Department of Neurobiology, University of Alabama, Birmingham, Alabama 35294
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bordey, Angélique and
Harald Sontheimer.
Differential Inhibition of Glial K+ Currents by 4-AP.
J. Neurophysiol. 82: 3476-3487, 1999.
Spinal cord astrocytes express four biophysically and
pharmacologically distinct voltage-activated potassium (K+)
channel types. The K+ channel blocker 4-aminopyridine
(4-AP) exhibited differential and concentration-dependent block of all
of these currents. Specifically, 100 µM 4-AP selectively inhibited a
slowly inactivating outward current (KSI)
that was insensitive to dendrototoxin (10 µM) and that activated at
50 mV. At 2 mM, 4-AP inhibited fast-inactivating, low-threshold (
70
mV) A-type currents (KA) and sustained,
TEA-sensitive noninactivating delayed-rectifier-type currents
(KDR). At an even higher concentration (8 mM), 4-AP additionally blocked inwardly rectifying, Cs+-
and Ba2+-sensitive K+ currents
(KIR). Current injection into
current-clamped astrocytes in culture or in acute spinal cord slices
induced an overshooting voltage response reminiscent of slow neuronal
action potentials. Increasing concentrations of 4-AP selectively
modulated different phases in the repolarization of these glial spikes,
suggesting that all four K+ currents serve different roles
in stabilization and repolarization of the astrocytic membrane
potential. Our data suggest that 4-AP is an useful, dose-dependent
inhibitor of all four astrocytic K+ channels. We show that
the slowly inactivating astrocytic K+ currents, which had
not been described as separate current entities in astrocytes,
contribute to the resting K+ conductance and may thus be
involved in K+ homeostatic functions of astrocytes. The
high sensitivity of these currents to micromolar 4-AP suggests that
application of 4-AP to inhibit neuronal A-currents or to induce
epileptiform discharges in brain slices also may influence astrocytic
K+ buffering.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three distinct K+ current
types, which are voltage dependent but Ca2+
independent, have been identified previously and characterized in
spinal cord (Chvatal et al. 1995; Macfarlane and
Sontheimer 1997
; Ransom and Sontheimer 1995
;
Sontheimer et al. 1992
) and hippocampal astrocytes
(Bordey and Sontheimer 1997
; Steinhauser et al.
1992
) by whole cell recordings. Two of them are outwardly rectifying and activate by membrane depolarizations. Pharmacologically and kinetically they resemble transient A currents
(KA) and sustained delayed-rectifying
K+ currents
(KDR), respectively (Bordey and
Feltz 1995
; Macfarlane and Sontheimer 1997
;
Sontheimer et al. 1992
). The third
K+ current is an inwardly rectifying
(KIR) current that is active at the
resting membrane potential and that increases with voltages more
negative then the resting potential (Ransom and Sontheimer 1995
; Ransom et al. 1996
). These three currents
appear to be expressed abundantly in rat, mouse and human astrocytes
(for review, see Barres 1991a
,b
; Ritchie
1992
; Sontheimer 1995
) and have been recorded in
culture (Barres et al. 1988
; Bevan and Raff
1985
; Bevan et al. 1987
; Glassmeier et
al. 1994
; Nowak et al. 1987
; Ransom and Sontheimer 1995
; Sontheimer et al. 1992
;
Steinhauser et al. 1994
; Tse et al. 1992
)
and acute slices (Akopian et al. 1997
; Bordey and
Sontheimer 1997
; Chvatal et al. 1995
;
Sontheimer and Waxman 1992
; Steinhauser et al.
1992
, 1994
) including slices from human biopsy tissues
(Bordey and Sontheimer 1998a
,b
; Labrakakis et al. 1997
; Patt et al. 1996
; Ullrich et al.
1997
). Differential sensitivity of these currents to various
K+ channel blockers, including tetraethylammonium (TEA),
4-aminopyridine (4-AP), cesium, and barium, has been reported. This
includes the demonstration that millimolar concentrations of 4-AP can
inhibit outward K+ currents in astrocytes (Bordey
and Sontheimer 1997
; Clark and Mobbs 1994
;
Nowak et al. 1987
; Sontheimer et al.
