Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E5 Canada
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ABSTRACT |
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Bekar, Lane K. and
Wolfgang Walz.
Evidence for Chloride Ions as Intracellular Messenger Substances
in Astrocytes.
J. Neurophysiol. 82: 248-254, 1999.
Cultured rat hippocampal astrocytes were used to
investigate the mechanism underlying the suppression of
Ba2+-sensitive K+ currents by GABAA
receptor activation. Muscimol application had two effects on whole cell
currents: opening of the well-known Cl channel of the
GABAA receptor and a secondary longer-lasting blockade of
outward K+ currents displaying both peak and plateau
phases. This blockade was independent of both Na+ (inside
and outside) and ATP in the pipette. It also seemed to be independent
of muscimol binding to the receptor because picrotoxin application
showed no effect on the K+ conductance. The effect is
blocked when anion efflux is prevented by replacing Cl
with gluconate (both inside and out) and is enhanced with more permeant
anions such as Br
and I
. Moreover, the
effect is reproduced in the absence of muscimol by promoting
Cl
efflux via lowering of extracellular Cl
levels. These results, along with the requirement for Cl
efflux in muscimol experiments, show a strong dependency of the secondary blockade on Cl
efflux through the
Cl
channel of the GABAA receptor. We
therefore conclude that changes in the intracellular Cl
concentration alter the outward K+ conductances of
astrocytes. Such a Cl
-mediated modulation of an
astrocytic K+ conductance will have important consequences
for the progression of spreading depression through brain tissue and
for astrocytic swelling in pathological situations.
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INTRODUCTION |
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Although activation of GABAA
receptors in both astrocytes and neurons results in
Cl currents, the consequences of this
activation are different. In neurons the
ECl is usually somewhat more negative
than the membrane potential and therefore a net
Cl
influx is evoked, leading to
hyperpolarization (Alvarez-Leefmans and Russel 1990
). In
astrocytes, the ECl is far more
positive than the membrane potential, and GABAA
receptor activation therefore will result in net
Cl
efflux and depolarization (Fraser et
al. 1995
). One exception is the situation in neonatal animals,
where neurons can have an ECl more
positive than the resting membrane potential, and
GABAA receptor activation results in
depolarization (Cherubini et al. 1991
). There is no
corresponding difference in the ECl of
astrocytes between neonatal and adult animals (Bekar et al.
1999
).
GABAA-receptor-mediated
Cl current is not the only effect of
GABAA receptor activation: in cultured astrocytes
and neonatal and adult astrocytes in situ and in one adult neuronal
preparation (cerebellar granule cells), a longer-lasting blockade of
K+ outward currents occurs concomitant with the
Cl
current (Bekar et al. 1999
;
Labrakakis et al. 1997
; Muller et al.
1994
; Pastor et al. 1995
). The mechanism of this
blockade and its link to the GABAA receptor is
unknown. However, an analogous situation exists with the astrocytic
kainate receptor, where cation currents are followed by
K+ channel blockade. In this case, it was found
that Na+ net influx through the kainate receptor,
with an increase in internal Na+ concentration,
is responsible for the secondary K+ channel
blockade (Robert and Magistretti 1997
).
Na+ increases alone were able to reduce outward
K+ currents.
In the present project, we investigate the mechanism that underlies the
K+ channel blockade after
GABAA-receptor-mediated
Cl currents in astrocytes. We used cultured rat
hippocampal astrocytes because we previously verified that the
GABAA receptors of this preparation have
properties similar to those of "complex" hippocampal astrocytes
in situ (Bekar et al. 1999
).
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METHODS |
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Cell culture
Primary cultures of hippocampal astrocytes were prepared from 1-day-old Wistar rat pups. Animals were anesthetized with methoxyflurane and decapitated. Hippocampi were removed aseptically, and cells were mechanically disaggregated using an 80-µm Nitex filter. The cells were seeded (105 cells/35 mm dish) onto round coverslips in DMEM Gibco BRL containing 20% low endotoxin horse sera (Hyclone) and incubated at 37°C in 5% CO2-95% humidified air. After 3 days, the culture medium was replaced by DMEM containing 10% low endotoxin horse sera. Cells then were fed twice a week in this manner. Cells were used for whole cell patch recordings when cells formed a confluent monolayer (~9-15 days). Cultures were checked periodically for purity: >85% of the cells are glial fibrillary acidic protein (GFAP) positive with the major other cell type being microglial cells.
