracellular K+ and kainate concentrations as in the
culture medium. For these, at 2-3 DIV elevation of K+
concentration significantly depolarized the membrane potential
(p = 0.005), whereas the kainate effect was not
significant. At 7-8 DIV both K+ (p < 0.001) and kainate (p < 0.001) were significantly
depolarizing and totally occluding (p < 0.001). The
horizontal line at 42 mV shows the equilibrium potential
for K+ with the extracellular concentration at 25 mM.
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A two-way ANOVA was used to estimate the effects of 1) culture medium
and 2) extracellular recording solution on the membrane potential. At
2-3 DIV the membrane potential was independent of the culture medium
but significantly dependent on the extracellular recording solution
(p = 0.002). This dependence was further analyzed with
a two-way ANOVA of the effects of 1) K+ concentration and
2) kainate concentration in the extracellular recording solution. Both
K+ and kainate had highly significant depolarizing effects
(p < 0.001 for both) and a significant interaction
(p = 0.026), suggesting that the depolarizing effect of
simultaneously increasing both K+ or kainate concentrations
to 25 mM and 50 µM, respectively, was not as
great as the sum of the depolarizations caused by increasing K+ or kainate separately (occlusion). Indeed, the combined
effect of K+ and kainate did not differ significantly from
the individual effects (total occlusion).
After 7-8 DIV both culture medium and extracellular recording solution
showed significant effects (p < 0.001 for both)
without interaction, i.e. their effects were additive
(two-way ANOVA). The effect of culture medium was further analyzed with
two-way ANOVA. Increased K+ (p = 0.004) or
kainate (p = 0.034) in the culture medium significantly hyperpolarized the membrane potentials without interaction. The effect
of recording solution was also further analyzed. After 7-8 DIV (as at
2-3 DIV), both increased K+ (p < 0.001)
and kainate (p < 0.001) concentrations were
significantly depolarizing. The combined effect of increased
K+ and kainate concentration was smaller than the sum of
the individual effects (p < 0.001) but larger than any
of the individual effects (p < 0.001, Tukey test),
which were not significantly different. Thus, at this point the effects
of K+ and kainate concentrations in the extracellular
recording solution were only partially occluding.
In conclusion, the immediate effects of increasing either
K+ (from 5 to 25 mM) or the kainate
concentrations (from 0 to 50 µM) in the extracellular
recording solution were depolarizations of similar magnitude that were
occluding and independent of the culture medium. Prolonged exposure to
high K+ or kainate concentrations in the culture medium
gave rise to hyperpolarization relative to cells grown in a
physiological K+ concentration without kainate. This
suggests that the neurons develop a compensatory hyperpolarizing
mechanism when exposed to depolarizing conditions for an extended period.
The membrane potentials of neurons in the different culture media may
influence mRNA expression. These membrane potentials were estimated
from measurements in extracellular recording solution with the same
K+ and kainate concentration as in the culture medium (Fig.
1, columns marked with a number sign). At 2-3
DIV elevated K+ concentration significantly depolarized the
membrane potential (two-way ANOVA, p = 0.005); on the
other hand, the kainate effect was not significant. After 7-8 DIV both
K+ (p < 0.001) and kainate
(p < 0.001) were significantly depolarizing, but their
combined effect was smaller than the sum of the effects of increasing
K+ or kainate separately (two-way ANOVA, p < 0.001); moreover, the combined effect was not significantly larger
than the individual effects (total occlusion, Tukey test).
No spontaneous synaptic potentials or currents were observed in any of
the recorded neurons at 2-3 or 7-8 DIV. In addition, no action
potentials were observed, even after depolarizations were induced with
K+ or kainate.
Kainate as Well as Depolarizing K+, Increase Cell
Viability--
The survival of mouse cerebellar granule neurons in
culture was strongly dependent on the concentration of K+
in the culture medium as indicated by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
(Fig. 2). 25 mM
K+ significantly (p < 0.001) increased the
viability of mouse cerebellar granule neurons in culture regardless of
the presence or absence of kainate both at 7 and 12 DIV. At 7 DIV
kainate significantly (p < 0.01) increased cell
viability in cultures grown in 5 mM but not in 25 mM K+, indicating that the effects of
K+ and kainate on viability were not additive. The effect
of kainate on cell viability was not detectable after 12 DIV.

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Fig. 2.
