kainate activates ca2+-permeable glutamate receptors and blocks voltage-gated k+ currents in glial...

Pfltigers Arch (1994) 426:310-319 E6i i i i hiin Journal of Physiology �9 Springer-Verlag 1994

Kainate activates Ca2+-permeable glutamate receptors and blocks voltage-gated K § currents in glial cells of mouse hippocampal slices

Ronald Jabs', Frank Kirchhoff 2, Helmut Kettenmann 2' 3, Christian Steinh~iuser 1

1 Institute of Physiology, Friedrich-Schiller University Jena, Teichgraben 8, D-07740 Jena, Germany 2 Institute of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany 3 Max-Delbrtick-Center for Molecular Medicine, Robert-R6ssle-Strasse 10, D-13122 Berlin, Germany

Received June 7, 1993 / Received after revision September 9, 1993 / Accepted September 25, 1993

Abstract. Glial cells in the CA1 stratum radiatum of the hippocampus of 9- to 12-day-old mice show intrinsic responses to glutamate due to the activation of a-amino- 3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/ kainate receptors. In the present study we have focused on a subpopulation of the hippocampal glial cells, the "complex" cells, characterized by voltage-gated Na + and K + channels. Activation of glutamate receptors in these cells led to two types of responses, the activation of a cationic conductance, and a longer-lasting blockade of voltage-gated K + channels. In particular, the transient (inactivating) component of the outwardly rectifying K + current was diminished by kainate. Concomitantly, as described in Bergmann glial cells, kainate also elevated cytosolic Ca ~+. This increase was due to an influx via the glutamate receptor itself. In contrast to Bergmann glial cells, the cytosolic Ca 2+ increase was not a link to the K * channel blockade, since the blockade occurred in the absence of the Ca 2§ signal and, vice versa, an increase in cytosolic Ca 2+ induced by ionomycin did not block the transient K + current. We conclude that glutamate receptor activation leads to complex and variable changes in different types of glial cells; the functional importance of these changes is as yet unresolved.

Key words: Glial cell - Hippocampus - Glutamate receptors - Kainate - Patch clamp - Potassium channels - Transmitter modulation

Introduction

The major excitatory neurotransmitter receptor in the mammalian central nervous system (CNS), the glutamate receptor, is expressed by both neurons and glial cells [3]. Based on pharmacological and electrophysiological studies, ionotropic glutamate receptors are classi- fied into subclasses, namely the N-methyl-D-aspartate

Correspondence to: C. Steinh~iuser

(NMDA) and the non-NMDA or u.-amino-3-hydroxy-5- methyl-4-isoxazole propionate (AMPA)/kainate receptors. The classical NMDA receptors are permeable to Ca 2+ in contrast to the AMPA/kainate receptors as analysed in marly neuronal preparations [12]. This Ca 2+ permeability is thought to be involved in long-term potentiation [11] and neurotoxicity [9], causing NMDA receptors to be a major focus of interest in neuroscience research. The AMPA/kainate receptors were shown to be involved in fast synaptic transmission [22]. Recently, a combination of molecular biological and electrophysiological studies revealed that AMPA/kainate receptors can be subdivided into high-affinity AMPA (GluR-1-4) and kainate (GluR-5-7, KA-1,2) receptors which form the ion channel complexes [27]. In heteromeric receptors the presence of the GluR-2 subunit, which is widely expressed in the CNS, infers the particular property of Ca 2+ impermeability to AMPA/kainate receptors.

AMPA/kainate-type glutamate receptors are expressed by a variety of glial cells as demonstrated in brain slices and in tissue culture. Glial precursor cells and oligodendrocytes in corpus callosum slices [2] and glial cells in the spinal cord (A. Chv~tal, A. Pastor, M. Mauch, E. Sykovfi and H. Kettenmann, unpublished ob- servations) respond to glutamate or kainate. In Bergmann glial cells, activation of AMPA/kainate receptors is accompanied by an influx of Ca 2+ [25]. As ex- pected, these cells do not express the GluR-2 subunit as analysed by in situ hybridization [4]. The elevation of Ca 2+, triggered by kainate or alternatively by application of the Ca 2+ ionophore ionomycin, leads to a transient blockade of a K + conductance [25].

