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The Journal of Neuroscience, September 15, 1998, 18(18):7127-7137
Involvement of Stretch-Activated Cl
Channels in
Ramification of Murine Microglia
Claudia
Eder,
Rolf
Klee, and
Uwe
Heinemann
Department of Neurophysiology, Institute of Physiology, Humboldt
University, D 10117 Berlin, Germany
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ABSTRACT |
A stretch-activated Cl
current
(ICl) was investigated in cultured
murine microglia using the whole-cell configuration of the patch-clamp
technique. After application of membrane stretch, a
Cl current appeared within seconds, and its
amplitude increased further within 3-8 min.
ICl underwent rundown, which was prevented by addition of 4 mM ATP to the intracellular perfusing
solution. The stretch-activated Cl current
exhibited outward rectification and did not show any voltage-dependent
gating. Lowering the concentration of extracellular Cl from 142 to 12 mM by equimolar
substitution of Cl with gluconate shifted the
reversal potential of ICl by 41.6 ± 1.8 mV in the depolarizing direction.
4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and
4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS)
blocked ICl in a voltage- and time-dependent
manner. At a test potential of +40 mV, a half-maximal blockade at 16.1 µM DIDS and at 71.0 µM SITS was determined
for ICl. At a concentration of 200 µM, 5-nitro-2-(3-phenylpropylamino)benzoic acid or
flufenamic acid blocked ICl by 88% and
75%, respectively. Each of these four Cl channel
blockers reversibly inhibited the ramification process of microglia,
whereas blockers of voltage-gated Na+ and
K+ channels did not affect the transformation of
microglia from their ameboid into the ramified phenotype. It is
suggested that in microglia functional stretch-activated
Cl channels are required for the induction of
ramification but not for maintaining the ramified shape.
Key words:
brain macrophages; ramification; stretch-activated
Cl current; SITS; DIDS; NPPB; flufenamic acid
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INTRODUCTION |
Microglia, the macrophages of the
brain, can be distinguished morphologically into ameboid and ramified
cells. Ameboid microglia appear in immature brain during late prenatal
and early postnatal periods. These cells have round somata with short
lamellopodial extensions. In contrast, microglia of normal adult CNS
exhibit a ramified morphology, characterized by long secondary and
tertiary branched processes arising from both poles of an elongated
flattened cell body (Ling and Wong, 1993
). Within the brain, ramified
microglia are known to be in an immunologically resting state. During
inflammation and several neurological disorders, microglia become
activated; the activation process is correlated with a loss of the
ramified shape of the cells (for review, see Kreutzberg, 1996; Streit, 1996).
The origin of ameboid and ramified microglia is still a matter of
controversy (for review, see Ling and Wong, 1993). The transformation of ameboid into ramified microglia during development was first demonstrated by Ling (1979). In vitro studies have shown
that the induction of ramification of microglia appears to be
influenced by astrocytes. Thus, ameboid microglia differentiate into
ramified cells during cultivation on top of an astrocytic monolayer
(Sievers et al., 1994; Tanaka and Maeda, 1996) or in mixed
microglia-astrocyte cultures (Liu et al., 1994). However, direct
contact between microglia and astrocytes is not a prerequisite for
these processes, because ramification can also be induced in microglia
during treatment with astrocyte-conditioned medium (Eder et al.,
1997a). Although the astrocytic factors responsible for triggering
these shape changes in microglia have not been identified, the
cytokines macrophage colony-stimulating factor and
granulocyte/macrophage colony-stimulating factor seem to play an
important role during processes of microglial transformation from their
ameboid into the ramified phenotype (Liu et al., 1994; Fujita et al.,
1996).
During depolarization of the cell membrane, ramified microglia exhibit
a voltage-gated sodium current and a voltage-gated outward potassium
current (Korotzer and Cotman, 1992; Sievers et al., 1994; Eder et al.,
1996, 1997a) that are not seen in unstimulated ameboid microglia (for
review, see Eder, 1998). However, it is not clear whether
Na+ and outward K+ channels are
required for shape changes in microglia. Expression of outward
K+ channels that is not accompanied by shape changes
of the cells can also be induced in microglia by treatment with low
doses of the astrocyte-conditioned medium (Eder et al., 1997a).
In this study we investigated the effects of several ion channel
blockers on the induction of ramification in cultured murine microglia.
We suggest a participation of stretch-activated chloride channels in
the ramification process of microglia. Properties of the
stretch-activated chloride current were studied in detail.