Viability of mouse cerebellar granule neurons
cultured in different media for 7 and 12 DIV. The respective
culture media are indicated below the columns.
Cerebellar granule neurons cultured in 25 mM K+
without kainate was set to 100% viability at 7 DIV. Each
column represents the mean ± S.E. of 10 cultures from
independent batches. Cultures grown in 5 mM K+
showed significantly (***, p < 0.001) lower viability
than cultures grown in 25 mM K+. Cultures grown
for 7 DIV in 5 mM K+ in the absence of kainate
showed significantly (p < 0.01) lower viability than
cultures grown in 5 mM K+ in the presence of
kainate.
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Depolarizing K+ Causes a Shift of mRNA Expression
from
6 to
5, Which Is Mimicked by Kainate in 12 DIV
Cultures--
Of the six GABAA receptor
subunit
mRNAs tested for, the
1,
2,
5, and
6 mRNAs were
expressed in detectable amounts, whereas
3 mRNA and
4
mRNA could not be detected.
The level of
5 mRNA was significantly affected by K+
and kainate (Fig. 3). At 7 DIV
5
expression was enhanced with 25 mM K+ in the
culture medium relative to 5 mM K+
(p < 0.001). At 12 DIV both high K+
(p < 0.01) and kainate (p < 0.01)
enhanced the expression of
5 to similar levels, but the combination
of K+ and kainate did not further increase
5.

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Fig. 3.
Expression of mRNA for the
5, 6, and
2 GABAA receptor subunits depends on
the concentrations of K+ and kainate in the culture
medium. The amount of amplified mRNA is shown as a function of
the number of PCR cycles. Band intensities are expressed relative to
those from a fixed amount of molecular weight marker. Each symbol
represents the mean ± S.E. of three cultures from independent
batches. The significance levels of the differences achieved by varying
the K+ and kainate concentrations as well as of their
interaction are indicated below each number of PCR cycles (two-way
ANOVA; *, p < 0.05; **, p < 0.01;
***, p < 0.001). Interactions could only be properly
estimated when at least three of the four intensities for a given
number of PCR cycles were above the detection limit. Significance
values for lower numbers of cycles are therefore shown in
parentheses.
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The
6 mRNA was reciprocally affected (Fig. 3). At 7 DIV
expression was significantly decreased in 25 mM
K+ compared with 5 mM K+
(p < 0.001). At 12 DIV both high K+
(p < 0.05) and kainate (p < 0.05)
inhibited
6 expression to the same extent and with additive effects.
The expression of
1 and
2 mRNA was not significantly
affected by the concentration of K+ or kainate in the
culture media (results not shown).
Depolarizing K+ and Kainate Specifically Decrease
Expression of the
2 Subunit mRNA--
Of the three
GABAA receptor
subunit mRNAs studied, the
2 and
3 were expressed in significant quantities, whereas expression of
1 was not detected. Elevation of the extracellular K+
concentration to 25 mM significantly (p < 0.001) decreased the expression of
2 mRNA in 7 DIV but not in 12 DIV cultures (Fig. 3). Kainate had no effect on neurons cultured for 7 days, but after 12 DIV it significantly (p < 0.05)
decreased the expression of
2 independently of the K+
concentration. Expression of
3 mRNA was not significantly
affected by increased K+ or the addition of kainate
(results not shown).
Neither Kainate nor the K+ Concentration Affects the
Expression of
or
Subunit mRNA--
Of the three
GABAA receptor
subunits, only
2 was detected. The
subunit mRNA was also found in significant amounts. Neither 25 mM K+ nor kainate had a significant effect on
expression of
2 mRNA or
mRNA (results not shown).
The GABA Concentration-Response Relationship Is Influenced by
K+ in the Culture Medium--
To investigate whether
differences in mRNA expression between cultures were reflected in
the functional properties of the derived membrane-bound receptors, we
determined GABA concentration-response relationships resulting from the
different culture conditions (Fig. 4).
The EC50 values and Hill coefficients are listed in Table
II. Neurons cultured for 10-12 DIV in 5 mM K+ without kainate had a significantly lower
EC50 value than neurons cultured in 25 mM
K+ without kainate (p = 0.005). The Hill
coefficients were not significantly different from each other.

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Fig. 4.