In the hippocampus two types of glial cells, the "passive" and the "complex" cells respond to glutamate and kainate [29]. In these cells, acti#ation of AMPA/kainate receptors leads to a subsequent blockade of K + currents [29]. The mechanism responsible for this interaction between receptor activation and K + channel activity is so far unknown.

In this study, we have focused on cells of the "complex" type which express time- and voltage-dependent

311

Na + and K + currents. We have investigated whether AMPA/kainate receptors in these cells are also Ca 2+ permeable as described in Bergmann glial cells and whether the influx o f Ca 2+ via the receptor channel represents the link between receptor activation and blockade of the K + conductance.

Materials and methods Preparation and recording set-up. Young mice (postnatal days 10-12) were ether anaesthetized, sacrificed by decapitation and their brains dissected out, washed and the hemispheres cut into 150-~tm-thick slices in frontal orientation using a vibratome (FTB, Piano, Marburg, Germany). Slice preparation was performed at 6~ in external solution (see below). Subsequently, slices were stored for at least 30 min in the same solution at 35~ For electrophysiological recordings slices were placed in a chamber mounted on the stage of a Zeiss microscope (Axioskop FS, Axioplan, Zeiss, Oberkochen, Germany) and fixed in the chamber using a U-shaped platinum wire with a grid of nylon threads [14]. The chamber was continuously perfused with carbogen-saturated external solution and substances were added by changing the perfusate. Cell somata in the hippocampal slice were visible with water immersion optics (Zeiss Achroplan 40 • and could be approached by the patch electrode.

The selected cells were located about 30 ~tm beyond the surface of the slice. Positive pressure was applied to the recording pipette while it was lowered under microscopic control. Thus, cellular debris was blown aside and the tip could be placed onto the surface of a cell soma. During recording, cells were filled with Lucifer Yellow (LY) by dialysing the cytoplasm with the patch pipette solution. Membrane currents were measured with the patch-clamp technique in the whole-cell recording configuration [18]. Current signals were amplified (EPC-7 amplifier, List Elec- tronics, Darmstadt, Germany), filtered at 3 or t0 kHz and sampled at 5 or 30 kHz by an interface (Battelle, Frankfm/]M., Germany) connected to an AT-compatible computer system which also served as a stimulus generator. The resistance of the patch pipettes was 5 - 6 Ms Limitations of voltage-clamp control in cells of slice preparations are discussed in detail elsewhere [24, 28]. The passive membrane properties of the different glial cells measured were rather variable reflecting an inhomogenous cell population. In most cases time constants of capacitance currents amounted to 0.4-1.7 ms and the series resistances varied between 8-18 Ms yielding a capacitance of CM = 45-100pF [28]. These capaci- tances corresponded to membrane surfaces which exceeded the cell areas by a factor of 4 - 3 3 assuming spherical shapes of the somata (diameter 10 ~tm). Voltages were corrected for liquid junc- tion potentials. In cells lacking inward rectification, membrane currents were leak subtracted and compensated for capacitance artefacts by adding corresponding de- and hyperpolarizing responses.

Solutions, reagents and electrodes. The standard external solution contained 124 mM NaCI, 5 mM KC1, 2 mM CaC12, 2 mM MgCI~, 24 mM NaHCO3, 1.25 mM NaHaPO4 and t0 mM glucose. In some experiments t0 mM BaC12 was added to block voltage-gated K + channels and NaC1 concentration was reduced to 109 mM to en- sure constant osmolarity. In all Ba 2+ experiments H~PO4 was omitted both in Ba2+-containing as well as in corresponding Ba 2+- free control solutions. By gassing the solutions with carbogen (5% COJ95% 02) the pH was adjusted to 7.4. The pipette solution contained 130 mM KC1, 0.5 mM CaC12, 2 mM MgC12, 5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), 10 mM 4-(2-hydroxy- ethyl)-l-piperazine-2-ethanesulphonic acid (HEPES) 3 mM ATP and, except for measurements of intracellular free Ca 2+ concentration ([Ca2+]0 changes, 0.1% LY. All experiments were carried out at room temperature (about 22~

Recording pipettes were fabricated from borosilicate capillar- ies (Hilgenberg, Malsfeld, Germany). Ca 2+ activity of the internal

solution was approximately t l nM. LY (CH, dilithium salt) was from Fluka (Neu-Ulm, Germany), all other reagents were pur- chased from Sigma (Munich, Germany).