Electrophysiological setup and protocols
For electrophysiological recordings, coverslips were placed in a
perfusion chamber. The chamber was perfused continuously with normal
Cl containing solution (see following text).
Cells were analyzed under optical control using the conventional whole
cell patch-clamp technique. Membrane currents were recorded in
voltage-clamp mode using the EPC-7 patch-clamp amplifier (List
Electronics), filtered at 3 kHz, and connected to a computer system
serving as a stimulus generator. Recording pipettes were made of
borosilicate capillaries (Hilgenberg, Germany) with resistances of 4-8
M
, and the reference electrode was a chlorided silver wire. The
voltage-clamp holding potential was
80 mV and cell membranes were
measured in current-clamp mode immediately after formation of whole
cell patch. Cell membrane capacitance and membrane input resistance
were calculated from the average of 10 10-mV depolarizing voltage steps
from the holding potential. Current-voltage (I-V)
relationships were calculated from data obtained in the first 10 ms of
the 100-ms voltage steps ranging from
160 to +80 mV in 20-mV
increments. These voltage step protocols were applied every 5 s
throughout the experiments.
All currents (pA) obtained using the whole cell patch-clamp technique
were normalized to cell size. To normalize values, all currents were
divided by the cell capacitance (pF) to give current densities. Cell
membrane capacitance is directly proportional to the size of the cell
or area of cell membrane (Sontheimer 1995). Additionally, all receptor currents were normalized to cell capacitance.
For calculation of conductance, traces represented in Fig.
1 and Table 3 currents obtained at +80
and 80 mV in the first 10 ms of the 100-ms voltage jumps for each
jump protocol conducted every 5 s were used. At
80 mV, where
presumably there is very little voltage-gated channel activity,
receptor conductance was calculated using an 80-mV driving force
(driving force for all the anions at
80 mV because the equilibrium
potential is at 0 mV due to symmetrical distribution). To calculate the
conductance at +80 mV, the current at
80 mV first was subtracted from
the total current at +80 mV (receptor current + voltage-gated
currents), leaving solely voltage-gated channel current. Receptor
currents at
80 and +80 mV are assumed to be equal because the anion
equilibrium potential is 0 mV (equal driving forces). Conductance then
could be calculated using a driving force of 170 mV (driving force for K+ at +80 mV;
90 mV equilibrium potential).
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When comparing muscimol receptor mediated effects on K+ channel conductance, all values were normalized to muscimol receptor current densities opposed to just cell capacitance. The rationale for normalizing in this manner is due to the fact that K+ conductance changes are linked directly to the size of the muscimol receptor current. Furthermore by normalizing to muscimol current density, one can rule out any variations in experimental conditions such as perfusion/diffusion speed, which would affect current size and desensitization rate.
Solutions
The composition of the different external solutions used is
listed in Table 1, and the internal
pipette solutions are listed in Table 2. Unless
otherwise mentioned, the cells always were incubated in normal
Cl containing solution and normal internal
solution was the pipette electrolyte. Kainic acid was obtained from
Sigma-Aldrich and picrotoxin and muscimol were purchased from Tocris
Cookson.
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Statistical methods
All values obtained are expressed as means ± SE. All statistical comparisons were performed using Excel software on an IBM compatible computer system. An unpaired two-tailed t-test, assuming equal variance, was performed on all series of data. Values of P < 0.05 were considered significant.
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RESULTS |
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Two effects of receptor activation
When cultured astrocytes in normal
Cl-containing solution were exposed to the
GABAA receptor agonist muscimol (200 µM), we consistently saw an increase in receptor inward conductance with a
concomitant longer lasting outward K+ conductance
decrease (Fig. 1). The peak conductance decrease always occurred with
the peak receptor inward conductance. The peak conductance blockade
averaged 35%, whereas the plateau conductance decrease averaged 15%
(Table 3; Fig. 1). The two effects of
GABAA receptor activation have been shown in
astrocytes of neonatal and adult hippocampal slices, demonstrating that
the effects are not a culture artifact (Bekar et al.