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MATERIALS AND METHODS |
Cell culture. Microglia were obtained from brain cell
cultures of newborn NMRI mice supplied by Hamann-Winkelmann
(Borchen, Germany). Mixed brain cell cultures were prepared as
described previously (Eder et al., 1995). Brain cortices were
enzymatically dissociated (15 min at 37°C with 0.25% trypsin, type
XI; Sigma, Deisenhofen, Germany), and a single-cell suspension was
achieved by repeated triturations. Cells were seeded into tissue
culture flasks at a density of 2-4 × 106/5 ml
in DMEM (Life Technologies, Gaithersburg, MD) supplemented with
10% heat-inactivated fetal calf serum (FCS) (Life Technologies) and
30% supernatant of L-929 fibroblasts as a source of macrophage colony-stimulating factor. After at least 10 d of incubation, microglia were harvested by shaking the cultures (30 min, 300 rpm) to
detach weakly adherent cells from the astrocytic monolayer. Isolated
microglia were seeded on glass coverslips in 24-well Costar plates
(3 × 104/1 ml). Patch-clamp recordings were
performed 1-5 d after the isolation procedure. To induce ramification
of microglia, astrocyte-conditioned medium (ACM) was added to cell
cultures.
Astrocytes from murine cortices were cultured as described by Hertz et
al. (1982). Cells were seeded at a density of 3-4 × 105/ml and were grown in DMEM supplemented with 10%
FCS. After 2 weeks in culture, the concentration of FCS was lowered to
2%. The supernatants of astrocytic cultures were collected twice per week and used as ACM.
In some cases one of the following ion channel blockers was added to
ACM immediately before cells were treated with the ACM: 1-10
µM tetrodotoxin (TTX), 1 mM
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) (all from Sigma), 10-500 nM charybdotoxin
(CTX), 10-500 nM kaliotoxin (KTX) (both from Latoxan,
Rosans, France), 200 µM flufenamic acid, 200 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)
(both from Biotrend/RBI), and 1-4 µM chlorotoxin
(Alomone Labs, Jerusalem, Israel). The effects of each drug were
investigated in a minimum of four different cell cultures (four to
eight coverslips per culture).
Electrophysiological recordings. Membrane currents were
measured using the whole-cell configuration of the patch-clamp
technique (Hamill et al., 1981). Current recordings were made
with an EPC-7 patch-clamp amplifier (HEKA, Lambrecht/Pfalz,
Germany). Data were recorded with a CED 1401 interface (Cambridge
Electronic Design Ltd., Cambridge, UK) and stored on line using the
software VCLAMP 6.0 (Cambridge Electronic Design) on an
IBM-compatible computer for subsequent analyses. Data were sampled at
10 kHz and low-pass-filtered at 3 kHz using an 8-pole Bessel filter.
Series resistance compensation was routinely used to reduce the
effective series resistance by approximately 70%. Patch electrodes of
3-4 M
were fabricated on a two-stage puller (Narishige PP-83,
Tokyo, Japan) from borosilicate glass (outer diameter, 1.5 mm, and
inner diameter, 1 mm) (Hilgenberg, Malsfeld, Germany). For
Cl current recordings, the electrodes were filled
with the following solution (in mM):
N-methyl-D-glucamine chloride (NMGCl) 120, EGTA 11, CaCl2 1, MgCl2 2, HEPES 10, pH 7.35. The
osmolarity was adjusted to 280 mOsm with D-glucose. In some cases
NMG+ was substituted by K+.
Mostly, the intracellular solution contained 4 mM NaATP
(Sigma). The extracellular solutions contained (in mM):
NaCl 130, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, pH
7.35. To prevent spontaneous activation of swelling-induced
Cl currents in microglia, the osmolarity of the
extracellular solutions was adjusted to 290-295 mOsm with D-glucose.
In some experiments, NaCl was substituted by sodium gluconate
(measurements were corrected for junction potentials). To study
Cl currents in isolation, 1 mM
BaCl2 (blocker of inward rectifying K+
currents), 1 µM TTX (blocker of voltage-gated
Na+ currents), and 100 µM
LaCl3 (blocker of voltage-gated H+
currents) were routinely added to the extracellular solutions. At the
concentrations given, neither of these drugs influenced stretch-activated Cl currents of microglia (data
not shown). In a few experiments, voltage-gated outward
K+ currents were blocked by 100 nM
kaliotoxin (Latoxan). All recordings were performed at room temperature
(20-23°C). Data are presented as mean values ± SD of the
number of experiments indicated.
Mechanical stimulation. In most experiments, membrane
stretch was applied via the recording patch pipette. After the
whole-cell configuration was established, the pipette was moved by
5 ± 1 µm. Microglia in culture are tightly attached to the
glass coverslips, and movement of the pipette resulted in obvious
stretching of the membrane. Displacement of the pipette for >5 µm
did not significantly increase amplitudes of stretch-activated
currents. In a few experiments, a fire-polished pipette was mounted on
a second manipulator and used for mechanical stimulation according to
the method described by Hu and Sachs (1996). Briefly, the pipette was
pressed against the cell using either vertical or horizontal
displacement of the pipette, which was <5 µm.