Microfluorimetric [Ca2+]~ measurements. Changes in [Ca2+]j were detected using the fluorescent Ca 2+ chelating dye fura-2 [17, 23]. Individual glial cells were recorded with pipettes containing 200 btM fura-2 instead of LY in the tip and normal pipette solution in the remaining part of the pipette. Single cell microfluorimetric measurements were obtained by a video image processing system with an intensified charge-coupled device (CCD) camera (Video- probe, ETM Systems, California, USA). Fura-2 fluorescence was elicited at 340 and 380 nm, respectively, and the intensities of con- secutive video frames were digitized, the fluorescence ratios were calculated and eight frames averaged. Every 2 s an averaged fluorescence ratio image was stored on the hard disk of an IBM-compatible personal computer (Intel 80386).

Results

Identification of glial cells in the slice

All recordings were obtained f rom glial cells located in the stratum radiatum of the hippocampus. To distinguish glial cells f rom neurons, a combinat ion o f morphologica l and electrophysiological criteria was applied. In the present study we focused on a subpopulat ion o f h ippocam- pal glial cells which are characterized by the expression of vol tage-gated K + and Na + channels, termed " c o m - plex" cells. The glial origin of this cell populat ion was previously shown by combining patch-clamp analysis and ultrastructural inspection (for details see [28]). These cells were characterized by their small soma diameter ( < 10 Ilm) and their inability to generate action potentials in the current clamp mode. We were able, with some training, to reliably select for " c o m p l e x " cells based on their typical morphologica l appearance in hippocampal slices. In bright field mic roscopy cell bodies displayed a lucent halo and dark cell boundaries. The resting potential o f this glial cell populat ion was - 7 4 _ + 9 m V (mean _+SD, n = 9 6 ) , i.e. much more negative as compared with neighbouring neurons which had resting potentials o f about - 6 5 m V [10]. We used de- and hyperpolar izing voltage steps ranging f rom - 1 6 0 to 20 m V to record the vol tage-gated K + and Na + channels [28]. The Na + current in these cells was at least one order o f magni tude smaller than that seen in neurons. Subsequently, we studied the properties o f responses activated by the glutamate receptor agonist kainate.

Reversal potential analysis of kainate-induced membrane currents revealed activation of a receptor conductance

At a holding potential o f - 7 0 m V application o f 1 m M kainate induced inward currents in all " c o m p l e x " cells measured (Fig. 1B) (797 + 376 pA, range 391 - 1 3 7 0 pA, n = 57). The kainate response significantly exceeded the corresponding currents induced by glutamate (by 1 1 9 7 _ + 874%, range 8 2 5 - 2 6 0 4 % , n = 4) as described

312

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Fig. 1 A-F. Reversal potential of kainate-induced receptor currents. A glial cell was analysed in standard bath solution. A Starting from a holding potential (V~) of - 7 0 mV the membrane was stepped tbr 50ms to depolarizing potentials up to 20 mV m 20-mV in- crements. The pattern of voltage pulses is shown in the inset. Cur- rents were leak subtracted and compensated for capacitance artefacts. B Starting from a 1,~ of - 7 0 mV the membrane was repetitively clamped to - 4 0 , -20 , 0, 20, 40, 60, 100, -100 , - I 3 0 and - 1 6 0 mV for 100 ms separated by 100-ms intervals. This stimulation sequence (shaded inset) was applied every 3 s before, during, and after kainate application. I/V curves were plotted from conesponding currents at the time points marked by arrows ( l - 3 , see upper insets). In addition, in the upper insets original current traces for a voltage step to 20 mY at 1, 2, and 3 are

t [aA]

i-1.2

given. In all cases data points were taken 10 ms after the onset of the voltage step (see current trace in the left inset, dashed line) to avoid contamination by Na + currents or capacitance artefacts. C [/V curves of kainate responses were calculated by subtracting current amplitudes at corresponding membrane potentials during kainate application from those before (2-1) or after addition of kainate (2-3). Nsets illustrate the original traces for the subtracted cur~ rents. D - F To isolate receptor activated currents 10 mM BaCla was added to the bath solution and membrane currents were analysed as described above. Note the residual K + current in BaCI2 solution (D) cansing a shift of the reversal potential in positive direction (K 2-1). The horizontal bars in the insets (B, C, E, F) mark zero current level for the original traces. Resting potential was - 7 4 mV in standard bath solution

