Differential Inhibition of Glial K+ Currents by 4-AP

Angélique Bordey and Harald Sontheimer

Department of Neurobiology, University of Alabama, Birmingham, Alabama 35294


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METHODS
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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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (tau 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 beta 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.


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INTRODUCTION
METHODS
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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 MOmega W and 4-6 MOmega 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 MOmega . 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.


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INTRODUCTION
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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.



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Fig. 1. 4-aminopyridine (4-AP) 100 µM and TEA differentially affect outward K+ currents activated after a conditioning hyperpolarization. A: whole cell traces (after off-line leak-subtraction) obtained by stepping the cell membrane from -70 to +80 mV after a prepulse to -110 mV. Control traces in a, and traces shown in presence of 4-AP (100 µM; b) and 4-AP with TEA (10 mM; c). B: peak current amplitudes were plotted against the applied membrane voltage. Resulting current/voltage (I-V) relationships for the control traces (); the traces in presence of 4-AP () and 4-AP plus TEA (open circle ) yield an activation threshold of -70 mV for all 3 currents. C: point-by-point subtractions of traces with 4-AP and traces with 4-AP plus TEA from control traces and from traces with 4-AP alone, respectively, reveals the 4-AP (100 µM)-sensitive (a-b) and TEA-sensitive (b-c) outward K+ currents, respectively. D: I-V curves of the 4-AP (100 µM, cross) and TEA-sensitive (stars) currents yield an activation threshold of -50 and -30 mV, respectively.

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.



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Fig. 2. 4-AP (100 µM) and TEA differentially affect outward K+ currents activated after a prepulse to -50 mV. A: whole cell traces (after off-line leak-subtraction) obtained by stepping the cell membrane from -70 to +80 mV after a prepulse to -50 mV. Control traces in a, and traces shown in presence of 4-AP (100 µM; b) and 4-AP with TEA (10 mM; c). B: I-V relationships for the traces displayed in A yield an activation threshold of -50 mV for the currents under control conditions () and -30 mV for the traces in presence of 4-AP alone (filled circles) and 4-AP plus TEA (open circle ). C: point-by-point subtractions of traces with 4-AP and traces with 4-AP plus TEA from control traces and from traces with 4-AP alone, respectively reveals the 4-AP (100 µM)-sensitive, slow inactivating (a-b) and the TEA-sensitive, sustained (b-c) outward K+ currents, respectively. D: I-V curves of the 4-AP (100 µM, cross) and TEA-sensitive (stars) currents yield an activation threshold of -50 and -30 mV, respectively.

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.



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Fig. 3. Additional effects 2 mM 4-AP on outward K+ currents in presence of 100 µM 4-AP. A: whole cell traces (after off-line leak-subtraction) obtained by stepping the cell membrane from -70 to +80 mV after a prepulse to -110 mV. Control traces in presence of 4-AP (100 µM) in a, and traces shown in presence of 4-AP (2 mM; b). Point-by-point subtraction of traces with 2 mM 4-AP from control traces (with 100 µM) reveals the 4-AP (2 mM)-sensitive currents. B: I-V relationships of the peak current amplitudes for the traces displayed in A yield an activation threshold of -70 mV for the currents obtained in presence of 100 µM 4-AP () and 2 mM () and for the 2 mM 4-AP-sensitive currents (cross). I-V curve of the amplitudes of the steady-state 4-AP-sensitive currents yield an activation threshold of -30 mV (stars). C: whole cell traces obtained by stepping the cell membrane from -70 to +80 mV after a prepulse to -50 mV. Control traces in presence of 4-AP (100 µM) in a and traces in presence of 4-AP (2 mM; b). Point-by-point subtraction of traces with 2 mM 4-AP from control traces reveals the 4-AP (2 mM)-sensitive currents. D: I-V relationships for the traces displayed in C yield an activation threshold of -30 mV for all 3 currents (in presence of 100 µM 4-AP: , 2 mM 4-AP: , and the 2 mM 4-AP-sensitive currents: cross).

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.



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Fig. 4. Effect of 4-AP on transient outward K+ currents. A: traces in a and b were obtained after point-by-point subtraction of traces with a prepulse to -110 mV from traces with a prepulse to -50 mV in presence of 100 µM (see Fig. 3, Aa and Ca) and 2 mM 4-AP (see Fig. 3, Ab and Cb), respectively. Resulting currents are transient outward K+ currents insensitive to 100 µM 4-AP but sensitive to 2 mM 4-AP. B: I-V relationships of the peak current amplitudes for the currents displayed in A yield an activation threshold of -70 mV for currents in both conditions.

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).



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Fig. 5. Inhibition of essentially all K+ currents including inwardly rectifying K+ currents (KIR) by 8 mM 4-AP. A: whole cell traces obtained by stepping the cell membrane from -140 to +80 mV (without leak subtraction) from a holding potential of -80 mV under control conditions (left) and in presence of 4-AP (8 mM; right). 4-AP-block of K+ current revealed the presence of large sodium currents (INa; inset). In this case, the cell membrane was stepped from -70 to +80 mV after a prepulse to -110 mV (with on-line leak subtraction). B: I-V curves of the peak amplitudes from current traces under control conditions () and in presence of 4-AP (open circle ) and from the peak inward INa amplitude (*).

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.



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Fig. 6. Astrocytic voltage spikes induced by current injection. A: under voltage clamp, current traces (without leak-subtraction) obtained by stepping the cell membrane from -140 to 80 mV. Off-line leak-subtraction reveals the presence of sodium currents (inset). B: under current clamp, voltage traces obtained by injection of 100-pA increment step currents. Inset: glial overshooting voltage response (spike) at greater magnification. C: superimposition of a 300-pA induced glial spike (---) and a 4-pA induced neuronal action potential (- - -). Glial spike was composed of 3 phases, including a depolarizing and repolarizing phase (phase 1) and a short (phase 2), and slow (phase 3) afterhyperpolarization. D: photographs of the astrocyte and the neuron from which the current responses were recorded.

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).



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Fig. 7. Modulation of the astrocytic voltage spike by 4-AP. A, C, E, and G: a 400-pA current was applied every 10 s in each current-clamp cell. B, D, F, and H: 100-pA step current injection were applied to the same cells as in the left panels, respectively. A and B: bath application of 4-AP (100 µM) reversibly affected the repolarization of the current-induced overshooting voltage response and the afterhyperpolarizations after this spike. C and D: 4-AP (2-4 mM) progressively and reversibly blocked the repolarization phase and the afterhyperpolarizations leading to block of the voltage peak response. E and F: 8 mM 4-AP had a similar but more dramatic effect as 4 mM 4-AP. Cell displayed KIR currents of small amplitudes (data not shown) and display a current-induced spike. G and H: under control conditions, the membrane response to current injection from -70 mV was passive presumably due to large KIR currents (same cell as in Fig. 5). 4-AP (8 mM) progressively blocked all K+ currents and led to generation of a spike in response to current injection.



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Fig. 8. Modulation of the current-induced spike by TEA (20 mM). Same protocols as in Fig. 7. Application TEA (20 mM) blocked the slow afterhyperpolarization (AHP) after the spike without affecting the repolarization phase and the fast AHP.

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.



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Fig. 9. Astrocytic spike recorded in acute spinal cord slices. A and C: 200-pA currents were injected into a spinal cord glial cell in an acute slice from a 9- and a 22-day-old rat, respectively. Cell was able to generate a spike response in the 9-day-old rat and a very slow response in the cell from the older animal. B and D: Lucifer-yellow-filled cell, the traces of which are represented in A and C, respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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