Pharmacological studies. Drugs were applied using a
four-barrel microperfusion pipette, positioned at a distance of
~30-50 µm from the recorded cell to permit a rapid exchange of
solutions. The flow rate was adjusted by hydrostatic pressure.
We added 1 mM 4-aminopyridine, 5 × 107 to 1 × 103 M DIDS,
1 × 106 to 5 × 103 M SITS, 200 µM NPPB, or 200 µM flufenamic acid to the extracellular superfusing
solution. Stock solution of 500 mM NPPB and 500 mM flufenamic acid (both dissolved in DMSO) were prepared
and stored frozen at 
20°C. Chlorotoxin (1 µM) was
dissolved in a 0.1% bovine serum albumin containing extracellular
solution.
The concentration-response curves for DIDS and SITS were approximated
using a Hill equation of the following form:
I[X] = Imax[X]n/([X]n + IC50n), in which
I[X] is the current amplitude during
superfusion with the drug, Imax is the maximal
current amplitude (before superfusion with the drug), n is
the Hill coefficient, and IC50 is the dissociation constant
that determines the concentration of the drug for a half-maximal current inhibition.
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RESULTS |
Induction of stretch-activated
Cl currents
Voltage ramps from 100 to +90 mV were applied for a duration of
400 msec every 10 sec. The holding potential was set to 60 mV. Using
a high K+-containing intracellular perfusing
solution that did not contain ATP, voltage-gated K+
currents were obtained in both ameboid and ramified microglia immediately after establishment of the whole-cell configuration. Figure
1A illustrates current
recordings of a microglial cell that expressed both inward and outward
rectifier K+ currents. Inward rectifier
K+ currents were observed at potentials negative to
75 mV, whereas outward K+ currents were detectable
at potentials positive to 30 mV. The distribution of inward and
outward K+ currents in murine microglia at various
functional states as well as kinetic and pharmacological properties of
these voltage-gated K+ currents has been
described elsewhere (for review, see Eder, 1998).
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Figure 1.
Induction of stretch-activated currents.
A, Current recordings under control (before stretching
of the membrane) and after application (3 and 7 min) of a membrane
stretch using a K+-containing intracellular
solution. B, Stretch-activated current isolated by
substraction of the control trace from the trace measured 3 min after
the stretch (shown in A). C, Time course
of induction of the stretch-activated current for the example shown in
A. Changes in amplitude (measured at +90 mV) are plotted
against time after establishment of the whole-cell configuration.
Arrows indicate the time at which the cell was stretched
by moving the patch pipette.
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Stretching of the cell membrane was induced by a movement of the
recording patch pipette by a few micrometers. A membrane stretch
resulted in the activation of an additional ionic current in both
ameboid and ramified microglia (Fig. 1A,B). In a few
experiments, stretching of the cell membrane was induced by a second
fire-polished patch pipette. No differences were seen between currents
evoked by both methods with respect to the time course of induction, kinetics, and pharmacological properties of the currents (see below).
As shown for the example in Figure 1, stretch-activated currents
activated very slowly. The amplitude of the currents increased steadily
during the first 3-8 min after moving of the patch pipette. However,
stretch-activated currents did not reach a steady state; rather the
currents decreased and disappeared almost completely within 7-15 min
after the membrane stretch. In contrast, recordings of voltage-gated
inward and outward K+ currents were stable for more
than 1 hr and did not show any kind of rundown. In Figure
1C, the time course of current induction is illustrated for
the example shown in Figure 1A. Amplitudes of the
current were measured at a potential of +90 mV. As shown in Figure
1C, in some cases stretch-activated currents could be activated again after their disappearance by application of a second
membrane stretch. However, currents induced by a second stimulus mostly
did not reach the amplitude of currents activated by a first stretch,
and they also disappeared within a few minutes. Currents could only
rarely be induced by a third membrane stretch. No differences were
detected in the time course of current induction and rundown of
stretch-activated currents between ameboid (n = 26) and
ramified (n = 14) microglial cells.
Martin et al. (1995) described a stretch-activated
K+ current in monocyte-derived macrophages that was
completely blocked by 1 mM 4-aminopyridine (4-AP). To
clarify whether a membrane stretch activates a similar
K+ current in microglia, we investigated the effect
of external 4-AP on stretch-activated currents in microglia. In these
experiments, voltage-gated outward K+ currents were
blocked by 100 nM KTX. The remaining stretch-activated current was unaffected by extracellular application of 1 mM
4-AP (n = 11; data not shown). In further experiments,
intracellular potassium ions were substituted by
NMG+. In ameboid and ramified microglia,
stretch-activated currents could also be evoked using
K+-free intracellular solutions (Fig.