A

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I I I [ I I I 0 3O 6O 9O 120 150 t [s]

313

Fig. 2A, B. Time courses of kainate-mediated receptor activation and K * channel blockade. A Kainate- induced membrane currents were analysed as described in Fig. 1B, C. The I/V curves on the upper insets are constructed from currents as marked by the arrows. The corresponding currents were subtracted as indicated. B The receptor-mediated conductance (squares) was estimated for each set of voltage jumps subtracting leak current in the standard solution from kainate-induced currents at a membrane potentials of -70 inV. The time course of the K + conductance (triangles) was determined for each stimulation sequence by subtracting receptor conductance from the corresponding whole membrane conductance calculated at t00 inV. Data were taken 10 ms after the onset of voltage steps. Resting potential of the cell was -77 mV

in a previous study using HEPES-buffered bath solutions [29]. Since kainate is no substrate for uptake systems [20] this current cannot be due to electrogenic transport processes, but identifies this response as receptor mediated. To determine the reversal potential of the kainate-induced current, the membrane was clamped from a holding potential of - 7 0 mV to - 4 0 , - 2 0 , 0, 20, 40, 60, 100, - 1 0 0 , - 1 3 0 and - 1 6 0 mV for 100 ms, separated by 100-ms intervals (Fig. 1B, shaded inset). This stimulation sequence was repetitively applied (every 3 s) before, during, and after kainate application (Fig. 1B, n = 29). Current/voltage (I/V) curves of the corresponding currents at the time points marked by arrows ( 1 - 3) are drawn in the upper insets. Under control conditions (i.e. before kainate application), large K § currents could be activated by depolarizing voltage steps (Fig. 1A, B). I /V curves of kainate responses were calculated by subtracting current amplitudes at corresponding membrane potentials during application of kainate from those before (Fig. 1C, 2-1) or after addition of kainate (Fig. 1C, 2-3). Data points were taken 10 ms after the onset of the voltage step. The I /V curve of the kainate-activated current revealed a large conductance increase with hyperpolarizing potentials. At potentials positive to 0 mV, the membrane conductance decreased in relationship to re- cords taken before kainate application (Fig. 1C, 2-1). In contrast, by subtracting the currents from those during kainate application, a nearly linear I /V curve was obtained after kainate application (Fig. 1C, 2-3). This be- haviour was similar to that described for the glutamate response in the same cell type and the kainate response in Bergmann glial cells. To isolate the receptor conductance at all membrane potentials the K + conductance was blocked by adding 10 mM BaC12 to the bath solution (Fig. 1 D - F , n = 6). Under these conditions only a residual K + current remained (Fig. ID). The I /V curve

of the kainate-induced current was determined by applying the stimulation protocol described above and subtracting currents at the peak of the kainate response from those in control solution. Taking the currents at the late phase as a control, the resulting I /V curve was linear and reversed near 0 mV (2 _+ 2 mV, n = 5, Fig. 1F, 2-3). We thus can conclude that kainate activates a cationic conductance similar to that described for kainate receptors and that this conductance increase can be isolated by blocking outwardly rectifying K + channels.

Kainate reversibly blocked voltage-gated K + currents

It was obvious that kainate elicited two effects with different time courses: an increase of the conductance in hyperpolarizing direction, and a longer-lasting effect, i.e. a reduction of currents activated by depolarization (Figs. 1B, 2A). About 2 min after kainate application, the conductance increase in the hyperpolarizing direction was terminated. The decrease of the conductance observed with depolarizing pulses was still obvious. The blocked conductance could be isolated by subtracting the currents of the control situation from those at the late phase of recording (Fig. 2A, inset 1-3). It activated at about - 4 0 mV, similar to the properties of a outwardly rectifying K + current.