1999
). Figure 2 illustrates both
kainate and muscimol effects on cultured hippocampal glial cells in
normal Cl
-containing external and
Na+-free external solutions. In normal
Cl
-containing external solution (Fig.
3A), application of 200 µM kainate (10 ± 1.5 pA/pF,
n = 10) or 200 µM muscimol (56 ± 15 pA/pF, n = 33) results in a receptor current and a secondary
blockade of outward currents.
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Role of Na+
To test the involvement of Na+ in the secondary blockade of the two agonists, we tested the responses in Na+-free external solution: the two responses to kainate are abolished completely, whereas the responses to muscimol are not significantly different (Table 3). These results suggest that Na+ is not a major factor involved in the secondary blockade of outward currents linked to GABAA receptor activation (Fig. 2B).
Need for Cl pore opening
Figure 3, A and
B, shows two ways in which we isolated binding of muscimol
to the GABAA receptor. First (Fig.
3A), we applied 200 µM muscimol in the presence of 200 µM of the noncompetitive GABAA receptor
antagonist picrotoxin. Picrotoxin does not effect binding of muscimol
to the receptor but rather inserts itself into the receptor channel
pore, effectively blocking the Cl conductance.
Picrotoxin effectively blocks both effects of muscimol, demonstrating
dependence on Cl
conductance through the
channel for the secondary blockade. The second way we isolated muscimol
binding to the receptor (Fig. 3B) was by replacing
Cl
with gluconate in both the pipette filling
solution and the external superfusion solution, which is impermeable
for the GABA receptor channel. Muscimol will bind under these
circumstances and open the channel, but no current carrier is available
to generate a current flux as the result of the conductance change.
Again, muscimol binding under these conditions induces neither an anion
flux nor a decrease in K+ current, reinforcing
the hypothesis that the secondary blockade is linked directly or
indirectly to Cl
net flux through the receptor
channel pore out of the cell.
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[ATP]i dependency
That this action is not due to modulation of a second-messenger effect of muscimol is supported by the observation that removal of ATP from the pipette solution had no effect on the secondary blockade (Fig. 3C). Therefore not only is the blockade of outward current not dependent, solely, on muscimol binding to the receptor, but it is also not dependent on phosphorylating mechanisms.
Effect of changing external Cl concentrations
Figure 4A
demonstrates that the secondary blockade of outward current involves
inhibition of a barium-sensitive K+ current.
Application of 10 mm barium causes a decrease of the membrane
conductance from 0.67 ± 0.07 to 0.21 ± 0.05 nS/pF
(n = 5). Muscimol application in the presence of barium
results in an inward receptor current (73 ± 20 nA/pF,
n = 5) that is not different from controls without
barium (56 ± 15 nA/pF, n = 33). However, the
secondary blockard is abolished in barium-containing solution (Fig.
4A). Experiments involving the reduction of the Cl concentration from 130 to 30 mM in the
external solution displayed interesting results. This reduction
mimicked the action of barium, decreasing the outward current at
depolarizing potentials as shown by the I-V relationship in
Fig. 4B. The membrane conductance was reduced from 0.73 ± 0.09 to 0.29 ± 0.04 nS/pF (n = 5). Figure 4C illustrates how the secondary reduction of outward
current associated with increased efflux of Cl
is occluded by barium. These experiments suggest that decreasing efflux
of Cl
results in the inhibition of a
barium-sensitive K+ outward current and that a
Cl
conductance does not play a role under these
circumstances.