2A). The
K+-free intracellular solution was used in further
studies. Moreover, to block inward rectifier K+
currents the extracellular solution contained 1 mM
BaCl2. Additionally, voltage-gated H+
currents of microglia were routinely inhibited by 100 µM
LaCl3, and voltage-gated Na+
currents were blocked by 1 µM TTX. Because
stretch-activated currents were not affected after the addition of
BaCl2 (n = 8), LaCl3
(n = 10), or TTX (n = 8) to the
extracellular superfusing solution (data not shown), these currents
could be investigated in isolation. In experiments using
K+-free intracellular solutions, cells were held at
10 mV (a potential close to the equilibrium potential for
Cl) to prevent a continuous flux of
Cl. Voltage ramps were applied from 120 to +90
mV for a duration of 400 msec every 10 sec.
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Figure 2.
Recordings of stretch-activated currents using a
K+-free intracellular solution. A,
Induction of the currents. Time (in minutes) after the membrane stretch
is indicated (c, control measurement before application
of the membrane stretch). B, Current recording in the
presence of 142 mM Cl
(Cl) or after lowering concentration of
extracellular Cl to 12 mM by equimolar
substitution with gluconate (gluconate).
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To test the selectivity of the current that was activated in microglia
in response to a membrane stretch, the concentration of extracellular
Cl ions
([Cl]o) was lowered from 142 mM to 12 mM. As shown in Figure
2B, lowering [Cl]o
by equimolar substitution with gluconate evoked a shift of the reversal
potential of the stretch-activated current by 41.6 ± 1.8 mV
(n = 8) to more positive potentials as well as a
reduction of current amplitude. Thus, it appears that the
stretch-activated current of microglia is carried mainly by chloride
ions.
Effect of intracellular ATP
An influence of intracellular ATP on Cl
channels, namely an induction of current activation (Gill et al., 1992)
or an inhibition of current rundown (Lewis et al., 1993), has been
described in several cell preparations (for review, see Strange et al.,
1996). In the presence of 4 mM intracellular ATP,
Cl currents did not appear spontaneously in murine
microglia. A small leak current was the only conductance detected when
no stretch was applied to the cell membrane (Fig.
3A). After stretching the membrane, Cl currents progressively increased in
amplitude, reaching an apparent plateau within 4-8 min. The rundown of
the stretch-activated Cl current
(ICl) observed without ATP was not
observed in cells perfused with a 4 mM ATP pipette
solution. ICl values reached their maximal
amplitudes and did not change further within recordings of >1 hr.
Figure 3A illustrates the development of
Cl currents with time after stretching of the cell
membrane using an ATP-containing pipette solution. The time course of
the changes in amplitudes of ICl is shown in
Figure 3B, whereas amplitudes were determined at a potential
of +90 mV.
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Figure 3.
Dependence of stretch-activated
Cl currents on intracellular ATP.
A, Current recordings during perfusion of the cell with
4 mM ATP. Currents are shown at 1 and 10 min after
establishment of the whole-cell configuration before the cell was
stretched (c) and at several times (in minutes)
after application of the membrane stretch. B,
Corresponding amplitudes of the current shown in A
(measured at +90 mV) are plotted against time after break-in. Membrane
stretch is indicated by the arrow.
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Pharmacology of stretch-activated
Cl currents
Several chloride channel blockers were tested for their ability to
block stretch-activated Cl currents of microglia.
Voltage commands were applied from the holding potential of 10 mV for
a duration of 400 msec. Cells were either depolarized to potentials
between 10 and +80 mV or hyperpolarized to potentials between 10
and 110 mV. Under control conditions (Fig.
4A), stretch-activated
Cl currents did not show any time-dependent
activation or inactivation behavior at all test potentials. The
stilbene disulfonate DIDS reduced the currents in the
nanomolar-micromolar concentration range. As shown in Figure 4, 1 mM DIDS applied extracellularly abolished stretch-activated
Cl currents (Fig. 4D), whereas
DIDS applied at lower concentrations induced a time- and
voltage-dependent block of the currents (Fig. 4B,C).
At hyperpolarizing potentials the block of ICl
by DIDS was less pronounced than at depolarizing potentials. To
determine concentration-response relationship for DIDS, the drug was
applied extracellularly to the cells at concentrations between 5 × 107 and 103 M. Amplitudes
of Cl currents evoked by a test pulse from the
holding potential of 10 mV to a potential of +40 mV were measured at
the end of the pulse where currents reached steady state. Current
amplitudes were measured and normalized to the maximal current
amplitudes for each cell, which were measured under control conditions
before application of DIDS. A concentration-response curve was fitted by the Hill equation and a half-maximal effective concentration (IC50 value) of 16.1 µM DIDS (Hill
coefficient 1.1) was determined for the stretch-activated
Cl current of microglia (n = 9)
(Fig. 4E). The inhibitory effects of DIDS were
reversible during washout.