Concentration-response relationships for
GABA-induced peak currents in neurons grown in different culture media
for 10-12 DIV. Peak currents were normalized to the peak current
of 2 mM GABA in each cell and shown as the means ± S.E. (n = 19-29 neurons). The line graphs result from
non-linear regression analysis of the concentration-response
relationships, and the corresponding EC50 values and Hill
coefficients are summarized in Table II.
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Table II
Characteristics of GABA-induced currents in cerebellar granule neurons
cultured in different media
The values were estimated by non-linear regression analyses of the
concentration-response relationships for GABA-induced peak currents
shown in Fig. 4. Numbers in parentheses are 95% confidence intervals
for n = 19-29 neurons tested/culture medium.
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Furosemide Inhibition of GABA-induced Currents Is Influenced by
Both K+ and Kainate in the Culture Medium--
Furosemide
has been shown to be a specific antagonist at GABAA
receptors containing
6 subunits (11). To estimate the relative contribution of
6-containing receptors we tested the sensitivity of
GABA-induced currents to inhibition by 100 µM furosemide
in 10-11 DIV neurons (Fig. 5). Both
culture conditions (p < 0.001) and GABA concentration
(p = 0.012) affected furosemide inhibition without
significant interaction (two-way ANOVA). Thus, furosemide inhibition
decreases with increasing GABA concentration and response levels.
Because the response levels obtained with 10 and 30 µM GABA were similar for cells grown in 25 mM K+,
with or without kainate and in 5 mM K+ with
kainate (Fig. 4, Table II), the furosemide inhibition was directly
comparable for these cells. For 5 mM K+ without
kainate, the response levels were higher; this would have a tendency to
decrease the observed inhibition. Yet, this group of cells was the most
sensitive to inhibition by furosemide.

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Fig. 5.
Furosemide inhibition of GABA-induced peak
currents in mouse cerebellar granule neurons cultured in different
media for 10-11 DIV. A, representative current traces
of the inhibitory effect of furosemide (100 µM) on peak
currents induced by 10 µM GABA in neurons cultured in 25 mM K+ with 50 µM kainate, 25 mM K+ without kainate, 5 mM
K+ with 50 µM kainate, and 5 mM
K+ without kainate, respectively. The increased current
variation in the beginning of the furosemide-modulated traces was
caused by transient voltage pulses used to monitor cell membrane
conductance and capacitance. These pulses were suspended ~5 s before
GABA application. B, each column represents the means ± S.E. (n = 4-14 neurons) for the inhibition of
GABA-gated peak currents by 100 µM furosemide. The
respective culture media are indicated below the
columns. The asterisks denote the significance
levels of the inhibition (*, p < 0.05; ***,
p < 0.001). The two GABA concentrations employed, 10 and 30 µM, corresponds to the following response levels
for each culture medium: 25 mM K+ with 50 µM kainate, 16 and 36%; 25 mM K+
without kainate, 17 and 34%; 5 mM K+ with 50 µM kainate, 18 and 37%; 5 mM K+
without kainate, 28 and 47% (calculated from the parameters in Table
I).
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|
At 10 µM GABA both K+ (p < 0.001) and kainate (p = 0.019) had negative effects on
the furosemide sensitivity (two-way ANOVA). For cells cultured in 5 mM K+ without kainate the control response
level was higher (28%) than that of other cells (16-18%). If cells
cultured in 5 mM K+ without kainate had been
tested at the same response level the measured furosemide inhibition
would have been enhanced and, thus, would strengthen the observed
significance of the K+ and kainate effects. For 30 µM GABA, only the effect of K+ was found to
be significant, but again, significance levels were probably
underestimated because neurons cultured in 5 mM
K+ without kainate were tested on a higher response level
than the other neurons.
 |
DISCUSSION |
Depolarizing Effects of K+ and Kainate--
Increased
extracellular K+ concentration as well as the addition of
kainate to the extracellular medium gives rise to depolarization. An
increased extracellular K+ concentration inhibits and
potentially reverses outward K+ membrane currents leading
directly to depolarization. Assuming an intracellular K+
concentration of 130 mM as in the intrapipette solutions,
increasing the extracellular K+ concentration from 5 to 25 mM K+ switches the equilibrium potential for
K+ , EK+, from
84 mV to
42 mV (45).