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Effect of replacing Cl with halide anions
If the secondary blockade of outward K+
channels is indeed a direct result of a decrease in the
Cl concentration, then replacement of
Cl
(both in the internal pipette solution and
in the external perfusion solution) with other halide anions (different
sizes) might show differing results. Furthermore the results should
agree with the anion selectivity isotherms proposed by Wright
and Diamond (1977)
. Figure 5
shows I-V relationships of cultured hippocampal astrocytes in three different salt solution. All three I-V
relationships were obtained in internal and external salt solutions
with symmetrical distribution of the major halide anion. The solutions
contained Cl
(chloride),
I
(iodide), or Br
(bromide) as the major anion. The I-V relationship obtained
in the Br
containing salt solution (Fig. 5) was
not significantly different from that in the Cl
containing solution. The I-V relationship in the
I
salt solution, however, was only
significantly different at +80 mV compared with the
Cl
solution. Results of the secondary
conductance blockade in response to muscimol application in the
different anion containing solutions are summarized in Table 3. When
comparing the percentage of peak conductance blockade normalized to
receptor current density (see METHODS),
Br
(0.40) and I
(0.38)
exhibited significantly larger blockade on the membrane outward
K+ conductance as compared with
Cl
ions (0.22).
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DISCUSSION |
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We investigated a long-lasting blockade of voltage-gated outward
K+ currents in cultured hippocampal rat
astrocytes after muscimol exposure in normal
Cl-containing external solution. This blockade
is identical to the one seen in adult and neonatal glial cells in situ
in external solutions buffered with bicarbonate (Bekar et al.
1999
; Muller et al. 1994
; Pastor et al.
1995
). It also is seen in cerebellar granule cells
(Labrakakis et al. 1997
).
The mechanism that connects this blockade with agonist binding to the
GABAA receptor has, to this date, remained
unknown. An analogous blockade associated with the ionotropic
AMPA/kainate receptors in glial cells is much better elaborated. These
AMPA/kainate receptors mediate a mixed
Na+/K+ current
(Seifert and Steinhauser 1995), resulting in increased intracellular Na+ concentrations.
Na+ itself has been found to be responsible for
the blockade of the outward K+ currents
(Borges and Kettenmann 1995
; Robert and
Magistretti 1997
). Our astrocytes exhibited such a
AMPA/kainate-evoked, Na+-dependent blockade of
the outward K+ channels, but the muscimol-induced
blockade was unaffected (Table 3) when Na+ was
removed from both internal and external solutions. Uptake of the
agonist cannot play a role because muscimol is not transported into the
cell (Kanner 1997
).
In symmetrical 130 mM Cl conditions
(ECl = 0 mV), the opening of the GABA
receptor channel would cause a Cl
efflux
(driving force of 80 mV). Cl
efflux is
necessary for blockade, suggesting that the blockade is dependent on a
decrease in the level of intracellular Cl
.
Muscimol binding alone without such a current is insufficient. Replacement of Cl
inside and outside the cell
with gluconate abolished both the receptor current and secondary
blockade. Direct blockade of Cl
efflux by
picrotoxin (Fraser et al. 1995
), prevented the secondary blockade and induction of efflux without affecting muscimol binding to
the GABAA receptor. Induction of
Cl
efflux without activating
GABAA receptors, through reduction of
extracellular Cl
mimicked the blockade. The
blockade is also independent of the presence of ATP in the pipette.
Thus the involvement of G proteins (unlikely in any event to play a
role in GABAA receptor activation) in the
secondary blockade of outward K+ currents is unlikely.
Evidence supporting the involvement of Cl ions
in the secondary blockade comes from experiments involving perfusion of
low [Cl
]o to the cell,
mimicking Cl
efflux changes that occur in
muscimol-receptor binding. Under these conditions, we saw a reduction
of outward currents without any effect on inward currents, similar to
the secondary response of muscimol. Furthermore reducing the
Cl
concentration in the presence of the broad
spectrum K+ channel blocker barium, no
significant effect on the membrane conductance was seen. This indicates
that Cl
efflux is inhibiting voltage-gated
outward K+ currents. Indeed the secondary
blockade caused by muscimol is occluded by barium, a blocker of a
number of K+ channels. The indication that a
reduction of the external Cl
concentration is
causing a significant Cl
efflux points to a
large Cl
conductance of the resting membrane, a
large carrier mediated efflux or both. Because we found no significant
change in the membrane conductance when the Cl
was reduced in the presence of barium, we have to exclude a significant Cl
conductance, at least under the
circumstances tested. Another possibility is the efflux via the
Na-K-Cl-Cl cotransporter, which is expressed by astrocytes and whose
mode of action depends on the combined driving forces of the
participating ions (Walz 1995
) and which would be
reversed by the reduction of external Cl
.