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Figure 4.
Effect of extracellularly applied DIDS on
ICl. Measurements of stretch-activated
Cl currents before (A) and
during superfusion of the cells with an extracellular solution
containing 50 µM (B), 100 µM (C), or 1 mM
(D) DIDS. E,
Concentration-response curve for DIDS. At each DIDS concentration the
percentage of current inhibition was determined in at least nine
cells.
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The inhibition of ICl by extracellularly applied
SITS is demonstrated in Figure 5.
Cl currents were blocked by SITS in a similar
voltage- and time-dependent manner as observed for the effects of DIDS
on ICl. To determine the concentration-response
relation for block of stretch-activated Cl current
by SITS, cells were superfused with SITS at concentrations between
106 and 5 × 103 M. An
IC50 value of 71.0 µM SITS (Hill coefficient
1.0) was calculated from effects of SITS on amplitudes of the
steady-state current evoked by a test pulse from 10 mV to +40 mV
(n = 8) (Fig. 5C).
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Figure 5.
Blockade of stretch-activated
Cl currents by extracellularly applied SITS.
A, Example of current recording using a voltage-step
protocol while superfusing the cell with control solution
(A) or a solution containing 50 µM
SITS (B). C,
Concentration-response curve for SITS (n = 8 for
each concentration).
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The effects of DIDS and SITS were also investigated using
K+-containing intracellular solutions. In these
experiments, 100 nM KTX was added to the superfusing
solution to block voltage-gated outward K+ currents.
Application of either 1 mM DIDS (n = 6) or
5 mM SITS (n = 5) completely blocked
stretch-activated currents (data not shown), suggesting that microglia
do not additionally express stretch-activated K+
currents.
Block of chloride currents by NPPB and flufenamic acid appeared to be
time- and voltage-independent (Fig.
6A,B). At a
concentration of 200 µM, NPPB reduced amplitudes of
ICl by 87.5 ± 2.4% (n = 6), whereas flufenamic acid diminished amplitudes of
ICl by 75.3 ± 7.1% (n = 6). The blocking effects of NPPB and flufenamic acid on
ICl were also reversible during prolonged
washout of the drugs.
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Figure 6.
Effects of NPPB, flufenamic acid, and chlorotoxin
on stretch-activated Cl currents.
A, Cl current measurement in the
absence and presence of 200 µM NPPB. B,
Measurements of stretch-activated Cl currents
before and during superfusion of the cells with 200 µM
flufenamic acid. C, Stretch-activated
Cl currents in the absence and presence of 1 µM chlorotoxin.
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It has been reported previously that Cl currents
in glioma cells are potently inhibited on superfusion with chlorotoxin
(Ullrich and Sontheimer, 1996; Ullrich et al., 1998). In contrast,
stretch-activated Cl currents of microglia were
unaffected by extracellular application of 1 µM
chlorotoxin. Neither kinetics (n = 6; data not
shown) nor amplitude (n = 6) (Fig. 6C) of
ICl was changed by the toxin.
Ramification of microglia
Cultured murine microglia were able to undergo dramatic shape
changes after exposure to ACM. Figure 7
shows examples of microglia that had been cultured either in normal
culture medium (untreated) or in ACM. Untreated microglia exhibited an
ameboid morphology during the whole time after isolation of the cells
from the astrocytic monolayer (Fig. 7A). In contrast,
addition of ACM to the cells evoked a transformation of microglia from
their ameboid into a ramified phenotype within a few hours. After
treatment with ACM, microglia exhibited long branched processes
with lamellopodial tips as shown in Figure 7B,C. These
ramified cells could be easily distinguished from ameboid cells by
visual inspection. Ramified microglia were defined as cells with
distinct processes at least greater than one cell diameter in length,
as described previously (Korotzer and Cotman, 1992). In the presence of
ACM, microglia retained their ramified morphology with time in culture.
In contrast, ramified cells returned to their ameboid morphology by
washing out ACM.
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Figure 7.
Cultures of ameboid and ramified murine microglia.
A, Untreated cultured microglia exhibited an ameboid
phenotype. B, C, Examples of ramified microglia that had
been treated with astrocyte-conditioned medium for 24 (B) or 48 (C) hr. Cells
were examined with a Zeiss Axioskop equipped with differential
interference optics.