The role of kainate is more complex. Kainate activates a subset of
ionotropic glutamate receptors (AMPA as well as kainate receptors)
giving rise to increased membrane permeability of Na+ and
K+ (and for some AMPA receptors also Ca2+),
resulting in membrane depolarization (39). Whether induced by increased
extracellular K+ concentration or by kainate,
depolarization opens voltage-gated Na+, K+, and
Ca2+ channels, further increasing membrane permeability of
Na+ and K+ and eliciting influx of
Ca2+. We have analyzed the effects of increased
K+ and kainate on membrane potential in order to correlate
these with cell viability and mRNA expression.
The observed effects of increasing the extracellular concentration of
K+ from 5 to 25 mM or adding 50 µM kainate to the extracellular medium are 2-fold. That
is, the acute effects of both are depolarizations of similar magnitude
that are mutually occluding and independent of the preceding culture
conditions. Prolonged exposure to elevated K+ or kainate in
the culture medium induces compensatory hyperpolarizing mechanisms; in
this respect the effects of K+ and kainate are additive.
Depolarization and compensatory hyperpolarization in concert determine
the membrane potentials in the culture media.
The mutual occlusion of the depolarizations induced by increased
K+ concentration and kainate in the extracellular recording
solution is predicted by the Goldman equation (45). This equation also states that the greater the concentration and membrane permeability of
a particular ionic species, the greater will be its role in determining
the membrane potential (45). From Fig. 1 it is apparent that, with 25 mM K+ and no kainate in the extracellular
recording solution, the membrane potential is, indeed, close to the
EK+. An effect of depolarizing K+
concentrations on expression of AMPA receptor subunits has been reported (46) that might influence the depolarizing ability of kainate.
Because we did not observe any interaction between the composition of
the culture medium and the depolarizations induced when adding kainate
to the extracellular recording solution, such an effect was not
important in our experiments.
How does this correlate with cell viability and mRNA expression?
This question is addressed in the following paragraphs. The interpretation is complicated by the fact that the measurements of
membrane potentials are made at two time points, but the derived effects (viability and mRNA expression) are influenced by the membrane potential experienced by the cell throughout the culture period and possibly with some delay.
Effect of Depolarizing K+ and Kainate on Cell
Viability--
During cerebellar development, excess granule neurons
are produced and subsequently lost via apoptosis (47). Also in culture, a proportion of the rat cerebellar granule neurons die relatively soon.
This apoptotic cell death is prevented or reduced if the extracellular
K+ concentration is elevated to depolarizing concentrations
(21). Our results show, in conflict with an earlier study (48), that also mouse cerebellar granule neurons survive significantly better when
grown in a depolarizing than in a physiological K+
concentration. Depolarizing concentrations of K+ have been
suggested to promote survival of cerebellar granule neurons in
dissociated cultures by increasing the intracellular Ca2+
concentration via L-type voltage-gated Ca2+
channels (21).
In the developing brain, excitatory amino acids appear to exert trophic
effects. NMDA has been shown to reduce apoptotic cell death in rat
cerebellar granule neuron cultures by stimulating brain-derived
neurotrophic factor (49). In cultures of rat cortical neurons kainate
has been reported to elevate intracellular calcium, presumably in part
via G protein activation, leading to enhanced nerve growth
factor-mediated increase in cell viability (50). Significant
neurotoxicity of kainate was not seen before 14 DIV (50). In the
present study, kainate increased the viability of mouse cerebellar
granule neurons cultured in physiological but not depolarizing
K+ at 7 DIV. In 12 DIV cultures no effect of kainate on
viability was detected. This profile seems not to correlate with
kainate depolarization, which increased from 2-3 DIV to 7-8 DIV. It
is possible that depolarization decreased again between 8 and 12 DIV or
that a neurotoxic effect of kainate developed and counteracted the
positive effect on cell viability. Alternatively, depolarization may
not be a main mediator of the effect of kainate on cell viability.
Effect of Depolarizing K+ and Kainate on the Expression
of GABAA Receptor Subunit mRNA--
The present study
provides, for the first time, a simultaneous semi-quantitative analysis
of the expression of 13 of the main mammalian GABAA
receptor subunit mRNAs (
1-6,
1-3,
1-3, and
) in
cultured mouse cerebellar granule neurons. The GABAA
receptor subunit mRNA species that were detected in this study
(
1,
2,
5,
6,
2,
3,
2,
) are generally in good
agreement with earlier studies of rat cerebellar granule neurons
in vivo (7, 20) and in vitro (20, 36). Most
importantly, our results show that the expression of some receptor
subunit mRNAs (
5,
6,
2) is strongly dependent on culture conditions.