1992
; Tse et al. 1992
). However, a detailed
pharmacological evaluation of the action of 4-AP on astrocytic
K+ channels is currently lacking.
4-AP has long been the standard drug to inhibit transient "A
currents," which were first described in molluscan neurons
(Connor and Stevens 1971b; Hagiwara et al.
1961
; Neher 1971
; Thompson 1977
)
and which have since been characterized extensively in neurons (Andreasen and Hablitz 1992
; Connors et al.
1982
; Everill et al. 1998
; Fisher and
Bourque 1998
; Hlubek and Cobbett 1997
; Li
and McArdle 1997
; Locke and Nerbonne 1997
;
Segal and Barker 1984
; Song et al. 1998
;
Storm 1990
; Zhou and Hablitz 1996b
) and
in numerous inexcitable cells (Baker et al. 1993
;
Brockhaus et al. 1993
; Gustafsson et al.
1982
; Howe and Ritchie 1988
; Kettenmann
and Ilschner 1993
; Lewis and Cahalan 1995
;
Mathie et al. 1998
; Wilson and Chiu
1990
). Theses currents inactivate rapidly (
t between 15 and
50 ms) and have an activation threshold between
60 and
75 mV. They
appear important in modulating the interspike interval and hence the modulation of high-frequency neuronal discharge (Connor and
Stevens 1971a
). From studies of recombinant K+
channels, it is now clear that currents resembling A currents can be
mediated by a number of cloned K+ channel subunits,
including Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3 (Baldwin et al.
1991
; Schroter et al. 1991
; Serodio et
al. 1994
, 1996
; Stuhmer et al. 1989
). In
addition, inactivating currents also can be obtained when
1 subunits
are coexpressed with subunits of the Kv1.x family that normally have
delayed rectifier properties (Heinemann et al. 1996
;
Rettig et al. 1994
). Despite the apparent heterogeneity
at the molecular level, most A currents share a relative sensitivity to
micro- or millimolar concentrations of 4-AP. Functionally, a large body
of literature implicates 4-AP-sensitive currents in the modulation of
high-frequency neuronal discharge (Connor and Stevens
1971a
; Golowasch et al. 1992
; Locke and
Nerbonne 1997
; Luthi et al. 1996
; Rudy
1988
; Segal et al. 1984
; Spigelman et al.
1992
; Wu and Barish 1992
; Zhang and
McBain 1995
; Zhou and Hablitz 1996
) as this was
originally proposed for molluscan neurons (Connor and Stevens
1971a
). In addition some studies propose that 4-AP also can
affect repolarization kinetics and thus alter spike waveform
(Bossu et al. 1996
; Locke and Nerbonne
1997
; Myers 1998
; Wu and Barish
1992
; Zhang and McBain 1995
).
The role of 4-AP-sensitive K+ currents in glial cells is
less clear. Glial K+ channels are believed to participate
in K+ homeostasis and may play a role in cell
proliferation/cell differentiation (Chiu and Wilson
1989; Dubois and Rouzaire-Dubois 1993
;
Puro et al. 1989
; Sontheimer 1995
;
Walz 1989
). Specifically, 4-AP has been shown to inhibit
cell proliferation of hippocampal astrocytes (Pappas et al.
1994
) but also other inexcitable cells including macrophages
and lymphocytes (Gallo et al. 1996
; Nilius and
Wohlrab 1992
; Pancrazio et al. 1993
;
Pappas and Ritchie 1998
; Pappone and
Ortiz-Miranda 1993
; Sobko et al. 1998
).