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It has been demonstrated in previous studies that an expression of both
voltage-gated sodium currents and voltage-gated outward potassium
currents was induced in ramified microglia that had been co-cultured
with astrocytes (Sievers et al., 1994). To test a possible involvement
of either of these channels in the induction of ramification of
microglia, the effects of specific blockers of these channels were
investigated. TTX, a blocker of voltage-gated Na+
currents, was added to the astrocyte-conditioned medium at
concentrations between 1 and 10 µM. TTX did not influence
ramification of microglia. As shown in Figure
8A,B, microglia showed
the same level of ramification as microglia treated with TTX-free
ACM 24 hr after treatment with TTX-containing ACM.
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Figure 8.
Effect of TTX and KTX on microglia. Ameboid
microglia were treated with ACM (A, C) and
TTX-containing (B) or KTX-containing
(D) ACM. Twenty-four hours after that treatment,
microglia exhibited a ramified morphology in both toxin-free and
toxin-containing ACM (A, same culture as in
B; C, same culture as in
D).
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Microglial ramification could also not be inhibited by the addition of
the potassium channel blockers KTX (Fig. 8C,D) or CTX (data
not shown) to ACM at concentrations between 10 and 500 nM. No differences in the phenotype were detected between microglia cultured with ACM either in the presence or in the absence of these
K+ channel blockers.
Effects of Cl channel blockers on ramification
of microglia
To investigate the role of stretch-activated
Cl channels in the process of ramification in
microglia, the Cl channel blockers DIDS, SITS,
NPPB, or flufenamic acid were added to the astrocyte-conditioned
medium. In the presence of either 1 mM DIDS or 1 mM SITS, ramification of microglia could not be induced. As
demonstrated in Figure 9, 24 hr after the
addition of DIDS- or SITS-containing ACM, microglia retained their
ameboid phenotype (Fig. 9A,C). Twenty-four hours after the
application of DIDS- or SITS-containing ACM, microglia were washed
several times with culture medium and treated for another 24 hr with
ACM that did not contain any Cl channel blocker.
Under these conditions, a transformation from their ameboid into a
ramified phenotype was induced in microglia as illustrated in Figure
9B,D. These microglial cells did not significantly differ in
their morphology from microglia that had been treated for 24 or 48 hr
exclusively with ACM. The level of ramification, i.e., the number and
size of processes and lamellopodial tips, was not quantified in our
study.
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Figure 9.
Effect of DIDS and SITS on ramification of
microglia. A, Addition of 1 mM DIDS to ACM
prevented the transformation of ameboid into ramified microglia.
B, After washout of DIDS-containing ACM, the
ramification of microglia was induced in the presence of ACM.
C, SITS (1 mM) applied to ACM inhibited
ramification of microglia. D, Microglia ramified in the
presence of ACM after washout of SITS.
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NPPB or flufenamic acid tested at a concentration of 200 µM also inhibited ramification of microglia, as shown in
Figure 10A,C. Microglia treated with NPPB- or flufenamic acid-containing ACM showed
an ameboid phenotype similar to that of untreated microglia. The
inhibitory effect of these Cl channel blockers on
ramification of microglia was also reversible. As demonstrated in
Figure 10B,D, after washout of NPPB- or flufenamic acid-containing ACM, microglia ramified in the presence of ACM to an
extent similar to that of ACM-treated microglia that had not been
pretreated with either of these Cl channel
blockers.
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Figure 10.
Inhibition of ramification of microglia by NPPB
and flufenamic acid. Cells were treated with ACM that contained 200 µM NPPB (A) or 200 µM
flufenamic acid (C). B, Microglia
exhibited a ramified morphology 24 hr after substitution of
NPPB-containing ACM with NPPB-free ACM. D, Ramified
microglia 24 hr after washout of flufenamic acid in the presence of
ACM.
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In further experiments, microglia were exposed to ACM containing either
1 mM DIDS, 1 mM SITS, 200 µM
NPPB, or 200 µM flufenamic acid for a duration of >24
hr. Microglial cells investigated for up to 5 d also did not
change their ameboid morphology in the continuous presence of the
Cl channel blockers (data not shown), suggesting
that these blockers did not delay morphological changes but prevented
ramification of microglia.
In contrast to DIDS, SITS, NPPB, and flufenamic acid, chlorotoxin did
not inhibit the process of ramification when added at concentrations of
between 1 and 4 µM to the ACM. Microglial cells exposed
to chlorotoxin-containing ACM ramified in a manner similar to microglia
exposed to chlorotoxin-free ACM (data not shown).