Thus, kainate induced two superimposed effects, an activation of a cationic conductance and a blockade of K § conductances. To analyse the time course of the two processes activated by kainate, the cationic conductance increase was determined by measuring the conductance increase evoked by hyperpolarizing voltage steps. The receptor conductance (filled squares in Fig. 2B) could be estimated by subtracting the leak current in standard solution from kainate-induced currents at - 7 0 mV, since

314

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B

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V h = -70 mV, I mM kainate

kainate insensitive component

Fig. 3A-E Kainate blocks a transient K + current. A A glial cell was voltage clamped at -70 mV and the membrane was stepped for 50 ms to de- (20, 10, 0, - t 0 , -20 mV) and hyperpolarizing potentials (-120, -130, -140, -150, - t60mV). B Sub- sequently, current traces were compensated for leak and capacitance currents. Pulse protocols are schematically drawn in the insets. C To isolate the transient current component, voltage steps described above were applied starting from a Vh of --40 inV. The remaining sustained components were subtracted from corresponding control currents (B-C) yielding the transient current (D). Kain-

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ate (t raM) was applied to the bath solution while the cell was clamped at -70 inV. Membrane currents were recorded as described above. Data were taken when the kainate response reached its plateau phase (see arrow 2 in Figs. tB, E or 2A). The kainate- sensitive current component (F) was isolated by subtracting residual responses in the presence of kainate (E) from corresponding control currents (B-E). Insets in C-F display I/V curves of corresponding current components. Under control conditions the resting potential of the cell was -75 mV. To eliminate Na + currents 1 gM tetrodotoxin (TTX) was added to the bath solution

at that membrane potential no voltage-dependent inward or outward currents are activated in "complex" glial cells (C. Steinhfiuser, K. Kressin and M. Weber, in preparation; see also Figs. 3A, 6A, 7A, B). With voltage steps applied in the depolarizing direction, two processes overlap, the cationic conductance increase and the blockade of the outward K + currents. To isolate the latter, the receptor conductance increase, as determined from the hyperpolarizing voltage steps, was subtracted from the conductance determined by a depolarizing voltage step and plotted as a function of time. Assuming a linear I /V curve of the receptor channel (as shown in the presence of Ba 2+, see Fig. 1E 2-3), K + conductance (filled triangles) was calculated by subtracting receptor conductance from whole membrane conductance determined at 100 mV for each stimulation sequence.

The onset of the conductance changes was similar for both processes, reaching max imum values about 30 s after adding kainate to the bath (Fig. 2B). As seen in Fig. 2B, the K + outward conductance decreased to 25 % as compared to control levels (24.5 _+ 5.6%, n = 9). Both processes were induced within a few seconds; while the cationic conductance increase was terminated 30 s after kainate washout, the effect on the K + conductance lasted for more than 2 min.

It can be concluded that in this type of glial cell the kainate response consisted of at least two components: a receptor-mediated conductance increase and a decrease of a voltage-dependent K § channel conductance. These

events overlapped in time of onset and and gave rise to the non-linearity of I /V curves under control conditions (Figs. 1C, 2A, 2-1).

Kainate blocked the transient outward K + current com- ponen t

To identify the type of K + channel affected by kainate, we compared voltage steps with high time resolution. Tetrodotoxin (1 pM, TTX) was added to the bath to block voltage-gated Na + channels. K + outward currents in complex hippocampal glial cells consist of at least two components: a transient "A-type" current and a non- inactivating (sustained) delayed rectifying current (Fig. 3). The sustained component was separated by applying depolarizing voltage steps to the cell starting from a holding potential of - 4 0 mV (Fig. 3C). Subsequently, this family of current traces was subtracted from the control (Fig. 3B, holding potential Vh = --70 mV) which isolated the transient K + current (Fig. 3D). To answer the question which of these components were blocked by kainate we analysed membrane currents during kainate application with high time resolution using the same pulse protocol as above (Vh = --70 mV). Subtracting the residual, kainate-insensitive current (Fig. 3E) from a control recorded before application (Fig. 3B) the current component blocked by kainate was isolated (Fig. 3F).

A D

1 mM kainate na

50 ~tM CNQX 6 O s 6 0 s

I mM kainate B E

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315

Fig. 4A-L Kainate responses are reversibly blocked by the non- N-methyl-d-aspartate (NMDA) receptor antagonist 6-cyano-7- nitroquinoxaline-2,3-dione (CNQX). Membrane currents and changes of intracellular free Ca ~+ concentration ([Ca2+]~) were evoked by application of 1 mM kainate for 30 s. Kainate-induced inward currents were simultaneously recorded in the whole-cell mode at a V~ of - 7 0 mV (top panel). Changes in [Ca2+]i as determined by the fluorescence ratio F34o/F3so a r e displayed in the

middle panel. The bottom panel shows corresponding pseudocolour video images before, during, and after the peak response. The colour scale inset refers to increasing [Ca 2+] levels from blue to red. A - C First application of kainate, D - F application of kainate in the presence of CNQX. G - I After removal of CNQX, kainate evoked again an inward current and a transient change in [Ca2+]i. Resting potential was - 7 9 mV at the beginning of the experiment