We set out to study in more detail the effects of 4-AP on K+ conductances in astrocytes under both current- and voltage-clamp conditions. These studies demonstrate that astrocytic K+ currents are blocked differentially by increasing concentrations of 4-AP with 100 µM blocking a slowly inactivating outward current that has not previously been recognized; 2 mM additionally blocked transient A-type currents and delayed rectifying K+ currents, and 8 mM additionally and surprisingly also inhibited inwardly rectifying K+ currents.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture
Primary spinal cord astrocyte cultures from
P0 to P1 Sprague-Dawley rat
pups were obtained as previously described (Macfarlane and
Sontheimer 1997). Pups were anesthetized by hypothermia and then decapitated and spinal cords were dissected from midcervical to
lumbar regions. Tissue was excised in filter sterilized complete saline
solution (CSS) containing the following (in mM): 137 NaCl, 5.0 KCl, 1 MgCl2, 25 Glucose, 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 2 CaCl2, adjusted to pH = 7.3 by NaOH. Tissue then was stripped of meninges and blood vessels,
minced, and incubated for 20 min at 37°C and
95%O2-5%CO2 in CSS with
0.5 mM (ethylenedinitrilo)tetraacetic acid (EDTA), 1.65 mM
L-cysteine, and 30 U/ml papain (Worthington, Freehold, NJ).
Enzyme solution was aspirated, and tissue was rinsed with culture
medium consisting of Earle's minimal essential media (EMEM) (GIBCO,
Grand Island, NY) supplemented with 20 mM glucose, 10% fetal calf
serum (FCS; HyClone, Logan, UT), 500 U/ml of penicillin/streptomycin, 1.0 mg/ml trypsin-inhibitor and 1.0 mg/ml BSA. Tissue was dissociated by trituration with a fire-polished Pasteur pipette. Cells were plated
on poly-ornithine/laminin-coated 12-mm glass coverslips (MacAlaster
Bicknell, New Haven, CT) at a density of 106/ml.
Cells were maintained at 37°C and
95%O2-5%CO2 in culture medium changed every 3-4 days. Cultures were >95% positively
immunoreactive for GFAP (rabbit monoclonal, INCSTAR, Stillwater, MN).
Coverslips with cultured cells were transferred to a recording chamber
mounted on the stage of an inverted Nikon Diaphot microscope equipped with Hoffman Modulation Contrast Optics. The chamber was perfused continuously with the following bath solution (in mM): 125 NaCl, 5 KCl,
1.2 MgSO4, 1.6 Na2HPO4, 0.4 NaHPO4, 10.5 glucose, 32.5 HEPES, and 1.2 CaCl2, adjusted to pH =7.4 (NaOH).
Slice preparation
The methods for preparation of thin spinal cord slices
used in this study were as previously described (Bordey et al.
1996a,b
). Animals were anesthetized with pentobarbital sodium
(50 mg/ml) and decapitated. The spinal cord was removed and chilled
(0-4°C) in 95%O2-5%CO2
saturated calcium-free artificial cerebrospinal fluid (Ca-free ACSF;
see composition in the following text). Next, a portion of the spinal
cord was isolated and glued with cyanoacrylate to a cutting chamber and
immersed in chilled and oxygenated ACSF. Transversal slices
(T4-L2) of 150-200 µm
were cut with a vibrating tissue slicer (Vibratome). After a recovery
period of
1 h in Ca-free ACSF, slices were placed in a flow-through
chamber continuously superfused with oxygenated ACSF at room
temperature and held in position by a nylon mesh glued to a U-shaped
platinum wire. The ACSF contained (in mM): 113 NaCl, 3 KCl, 1 NaH2PO4, 25 NaHCO3, 11 glucose, 2 CaCl2, and 1 MgCl2,
saturated by 95% O2-5%
CO2 to maintain pH at 7.4.