Depolarizing K+ and Kainate Hinders Maturation of
Subunit Expression--
Our results demonstrate that mouse cerebellar
granule neuron cultures maintained in depolarizing K+
express the
5 subunit as a substitute for
6, the latter prominent in cultures grown in physiological K+. At 12 DIV the effect
of high K+ is mimicked by kainate in the culture medium,
occluding the effect of K+ on
5 mRNA. Although
kainate is significantly depolarizing already at 7-8 DIV (and maybe
earlier), the changes of mRNA expression are likely to be delayed
relative to changes in membrane potential. Thus, on this basis, it is
feasible that the reciprocal changes in
5 and
6 mRNA
expression are mediated more or less by differences in membrane potential.
The
5 subunit has not been found (4, 26, 27) or found in very low
levels (single cell bodies) (7) in rat cerebellum in vivo.
In rat cerebellar granule neurons cultured in 12.5 or 25 mM
K+, however,
5 subunit mRNA has been found (24, 35).
During development of the rat central nervous system, the
5 gene
expression in particular seems to undergo a prominent peak in the early
brain (5, 6), i.e. the expression of the
5 gene might, in
some brain areas, indicate undeveloped neurons. This is opposite to the
6 subunit, which is reported to mark maturation of cerebellar granule neurons in vivo (5, 7, 22).
Several studies show that the level of the
6 subunit mRNA and
protein increases during the development of rat cerebellar granule
neurons cultured in 25 mM K+ (e.g.
Refs. 23, 24, and 51). Depolarizing K+ concentrations do
not diminish the
6 level (35). Actually, one study shows that
6
expression is maintained by depolarizing K+ (36).
In contrast to this but in agreement with the present study, cultures
of mouse cerebellar granule neurons grown in 25 mM
K+ for 11-15 DIV have been shown not to express the
6
gene, whereas cultures grown for the same period in 5 mM
K+ did (37). These results suggest a species-specific
difference regarding regulation of
6 expression, which, however, may
not apply to
5 subunit expression. In accordance with our results for mouse cerebellar granule neurons, Harris et al. (35)
find that in rat granule neurons the level of
5 mRNA was higher
after 5 DIV in 25 mM K+ than in 12.5 mM K+.
In the present study, the effect of K+ on
5 and
6
expression was mimicked by kainate at 12 DIV but not 7 DIV. To our
knowledge, no AMPA or kainate receptor-mediated effects on
5
expression in mouse or rat cerebellar granule neurons have been
reported. The
6 gene expression in rat cerebellar granule neurons
has been found to be increased by glutamate. This effect was not
sensitive to the NMDA receptor antagonist MK801
((5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), suggesting involvement of AMPA/kainate and/or metabotropic glutamate receptors (35). In light of the proposed species-specific difference it
may not be surprising that we found the opposite effect on
6
expression when using kainate, but this could also be due to activation
of metabotropic glutamate receptors by glutamate in the rat experiments.
The Effect on
2 Subunit Expression Does Not Correlate with
Membrane Potential--
Compared with the
5 and
6 subunits the
effects of K+ and kainate on
2 mRNA expression
showed a very different pattern; at 7 DIV only change of K+
concentration was effective, whereas at 12 DIV only kainate had a weak
effect. As for
5 and
6 mRNA, the effect of increased K+ at 7 DIV could potentially be due to depolarization, but
because the K+ effect had vanished after 12 DIV we can
postulate that either the mechanism had changed or it was never
mediated through membrane potential. The lack of K+ effect
after 12 DIV excludes the possibility that the kainate effect was
mediated by depolarization. If expression of
2 mRNA is
influenced by membrane potential at all, the effect seems to be
transient. To our knowledge, there are no reports on the effect of
kainate on the expression of GABAA receptor
subunits.
mRNA Expression--
Interestingly, it has been reported
that rat cerebellar granule neurons show very low expression of
mRNA when cultured in 5 mM K+. However,
mRNA expression increases significantly in culture medium with 25 mM K+- or in Mg2+-free medium
containing NMDA (52). As for
6 mRNA, depolarizing medium is
important for maintenance of
mRNA expression (36). The lack of
effect of depolarization on
mRNA expression in mouse cerebellar granule neurons found in the present study could indicate a
species difference in regulation of expression of the
subunit, although it could also be due to differences in culture conditions.