There is evidence for Cl-dependent
modulation of other physiological processes: Adams and Oxford
(1983)
showed that inorganic anions reduce and slow delayed
rectifying K+ channel conductance in the squid
giant axon. They found that F
,
Br
, and Cl
all
reversibly reduced outward K+ conductance
(Adams and Oxford 1983
). However, this effect of Cl
also might be explained by the formation of
a junction potential when changing
Cl
-containing solutions. Changing from 130 to
30 mM Cl
always shifted the membrane potential
more negative, as obtained in current-clamp mode on the EPC-7. As a
result, the voltage jump protocol may not depolarize the membrane to
the same extent, resulting in smaller outward currents relative to the
control situation. A strong argument against this hypothesis is that
the activation kinetics (normally around
40 mV) of the resulting
I-V relationship would appear more positive than control,
which was never the case. Furthermore the reversal potential of the
I-V relationship does not shift more positively,
demonstrating that the effect is solely on outward currents at
depolarized potentials more positive than 0 mV. If it was a result of a
junction potential, the I-V relationships would look
identical but shifted more positively. Similar studies in the squid
giant axon also showed that reduced or slowed K+
conductance by F
or Br
did not shift I-V relationships more positively
(Adams and Oxford 1993
).
Although there was no significant difference in resting membrane
currents, the secondary conductance decrease in response to muscimol is
significantly larger when both intra- and extracellular Cl is replaced by either
Br
or I
. Although
studies have shown anions to effect G-protein-mediated responses
(Higashijima et al. 1987
; Lenz et al.
1997
), Br
and I
in these studies always showed a lesser ability to affect G proteins than Cl
(Lenz et al. 1997
). In
contrast, the secondary response in our study shows an increase in size
during replacement of Cl
with
Br
or I
, suggesting,
again, that the secondary response is not dependent on
G-protein-mediated effects. The effects of the different halides in our
glial cell culture system display activity dependent characteristics (Br
= I
> Cl
; conductance decrease) consistent with
sequences 1 and 2 of the selectivity isotherms proposed by
Wright and Diamond (1977)
, indicating interaction of the
halide anions with a selective site, likely, on voltage gated outward
K+ channels.
Physiological significance
Our results suggest that the intracellular
Cl concentration is modulating the conductance
of outward K+ channels. In cultured astrocytes
with symmetrical Cl
distribution (130 mM inside
and outside), muscimol causes a decrease in the internal
Cl
concentration through
Cl
outflux. This decrease is large enough to
cause a 35% blockade of K+ outward conductances.
Because the same phenomenon is seen in astrocytes in situ (Bekar
et al. 1999
), it is probably not a cell culture artifact.
However, it is not clear under what conditions this mechanism is
playing a physiological role.
The normal physiological Cl concentration of an
astrocyte in situ and in vitro is probably between 30 and 40 mM
(Walz 1995
). At this Cl
concentration level, it is not clear how large the modulatory effect
will be because the outward driving force for
Cl
will be diminished.
It might well turn out that the largest changes seen in the internal
astrocytic Cl concentration are not due to GABA
binding but involve Cl
concentration increases
due to pathological swelling and spreading depression waves when
massive anion channel opening occurs (Phillips and Nicholson
1979
). Whatever the circumstances, we established that
intracellular changes in the Cl
concentration
per se have to be considered as influencing important physiological
processes. Because this secondary blockade also is seen in granular
cerebellar neurons, this mechanism might not be unique to astrocytes.
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ACKNOWLEDGMENTS |
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M. Lang is thanked for excellent technical assistance.
This project was supported by an operating grant from the Medical Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests: W. Walz, Dept. of Physiology, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, SK, S7N 5E5 Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 November 1998; accepted in final form 5 March 1999.
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REFERENCES |
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