Electrophysiology
Recordings were obtained using the whole cell patch-clamp
technique (Edwards et al. 1989). Patch pipettes were
pulled from thin-walled borosilicate glass (1.55 mm OD, 1.2 mm ID, WPI,
TW150F-40) on a PP-83 puller (Narishige, Japan). Pipettes had
resistances of 2-3 M
W and 4-6 M
W for culture and slice
recordings, respectively when filled with the following solution (in
mM): 145 KCl, 0.2 CaCl2, 1.0 MgCl2, 10 ethylene glycol-bis (-aminoethyl
ether)-N, N,N',N'-tetraacetic acid (EGTA), and 10 HEPES
(sodium salt), pH adjusted to 7.2 with tris(hydroxymethyl)aminomethane
(Tris). Culture and slice recordings were performed using an Axopatch
1-B amplifier and an Axopatch-200A (both Axon Instruments, Foster City,
CA), respectively. Current signals were low-pass filtered at 5 kHz and
digitized on-line at 25-100 kHz using a Digidata 1200 digitizing board
(Axon Instruments) interfaced with an IBM-compatible computer system.
Drug solutions were bath-applied after 5-6 min of recording when a stable access resistance was obtained. Solutions equilibrated within 3-4 min, as determined by the stability of test responses delivered at 15 s-intervals. Dendrotoxin was both bath and pressure-applied from a patch pipette with 10-20 psi at ~20 µm away from the cell body of the recorded cell.
Data analysis
Data acquisition, storage and analysis were done using PClamp
versions 6 and 7 (Axon Instruments). For all measurements, capacitance compensation and series resistance compensation (60-80%) were used to
minimize voltage errors. Settings were determined by compensating the
transients of a small (5 mV) 10-ms hyperpolarizing voltage step from a
holding potential of 70 mV; the membrane capacitance reading of the
amplifier was used as value for the whole cell capacitance. The average
membrane capacitance (Cm) measured in 141 recordings was 24.4 ± 14.0 (SD) and the average series
resistance (Rs) was 6.8 ± 2.6 M
. Rs was compensated by 69.5 ± 5.9%. Alternatively, membrane resistance
(Rm) was determined from the average
response to 50 hyperpolarizing (10 mV) current pulses (20 ms). The time constants for charging the capacitive transients resulted from a
monoexponential fit for glial cells and a biexponential fit for
neurons. The value of the resting potential was determined in the first
2 min of whole cell recording while Cm
and Rs were measured after 5 min of
recording when stable values were obtained. Values of the series
resistance and cell membrane capacitance were monitored throughout the
recording. For leak current subtraction, the membrane resistance was
determined with Clampfit (Axon Instruments) and a subsequent leak
subtraction was performed off-line. Peak currents were determined using
Clampfit (Axon Instruments), and statistical values (mean ± SD,
with n being the number of cells tested) were evaluated with
a statistical graphing and curve-fitting program (Origin, MicroCal).
All chemicals were from Sigma (St. Louis, MO) unless otherwise noted.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Voltage- and current-clamp recordings were obtained from 141 cultured spinal cord astrocytes. These were primarily process-bearing, stellate astrocytes with a mean resting potential
(Vr) determined at the time of
establishment of whole cell recordings of 75.7 ± 4.6 (SD) in 5 mM extracellular K+ concentration.
Differential effects of 4-AP on outward K+ current amplitudes
Astrocytes were voltage-clamped either at 70 or
80 mV, and the
membrane then was stepped for 300 ms from
70 to +50 mV (20-mV increments) after a prepulse to
110 mV. Representative examples of a
family of currents elicited by this stimulation protocol is shown in
Fig. 1Aa. Outward currents
activated at potentials more positive than
70 mV and increased with
increasing voltages. Currents showed two components: an initial
transient that inactivated within 30-50 ms and a sustained component
that did not inactivate further for the duration of the voltage step.
The voltage dependence of these currents is more readily visible in the
current/voltage (I-V) curves, in which peak current
amplitudes were plotted as a function of applied potentials (Fig.