A 1 mM kainate, 2 mM Ca 2+ D

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B ~1 -~'~ ~,11111 I

L2 * -* 100 200 I �9 II �9 I

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1 mM kainate, Ca 2+ free

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L ~ 0.2 100 200 I i I I I

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Fig . 5A-F, Kainate-induced changes of [Ca2+]i are abolished in nominal Ca2+-free bathing solution. In the top panel (A, D) inward currents induced by kainate at a Vh of --70 mV are shown. The middle panel (B, E) demonstrates the changes in [Ca2+]i as determined by the fluorescence ratio F34o/F38o. On the bottom (C, F) corresponding pseudocolour video images before, during, and after the peak response are shown. The colour scale inset refers to increasing [Ca 2+] levels from blue to red. Only in the presence of Ca ~+ in the bath solution kainate induced a transient increase in [Ca2+]~ (B, C) while the [Ca2+]i response was abolished in nominal Ca2---free solution (E, F). The inset shows the fluorescent image of the fura-2-filled cell under study. The bar denotes 10 ~tm. Resting potential was - 7 9 mV in standard bath solution

Proper t ies o f this ka ina te - sens i t ive current are s imi la r to the t ransient current shown in Fig . 3D. The ka ina te - induced b l o c k o f the t ransient c o m p o n e n t was i ncomple t e in on ly a few cases, whi le in the ma jo r i ty o f cel ls 1 m M kaina te comp le t e ly abo l i shed this current (see Fig. 3 ver- sus Figs . 1, 7).

Kainate activates Ca2+-permeable receptors in "complex" glial cells

To inves t iga te whether changes in [Ca2+]~, act ing as a second messenger , were r e spons ib le for the b l o c k a d e of the K + current , [Ca2+]~ levels were measu red dur ing

316

A

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Fig. 6A-C. Increase in [Caa+]i is not caused by activation of voltage-gated Ca z+ channels. To isolate voltage-gated Ca 2+ currents, Na § and K + channels were blocked adding 1 btM TTX and 10 mM BaC12 to the bath solution (B). Membrane currents were analysed according to the protocol described in Fig. tA. The membrane potential was depolarized up to 20 mV (50 ms, 20-mV increment, see inset) and voltage-activated ionic currents were recorded (A, B). Vh was -70 inV. Membrane currents are leak subtracted and compensated for capacitance currents. No inward (Ca 2+) currents could be recorded under these conditions (B). C KC1 (50 raM) was added to the normal bath solution and membrane potential was measured in the current-clamp mode (upper panel) while [Ca2+]~ was simultaneously recorded with the fura-system (lower panel). Note that no increase in [Ca2+]i was recorded although the cell was depolarized up to - 4 mV in high external K + solution

application of kainate using a fura-2-based video imag- ing system [23]. Hippocampal glial cells were voltage clamped (Vh = - 7 0 mV) and filled with the dye fura-2 by dialysis from the patch pipette. Thus, ion currents and changes of [Ca2+]~ could be measured simultaneously (Figs. 4 - 6 ) . Changes in the fluorescence ratio F34o/F38o (range 0 . 3 - 0 . 6 for resting level and 0 .6 -1 .3 for elevated [Caa+]i) were recorded as a measure for [Ca2+]~. In all cells tested (n = 16) application of 1 mM kainate induced a significant and reversible increase in [Ca2+]i (Fig. 4 A - C ) . The rise time of [Ca2+]i was about 20 s. Then, 1 - 2 rain after the application of kainate [Ca2+]~ reached the base level. Generally, repetitive application of kainate led to smaller changes and longer-lasting, elevated [Ca2+]~ levels. Co-application of the specific non- NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 gM), not only blocked the kainate-induced inward current (block to 3.4-+2.7%, range 0 - 6.5%, n = 4) but also reversibly blocked the ac- companying shift in [Ca2+]i (Fig. 4 D - F ) .

To decide whether Ca 2+ was released from intracellular stores or due to a transmembrane influx through receptor channels, we applied kainate in nominal Ca 2+- free bathing solution (Fig. 5, n = 3). We changed to the Ca2+-free solution, 8 - 1 0 min before testing kainate. Un- der these conditions kainate did not induce an increase [Ca2+]i (Fig. 5E), whereas the kainate-induced inward current remained (Fig. 5D).