The effects of Cl channel blockers were also
investigated in ramified microglia that had been treated first with ACM
for 24 hr before Cl channel blockers were added to
the ACM. Ramified microglia were not influenced by each of the
Cl channel blockers (data not shown). Neither 1 mM DIDS-containing ACM nor 1 mM SITS-containing
ACM evoked changes in morphological characteristics of ramified
microglia. Similarly, microglia retained their ramified phenotype
after the addition of either 200 µM NPPB or 200 µM flufenamic acid to the ACM.
| |
DISCUSSION |
Properties of stretch-activated Cl currents
in microglia
Stretching of the cell membrane activated an outwardly rectifying
current in microglia, the macrophages of the brain. The existence of
stretch-activated ion channels in macrophages has also been
demonstrated by Martin and coworkers (1995). The stretch-activated K+ current described in monocyte-derived macrophages
also exhibited an outwardly rectifying behavior (Martin et al., 1995).
However, in contrast to observations made in monocyte-derived
macrophages, substitution of intracellular K+ by
NMG+ did not alter stretch-activated currents in
microglia. Moreover, stretch-activated currents of monocyte-derived
macrophages and of microglia differ also in their pharmacological
profile (see below), suggesting the presence of different types of
stretch-activated ion channels in these two types of macrophages.
In mammalian cells, a wide variety of stretch-activated ion channels
has been described, namely nonselective ion channels, in which current
is carried by several cations and channels that are selective to
either potassium or chloride ions (for review, see Morris, 1995).
Because in microglia reduction of extracellular Cl
ions resulted in a decrease in amplitude and a large shift of the
reversal potential of stretch-activated currents to more depolarized potentials, these stretch-activated currents are mainly carried by
chloride ions.
Chloride currents of microglia activated slowly with time after
stretching of the cell membrane. Presumably, membrane stretch does not
directly induce Cl current activation in
microglia, because the currents reached their maximal amplitudes only
several minutes after the initial application of the stretch. It is
more likely that a mechanical deformation of the cell membrane triggers
some internal signals that are required for Cl
channel opening.
Stretch-activated Cl currents of microglia showed
a rundown immediately after reaching their maximal amplitudes. The
rundown of the current was prevented by addition of ATP to the
intracellular solution. A similar dependence on intracellular ATP has
been described for Cl channels of several cell
preparations (for review, see Strange et al., 1996). However, in
contrast to observations in NIH/3T3 fibroblasts (Gill et al., 1992) and
pancreatic duct cells (Verdon et al., 1995), ATP was not required to
induce Cl current activation in microglia.
At least four different Cl channel blockers
potently inhibited stretch-activated Cl currents
of microglia. NPPB and flufenamic acid inhibited ICl in a
time- and voltage-independent manner, whereas DIDS and SITS evoked a
time- and voltage-dependent blockade of the current, with the deepest
block seen at depolarizing potentials. This finding together with the
IC50 values for current blockade are in good agreement with
data reported for Cl currents in T lymphocytes and
osteoclasts (Lewis et al., 1993; Steinert and Grissmer, 1997). In
contrast to observations made for Cl currents in
oocytes (Ackerman et al., 1994), extracellularly applied
La3+ did not influence Cl
currents in microglia and could therefore be used to inhibit voltage-gated outward proton currents of the cells (Eder et al., 1995).
In previous studies, Cl channels have been
detected in rat (Visentin et al., 1995; Schlichter et al., 1996),
bovine (McLarnon et al., 1995), and human (McLarnon et al., 1997)
microglia. It is difficult to compare properties of
Cl currents described in the present paper with
those of bovine and human microglia, because Cl
channels observed in these preparations were measured exclusively in
excised patches, whereas corresponding whole-cell currents were not
detected (McLarnon et al., 1995, 1997). In agreement, Cl currents were also not seen in whole-cell
measurements of murine microglia when no stretch was applied to the
cell membrane. However, McLarnon and coworkers (1995, 1997) reported an
inactivation of Cl currents that became faster
with increased cell depolarization, whereas we did not observe any
time-dependent inactivation of the currents at test potentials up to
+80 mV. Stretch-activated Cl currents of murine
microglia share similarities with Cl currents that
appeared spontaneously or after cell swelling in rat microglia
(Visentin et al., 1995; Schlichter et al., 1996). These outwardly
rectifying currents also do not show any voltage-dependent gating, and
they are sensitive to the Cl channel blockers NPPB
and flufenamic acid. Thus, it might be possible that in microglia the
same type of Cl channels can be activated by
osmotic or mechanical stimuli. It has been shown for T
lymphocytes that the activation of mini Cl
channels, which was seen in the presence of either hypo-osmotic external or hyperosmotic internal solutions, could also be induced by
application of positive pressure to the cell membrane via the patch
pipette (Lewis et al., 1993). In contrast, distinct ion channels became
activated in chick heart cells by either direct mechanical strain or
hypotonic swelling (Hu and Sachs, 1996).