1B). These show outward rectification and a relatively steep
voltage dependence.
|
When a low concentration of 4-AP (100 µM) was bath applied, it
reduced both the peak and steady-state components of the outward currents (Fig. 1A, b vs. a). The 4-AP block did
not affect the activation threshold of the residual current which was
still close to -70 mV (Fig. 1B, ). 4-AP inhibition was
more readily visible after point-by-point subtraction of the current
traces in the presence of 4-AP from the control traces in the absence
of the drug (Fig. 1C, a-b). The I-V
relationship of the 100 µM 4-AP-sensitive current shows an activation
threshold of
50 mV (Fig. 1D, cross). This effect of 100 µM 4-AP was observed in 19/19 astrocytes tested. Subsequent addition
of tetraethylammonium (TEA) at 10 mM decreased the steady-state
components of the outward current without affecting the peak of the
transient current component (Fig. 1A, c compared with b). The TEA-sensitive current was isolated by
subtracting the currents in presence of TEA and 4-AP from the currents
with 4-AP alone (Fig. 1C, b-c). The
I-V plot of the TEA-sensitive current indicates that
TEA-sensitive currents have a more positive activation threshold that
was close to
30 mV (Fig. 1D, stars).
Because 4-AP-sensitive, transient A currents require a relatively
negative prepulse potential to be activated, these currents can be
inhibited selectively by stepping the membrane to a more depolarized
potential before the test voltage (Rogawski 1985; Rudy 1988
). This was done in Fig.
2, for the same cell shown in Fig. 1. The
prepulse potential was
50 mV instead of
110 mV. At this more
depolarized potential, the transient current component was diminished
greatly. The residual outward currents activated at
50 mV as shown in
Fig. 2, Aa and Ba (
). Under these conditions, application of 4-AP (100 µM) still decreased outward
K+ currents (Fig. 2A, b compared with
a). The residual current, which was insensitive to 100 µM
4-AP, had an activation threshold of
30 mV and did not inactivate
during the voltage step (Fig. 2, Ab and B,
).
By contrast, the 4-AP-sensitive current obtained by subtraction, was
slowly inactivating (Fig. 2C, a-b) and the I-V plot yielded an activation threshold of
50 mV for this
current component (Fig. 2D, cross), which is henceforth
referred to as slowly inactivating K+ current
(KSI). Combined application of 10 mM
TEA in the presence of 100 µM 4-AP depressed the outward currents by
an additional 50% (Fig. 2A, c compared with
b). The subtracted TEA-sensitive current was sustained (Fig.
2C, b-c) and activated at
30 mV
(Fig. 2D, stars). This current is henceforth referred to as
KDR and is analogous to the neuronal
delayed rectifier.
|
We next assessed the concentration dependence of 4-AP block on outward
K+ currents by increasing the 4-AP concentration
applied. In 11 cells we directly compared the effects of 100 µm 4-AP
and 2 mM 4-AP and an example recording is shown in Fig.
3. When the same cell was depolarized
from 70 to 50 mV after a prepulse to
110 mV, 2 mM 4-AP depressed
both the peak and steady-state currents (Fig. 3A,
b compared with a) but still did not completely
eliminate the transient current component. Thus the subtracted 2 mM
4-AP-sensitive current displays a transient, fast inactivating
component (see later for isolation) and a sustained component (Fig.
3A, c = a
b).
The I-V relationship of the control currents (in presence of
100 µM 4-AP), the currents in presence of 2 mM 4-AP, and the 2 mM
4-AP-sensitive currents measured at the peak current amplitudes, all
yield an activation threshold of
70 mV (Fig. 3B,
,
,
and cross, respectively). When measured at the steady-state current amplitude (end of the pulse), the I-V curve of the 2 mM
4-AP-sensitive current gave an activation threshold of
30 mV (Fig.
3B, stars), similar to that of the TEA-sensitive current
[Figs. 1 and 2, both C (b-c)]. When
the same cell was depolarized from
70 to 50 mV after a prepulse to
50 mV, thus removing the transient K+ current,
increasing the 4-AP concentration from 100 µM to 2 mM further reduced
the sustained current. The subtracted current (Fig. 2C,
a-b) activated at
30 mV (Fig. 2D,
cross), a value identical to that of the TEA-sensitive current,
KDR, displayed in Fig. 2C (b-c). Higher concentrations of 4-AP e.g.,3 mM
(data not shown, n = 5) and 4 mM (n = 6) had essentially identical effects.
|
Figure 4 shows the effect of 100 µM and
2 mM 4-AP on the transient, fast-inactivating current components only,
along with their respective I-V curves (Fig. 4C).