To estimate the possible contribution of voltage- gated Ca ~+ channels to [Ca2+]~ changes, we selected conditions to isolate and facilitate Ca ~+ currents by applying 10raM Ba 2+ and i btM TTX to the bath solution (Fig. 6B). The membrane potential was stepped for 50 ms up to 20 mV (20-mV increment) starting from Vh = - 7 0 inV. No inward currents could be activated

under these conditions indicating that Ca 2+ channels were not present in the "complex" ceils (Fig. 6B). Fur- thermore, the possibility that Ca 2+ enters the cell via activation of voltage-gated channels can be excluded since elevation of the external K + concentration ([K+]o) from 5 to 50 mM did not lead to a transient rise in [Ca2+]i (Fig. 6C). In the current-clamp mode (n = 2) the cells were strongly depolarized (from resting potential of - 8 0 and - 6 5 mV to - 2 2 and - 4 mV, respectively) while in the voltage-clamp mode (Vh = - 7 0 mV, n = 1) the increase in [K+]o induced a large inward current (294 pA). In none of the three cells could an increase in [Ca2+]i be recorded (Fig, 6C).

These results confirmed that the kainate-induced increase in [Ca2+]i was not due to influx through voltage- gated Ca 2+ channels in this type of glial cells. They are compatible with the assumption that kainate triggered a Ca 2+ entry through the receptor channel.

Kainate-induced block of K + channels was independent of [Ca2+]~

In Bergmann glial cells activation of glutamate receptors leads to a block of K + currents mediated by an increase in [Ca2+]i. To test whether this mechanism was also present in complex hippocampal glial cells, kainate responses were investigated in nominal Ca2+-free external solution. In all cells measured (n = 7), the kainate-induced block of the transient outward current remained unchanged (Fig. 7), although no increase in [Ca2+]i was recorded under these conditions (Fig. 5 D - F ) . In addition, elevation of [Ca~+]i by applying the Ca z+ ionophore ionomycin (50 l.tM, n = 11), did not cause a reduction of the transient K + current (not shown). In con-

A 2 mM CaC12 B 0

317

control

0.5 nA 1__ D 10ms E

Ca 2+ - free

c o n t r ~ ! l

Fig. 7A-F. Kainate-induced block of K ~ conductance is (g~) not affected by low extracellular Ca 2+. Two glial cells were analysed in standard external solution (A-C) and nominal Ca2+-free solution (D-F). A, D The characteristic current pattern induced as the membrane was stepped for 50 ms to de- and hyperpolarizing potentials (range -160 to 20 mV, 20-mV increment). The pattern of voltage steps is shown in the inset. Subsequently, the stimulation sequence described in the legend of Fig. tB was used to analyse

F

1 mM kainate 0.5 nA~

0.2s

I mM kainate

membrane currents before (B, E) and about 1.5 min after kainate application (since at this time the reduction of g~ was still obvious while the receptor conductance was already mainly reduced) (C, F). Note that the kainate-induced block of the transient current remained in Ca2+-free solution. Resting potential of the two cells was -74 mV (A-C) and -69 mV (D-F). Vh was -70 mV in all cases

trast, in some cases a slight conductance increase was observed. Thus, it can be concluded that in the complex glial cells the kainate-induced block of K + conductance was not mediated by [Ca2+]i and that these transient K + channels were not sensitive to changes in [Ca2+]i.

Discussion

Properties of glutamate receptors in the "complex" gIial cells of the hippocampus

The glutamate receptor was characterized in a selected population of glial cells ( " complex" cells) in the stratum radiatum of the hippocampus. Previously, it was demonstrated that glutamate induced a receptor-mediated conductance increase as well as a block of voltage-dependent K + currents in these cells [29]. In the present study we have shown that receptor activation by the non- N M D A receptor agonist kainate could be blocked by the AMPA/kainate receptor antagonist, CNQX. Thus, it can be concluded that the "complex" glial cells express glutamate receptors of the AMPA/kainate subtype.