With respect to their kinetic and pharmacological properties,
stretch-activated Cl currents of microglia closely
resemble Cl currents that activated either
spontaneously or in response to osmotic stress in other types of immune
cells (for review, see Gallin, 1991; DeCoursey and Grinstein,
1998), including monocytes (Kim et al., 1996), monocyte-derived
macrophages (Nelson et al., 1990), neutrophils (Stoddard et al., 1993),
and lymphocytes (for review, see Garber and Cahalan, 1997).
Functional role of stretch-activated Cl
currents in microglia
In this study we demonstrate an involvement of chloride channels
in ramification of microglia. At least four distinct chloride channel
blockers reversibly inhibited the transformation of microglia from
their ameboid into the ramified shape. Because ramified microglia were
not affected by Cl channel blockers, we conclude
that Cl channels are required for the induction of
ramification, but they are less important for maintaining morphology of
microglia. Membrane stretch during sprouting of processes might be the
trigger for Cl channel activation in microglia.
Moreover, because morphological changes of microglia are accompanied by
cytoskeletal reorganization (Abd-El-Basset and Fedoroff, 1995; Ilschner
and Brandt, 1996), Cl channels might also become
activated or their rate of activation might be increased during the
process of cytoskeletal reorganization. It has been demonstrated that
disruption of F-actin leads to Cl channel
activation in astrocytes (Lascola and Kraig, 1996) and astrocytoma
cells (Ullrich and Sontheimer, 1997) and potentiates the rate of
Cl channel activation under hypo-osmotic
conditions in B lymphocytes (Levitan et al., 1995; Garber and Cahalan,
1997). Further investigations will be required to clarify whether
Cl currents in microglia can be activated or
modulated by cytoskeletal disruptive agents.
A functional role of Cl currents in microglia
could also be related to changes in membrane potential. Conceivably, a
shift of the resting membrane potential to more positive values near the Cl reversal potential might be a prerequisite
for the induction of ramification in microglia. Membrane depolarization
induced by Cl channel activation could lead to
additional activation of other ion channels in microglia, e.g.,
voltage-gated K+, H+, or
Ca2+ channels (for review, see Eder, 1998). It is
thus possible that Ca2+ influx via voltage-gated
Ca2+ channels triggers
Ca2+-dependent intracellular processes necessary for
microglial shape changes.
It is also possible that an activation of Cl
channels in microglia is required for triggering tyrosine
phosphorylation signaling pathways as has been demonstrated for T
lymphocytes (Phipps et al., 1996). It has been shown that tyrosine
phosphorylation is induced during the process of ramification in
microglia. Thus, herbimycin A can inhibit the induction of ramification
(Liu et al., 1994). Furthermore, in the presence of the tyrosine kinase inhibitor genistein, ramified microglia shortened their processes (Tanaka and Maeda, 1996).
For other types of immune cells, an involvement of
Cl channels in regulatory volume decrease has been
proven by several researchers (for review, see DeCoursey and Grinstein,
1998). Volume regulatory processes could also be involved in microglial
ramification. Thus, cell volume of microglia might be adjusted by
chloride efflux via stretch-activated Cl channels
followed by extrusion of water.
It is still unclear why voltage-gated Na+ and
K+ channels become upregulated in ramified
microglia. It has been shown that in ramified microglia voltage-gated
Na+ currents can be effectively blocked by TTX
(Korotzer and Cotman, 1992), and voltage-gated outward
K+ currents are highly sensitive to CTX and KTX
(Eder et al., 1996). However, in the presence of each of these
blockers, microglia were also able to ramify, suggesting that neither
Na+ nor voltage-gated outward K+
currents are required for the regulation of shape changes in microglia.
Because voltage-independent Ca2+-activated
K+ channels of microglia are inhibited by CTX in a
concentration range similar to that of voltage-gated outward
K+ channels (Eder et al., 1997b), it is also
unlikely that these channels play an important role during processes of
ramification in microglia. An investigation of the role of inward
rectifier K+ channels and outward
H+ channels in microglia during processes of
ramification is more difficult, because so far no specific inhibitor
exists for each of these channels.
| |
FOOTNOTES |
Received March 13, 1998; revised June 26, 1998; accepted June 30, 1998.
This work was supported by a fellowship of the Alexander von Humboldt
Foundation (C.E.) and Grant SFB 507/C3 of the Deutsche Forschungsgemeinschaft (C.E., U.H.). We thank Sieglinde Latta for the
excellent preparation of cell cultures and Astrid Düerkop for
technical assistance. We are very grateful to Dr. Thomas E. DeCoursey
for many helpful discussions and comments on this manuscript.
Correspondence should be addressed to Claudia Eder, Department of
Neurophysiology, Institute of Physiology, Humboldt University, Tucholsky Strasse 2, D 10117 Berlin, Germany.
| |
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Top
Abstract
Introduction