Isolation of these currents was obtained through point-by-point
subtraction of the currents obtained with a prepulse to 110 mV (Fig.
3A) from the currents obtained with a prepulse to
50 mV
(Fig. 3C) in presence of 4-AP at either 100 µM
(traces a) or 2 mM (traces b). Currents activate at
70 mV (Fig. 4C) and were termed
KA in analogy with neuronal A-type
currents. They were insensitive to 100 µM 4-AP but were reduced by
>50% in 2 mM 4-AP. Dendrototoxin (Brau et al. 1990
; Hopkins et al. 1999
) bath-applied at 200 nM or
pressure-applied at 10 µM had no effect on either
KSI or
KA.
|
Application of even higher concentration of 4-AP (8 mM) induced
essentially a complete block of KA,
KDR, and
KSI (n = 5). Surprisingly, in cells displaying large inward currents, 4-AP (8 mM)
also completely blocked inward K+ currents (Fig.
5A, n = 4) and
revealed the presence of inward sodium currents
(INa) that were masked previously by
the much larger inward K+ currents (Fig.
5A, inset). The inward K+ currents
were barium sensitive (10 µM, data not shown) and
Cs+ sensitive (1 mM, not shown) and showed
kinetic features identical to those previously described for inwardly
rectifying K+ currents
(KIR) in astrocytes (Ransom and
Sontheimer 1995; Ransom et al. 1996
).
I-V plots of the recorded peak currents are displayed in
Fig. 5B under control conditions (
) and in presence of
4-AP (
) with the I-V curve of
INa (stars).
|
Differential effects of 4-AP on current-induced voltage responses
Under current-clamp conditions, depolarizing current pulses
induced a transient voltage response corresponding to a single spike in
spinal cord glial cells as shown for a representative cell in Fig.
6. The physiological relevance of these
spikes is enigmatic and has been debated in several recent studies
(Bordey and Sontheimer 1998a,b
; Labrakakis et al.
1997
). For purposes of this study, we are not concerned with
the biological relevance of this response but rather are interested in
the relative contribution of the four above-characterized
K+ current components to the overall waveform of
this experimentally induced spike. Note, however, that in the following
text we show recordings of such spikes from astrocytes in acute spinal
cord slices to dismiss the notion that these responses were an artifact of cell culture.
|
Under voltage clamp, the cell membrane was stepped from 140 to 80 mV
from a holding potential of
70 mV (Fig. 6A). In Fig. 6B, 100-pA increment step currents were injected from a
70
mV potential under current clamp. Off-line leak subtraction revealed the presence of inward voltage-activated sodium currents (Fig. 6A, inset). The inset in Fig. 6B shows
the glial spike at greater time resolution. As previously described
(Bordey and Sontheimer 1998a
), these spikes are rapidly
activating but require
10 ms for repolarization, which is always
incomplete. In Fig. 6C, a glial spike and a neuronal action
potential induced in a spinal astrocyte and neuron on the same
coverslip by a 300- and a 4-pA current injection, respectively, were
superimposed to illustrate the difference in waveform. The glial spike
(Fig. 6C, solid line) was composed of three phases: a peak
consisting of a depolarizing and repolarizing phase, a short
after-hyperpolarization (AHP), and a slow, prolonged AHP. Photographs
of the recorded astrocyte and the recorded neuron are shown in Fig.
6D.