In normal K+-containing bath solution kainate application led to a complex electrophysiological response involving an increase in receptor conductance and a decrease of K + conductances as discussed below. After blocking the K + conductances of the "complex" glial cell by applying Ba 2+, the kainate-induced conductance increase could be isolated. The reversal potential of this current component was about 0 mV suggesting

a cationic conductance as described for AMPA/kainate receptors. As described for kainate responses observed in other cell types, the I/V relationship was linear.

Receptor activation and K + channel blockade

In addition to the activation of a cationic conductance, glutamate receptor activation by kainate led to a transient decrease of a voltage-gated K + conductance in the "complex" cells. Since the K + currents in most cases were only activated in the depolarizing direction, the receptor-induced conductance change could be dis- tinguished f rom the effect on K + currents. It was demonstrated that kainate mainly affected a transient outward K + current. Such a link between glutamate receptor activation and an effect on K + channels has been previously described, e.g. in horizontal cells of the retina [21] and in hippocampal neurons [6, 7]. Moreover, AMPA/kainate receptor activation in Bergmann glial cells also led to a transient reduction of the large passive and time- independent K + currents [25].

Link between glutamate receptor activation and K + current blockade

Our experiments suggest that the K + channel blockade was not mediated by an increase in [Ca2+]i . In contrast to Bergmann glial cells, these results imply that the block of transient outward K + channels by glutamate

318

receptor activation was independent of changes in [Ca2+]i. Such a CaZ+-independent link has also been described for hippocampal neurons [6]. In that study, how- ever, this effect was not mediated by ionotropic receptors, but rather by the metabotropic glutamate receptor activated by trans-l-amino-l ,3-cyclopentanedicarboxyl- ate (trans-ACPD). In contrast, Kaneko and Tachibana [21] suggested that glutamate might directly act on K + channels. In their system (horizontal cells of carassius auratus retina), glutamate reduced the number of avail- able inwardly rectifying K + channels. The delayed removal of K + current blockade in our preparation rather suggested that a second messenger system distinct f rom Ca 2+ is involved. One possible mechanism could be mediated via cyclic AMR Baba et al. [1] demonstrated that glutamate inhibits adenosine 3' ,5 '-cyclic monophosphate (cAMP) formation in cultured astrocytes. As a next step in that cascade, one could speculate that K + channels could be modulated by phosphorylation via a cAMP- dependent protein kinase [5]. It seems unlikely that the K + channel block is mediated via the nitric oxide (NO)/ guanosine 3' ,5'-cyclic monophosphate (cGMP) pathway as neither the NO donor nitroprusside, the scavenger haemoglobin, nor the NO synthase inhibitor N~-nitro-L - arginine affected glial K + currents in the hippocampal slice (K. Kressin and C. Steinh~iuser, unpublished results). Further experiments are needed to elucidate the link between glutamate receptor activation and K § channel blockade.

Molecular properties of the glutamate receptor in "complex" glial cells

So far, nothing is known about the molecular structure of glutamate receptors in "complex" glial cells of the hippocampus. Our data suggest that the receptor is an AMPA/kainate receptor. We could demonstrate that activation of this receptor leads to an increase in Ca 2+ by influx through the receptor channel itself. In contrast to Bergmann glial cells the I/V relationship was linear in this type of hippocampal glial ceils. We conclude that this glial glutamate receptor has properties very similar to recombinant high-affinity kainate receptors containing the edited forms of GluR-5 or GluR-6 subunits in combination with KA-2 subunits [15, 19]. Recently, it was shown that the GluR-6 and KA-2, but not GluR-5, subunits are expressed in the hippocampus [16, 19].

Functional implications

K + channels in glial cells have been shown to be involved in functional mechanisms. First, they control the spatial buffer currents which are thought to be responsible for K + clearance in the brain [26]. Second, it has been demonstrated for Schwann cells that K + channel activity is involved in the control of proliferation. Block- ade of K + channel activity directly interferes with the proliferative state [8]. Indeed it has been demonstrated that glutamate receptor activation results in a reduction

of astrocytic proliferation in culture [13]. Thus, glutamate receptors could interfere with these two func- tionally important mechanisms. It is open for future research to demonstrate that these mechanisms are indeed operative in the intact CNS.

Acknowledgement. This research was supported by the German Federal Ministry for Research and Technology (grants to H. K. and C. S.), the Gertrud Reemtsma Foundation (stipend to R. J.), Carl Zeiss Jena and the Deutsche Forschungsgemeinschaft (grant SFB 317). The authors thank I. Krahner for excellent technical assistance.

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