GABA Sensitivity and Inhibition by Furosemide Reflect the
6
mRNA Level--
The presence of
6 subunit in the
GABAA receptor complex has functional implications with
regard to GABA and furosemide sensitivity. Provided that a proportion
of the
6 mRNA is normally translated into protein and
incorporated into receptor complexes in the cell membrane, a
correlation between mRNA expression and these functional properties
would be expected.
Recombinant expression of
1
2
2,
6
2
, and
1
6
2
2 combinations in human embryonic kidney 293 cells (24)
and oocytes (30) have demonstrated a higher EC50 value for
GABA in the
1
6
2
2 combination (34 and 107 µM,
respectively) than in the
1
2
2 (14 and 41 µM,
respectively) and
6
2
2 (2 and 6.7 µM,
respectively) combinations. A relatively modest expression of
6
subunit would probably result primarily in
1
6
2
2 receptors,
whereas a relatively high level of
6 would favor
6
2
2
receptors with high sensitivity to GABA. In agreement with this we
found that only the neurons with the highest level of
6 mRNA
expression, namely those cultured in physiological K+
concentration without kainate, differed with regard to GABA
sensitivity. These neurons had the lowest EC50,
e.g. significantly lower than neurons grown in 25 mM K+ (Table II, Fig. 4).
Sigel and Baur (30) show that oocytes expressing the
1
6
2
2
combination with regard to furosemide inhibition were more like the
6
2
2 combination. Furthermore, it was possible to gradually vary the IC50 for furosemide between the values for the
1
2
2 and the
6
2
2 combination by adjusting the
1/
6 cDNA ratio. These observations suggest that furosemide
inhibition is a more sensitive indicator of
6 protein in the
receptors than GABA EC50. In agreement with this notion we
found that furosemide inhibition at 10-11 DIV was significantly
dependent on both K+ and kainate; this was also the case
for
6 mRNA expression at 12 DIV. In addition, the rank order of
6 mRNA expression at 12 DIV and furosemide inhibition is in good
agreement; that is, 5 mM K+ without
kainate > 5 mM K+ with kainate > 25 mM K+ without kainate > 25 mM
K+ with kainate. The kainate effect on
6 expression was
not significant at 7 DIV but increased to a significant level between 7 and 12 DIV. Thus, the functional properties of the GABAA
receptors at 10-11 DIV seem to be in accordance with development of
mRNA levels from 7 to 12 DIV.
Our Cerebellar Granule Neurons Were Electrically
Silent--
Mellor et al. (37) report spontaneous miniature
synaptic currents in mouse cerebellar granule neurons cultured and
recorded in 5 mM K+, but not in 25 mM K+, at 15 DIV. Action potentials were also
reported subsequent to adequate depolarization, but for neurons
cultured and recorded in 25 mM K+, an initial
hyperpolarizing current injection was required to reactivate the
inactivated Na+ channels. In contrast, we did not observe
spontaneous synaptic potentials or action potentials in any of the
recorded neurons at 2-3 or 7-8 DIV. Because the membrane potentials
in our neurons cultured and recorded in 5 mM K+
without kainate were more negative (
56 ± 3 mV at 2-3 DIV and
56 ± 1 mV at 7-8 DIV) than those of Mellor et al.
(37) (
50 ± 2 mV at 15 DIV), inactivation of voltage-gated
Na+ channels is not likely. The most probable reason for
the lack of electrical activity is that necessary features such as
synapses and high density of voltage-gated Na+ channels
might be less developed at 7-8 DIV.
Concluding Remarks--
Both a depolarizing K+
concentration and kainate in the culture medium have the potential to
enhance survival of mouse cerebellar granule neurons, but this happens
at the expense of maturation of the GABAA receptor subunit
expression. This is normally not desirable, but with sufficient
knowledge of the effects of K+ and kainate these parameters
may perhaps be exploited to adjust the subunit expression in a
preferred direction. Species-specific effects on expression of some
subunits make extrapolation from one species to another difficult. The
actions of extracellular K+ and kainate described here can
probably in part be attributed to alterations of the membrane
potential. To clarify the exact mechanism of action, more detailed
experiments employing subtype-specific glutamate receptor antagonists
and Ca2+ channel blockers are required.