Figure 7 summarizes the relative contribution of 4-AP-sensitive currents and their relative dose dependence to the glial spike in three representative current-clamped astrocytes. In Fig. 7, A, C, E, and G, successive current injections were applied every 10 s to monitor the drug effect. In Fig. 7, B, D, F, and H, left and right, 100-pA step current injections were applied under control conditions and in the presence of 4-AP. Application of 4-AP (100 µM) primarily inhibited the short AHP and decreased the slow AHP amplitude (Fig. 2, A and B), whereas higher 4-AP concentrations (2 mM, n = 15; 4 mM, n = 8) blocked the repolarizing phase leading to a block of the voltage peak response (Fig. 2, C and D). 4-AP (100 µM) also led to a 2-mV depolarization, as visible by the voltage shift in the presence of 4-AP. On average, a 2.30 ± 0.48 mV (n = 8) depolarization was observed with 100 µM 4-AP that did not increase further in 2 mM 4-AP. In Fig. 7, E and G, progressive effects of 8 mM 4-AP (n = 5) on a 400-pA current injection were shown. In cells displaying small inward currents (see Fig. 6B for a representative example), 8 mM 4-AP had similar effects as 4 mM 4-AP (Fig. 7, E and F). In cells displaying large inward currents (same cell as in Fig. 5A), 4-AP lowered the resting K+ conductance of cells to the extent that cells initially showing little to no voltage transients could now be induced to show voltage transients in response to current injection (Fig. 7, G and H). This was due to the almost complete block of outward and inward K+ currents, thus unmasking the much smaller Na+ currents in these cells, which now became a large enough conductance to depolarize the glial membrane. Using the same protocols, we tested the effects of TEA (20 mM) (Fig. 8). Application of TEA blocked the slow AHP without affecting the short AHP (n = 8).
|
|
Current-induced spike in situ
To ascertain whether these astrocytic action potentials were
an artifact of cell culture (Sontheimer et al. 1992), we
obtained recordings from astrocytes in acute spinal cord slices. In
young rats of 9-11 day postnatal (P), five of eight recorded cells
could generate a spike in response to depolarizing current injections that was identical to that observed in cultured cells (Fig.
3A). The Lucifer-yellow-filled cell displayed a stellate
shape with two processes going slightly deeper into the slice than
other processes (Fig. 9B). In
older animals, P18-P22, only two of eight cells could generate a slow
spike (Fig. 9C). The Lucifer-yellow-filled cell is displayed
in Fig. 9D.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we characterized four biophysically distinct
K+ current types expressed in spinal cord
astrocytes. These are: delayed rectifying K+
currents (KDR), transient A-type
K+ currents
(KA), slowly inactivating outward
currents (KSI), and inwardly
rectifying K+ currents
(KIR). Of these, the
KSI has not been previously described in astrocytes and was revealed in our study through its differential sensitivity to 4-AP. Indeed, the key finding of this study is that all
four K+ astrocytic currents are sensitive to
4-AP, each showing a distinctly different sensitivity for current
inhibition by 4-AP. KIS was most
sensitive and was completely inhibited by 100 µM 4-AP.
KA and
KDR were each inhibited by
intermediate 4-AP concentrations of 2-4 mM. As previously described,
these two currents could be isolated by changing the prepulse
potentials between 110 mV, activating both, and
50 mV, activating
only KDR. Last and unexpectedly, KIR currents were inhibited completely
by higher 4-AP concentrations e.g., 8 mM.
KIR currents showed typical
sensitivity to Cs+ and Ba2+
but had previously been thought to be 4-AP insensitive (Ransom and Sontheimer 1995
).
Importantly, these currents also showed different thresholds for
activation. Although KDR did not
activate until the membrane was depolarized to 30 mV, both
KIS and
KA showed activation thresholds close
to the cells resting potential. Thus
KIS activated at potentials more
positive than
60 mV and thus is likely to contribute to the resting
conductance of astrocytes. Indeed, we found in current-clamp experiments that 100 µM 4-AP depolarized astrocytes by 2.3 mV, evidence of KSI contributing to the
resting conductance of astrocytes. On the basis of this data, we
suggest that KSI rather than
KDR may be a K+
conductance that is important in the context of
K+ buffering by astrocytes. This conclusion,
however, will have to await a more detailed characterization of
KSI with specific emphasize on its
role in K+ buffering.
We used current-clamp experiments to delineate the contribution of
these four K+ conductances to stabilization and
repolarization of the astrocytic membrane potential. Therefore we
injected currents of increasing amplitude and recorded the resulting
voltage response while selectively inhibiting subsets of
K+ currents. Owing to the expression of
voltage-activated Na+ channels, spinal cord
astrocytes show overshooting voltage spikes, reminiscent of a slow
neuronal action potential (Kehl et al. 1997). The
biological significance of these glial spikes is an enigma (Bordey and Sontheimer 1998a
; Labrakakis et al.
1997
) and for purposes of our study not germane. However, they
provided a means to assess the contribution of selective K+
currents to the repolarization of the astrocytic membrane. Moreover, we
show for the first time that such spikes can be recorded in astrocytes
in acute spinal cord slices, dismissing concerns that these responses
may be an artifact of cell culture. Not unexpectedly, our current-clamp
recordings showed that the relative magnitude of
KIR currents determines the current required
to initiate glial spikes. Indeed, blockade of
KIR by 8 mM 4-AP revealed small
Na+ currents that were sometimes masked by the much larger
resting K+ conductance mediated by
KIR channels. Interestingly, repolarization was only marginally affected by 100 µM 4-AP but virtually was eliminated by 2 mM 4-AP, suggesting a prominent role for
KA but not KIS in
the repolarization of the astrocytic membrane after a large
depolarization. On the basis of our results, we propose that
KDR is primarily responsible for the slow
AHP of the astrocytic membrane after large membrane depolarizations
(more than
30 mV), which is consistent with these currents showing a
positive activation threshold and a slow but sustained activation.
KIR and KIS on the other hand constitute the major resting K+
conductances. Given their opposite rectification modes, we would suggest that KIR serves K+
uptake, whereas KIS may serve K+
release in the context of spatial K+ buffering
(Newman 1993
; Newman and Reichenbach
1996
; Orkand 1991
; Orkand and Opava
1994
). Given the peculiar voltage requirements for its
activation, the role of KA currents remain
elusive. These currents have previously been implicated in growth
control of macrophages and lymphocytes (Gallo et al.
1996
; Nilius and Wohlrab 1992
; Pancrazio
et al. 1993
; Pappas and Ritchie 1998
;
Pappone and Ortiz-Miranda 1993
; Sobko et al.
1998
) and also hippocampal astrocytes (Pappas et al.
1994
) and may serve a similar role in spinal cord astrocytes.
Owing to its modulatory effect of neuronal spike discharge, 4-AP has
been a useful drug to mimic epileptiform activity in hippocampal and
cortical brain slices (Avoli 1996; Chesnut and Swann 1988
; Perreault and Avoli 1992
;
Rutecki et al. 1987
; Schwartzkroin 1986
;
Szente and Baranyi 1987
; Traub and Jefferys
1994
) and has become one of the mainstays in epilepsy research.
The concentrations used in some of these studies (Armand et al.
1999
; Arvanov et al. 1995
; Gean et al.
1990
; Hoffman and Prince 1995
; Holsheimer and Lopes da Silva 1989
; Ikegaya et al. 1998
;
Mattia et al. 1993
, 1995
; Schuchmann et al.
1999
; Watts and Jefferys 1993
), 100 µM to 2 mM, also would inhibit astrocytic KIS
currents. On the basis of our data, one must expect a concomitant,
albeit small astrocytic depolarization, possible in concert with
reduced K+ fluxes across astrocytic membranes. Because
K+ is known to rise during epileptiform activity, it will
be important to dissect the role that inhibition of astrocytic
KIS channels have in the context of 4-AP-induced
epileptiform discharges.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank S. Nee-MacFarlane for providing cultured cells.
This work was supported by National Institutes of Health Grants RO1-NS-31234 and P50-HD-32901.
![]() |
FOOTNOTES |
---|
Address for reprint requests: H. Sontheimer, Dept. of Neurobiology, The University of Alabama at Birmingham, 1719 6th Ave. S., CIRC Rm. 545, Birmingham, AL 35294.
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 9 November 1998; accepted in final form 24 August 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|