GLIA6:11&126 (1992)

Calcium Dependence of Serotonin-Evoked Conductance in C6 Glioma Cells DAVID MANOR, NAVA MORAN, AND MENAHEM SEGAL Department of Neurobiology, The Weizmann Institute, Rehouot 76100, Israel

KEY WORDS

5-HT receptor, Intracellular Ca2+,K' conductance, C1- conductance, Patch-clamp

ABSTRACT Whole-cell membrane currents and imaging of intracellular calcium concentrations ([Ca"],) were used to investigate the role of calcium in a response to serotonin of C6 glioma cells. Activation of a high-affinity serotonin receptor induced a transient rise in calcium concentration in these cells and activated a predominantly potassium conductance, with a small chloride component. Perfusion of the cytoplasm with an internal solution containing high calcium concentration induced similar but prolonged increase of membrane conductance. The responsiveness of C6 cells to serotonin was negatively correlated with the concentration of the unbound calcium chelator BAPTA when BAPTA-buffered calcium-containing intracellular solutions were used. Responses to serotonin persisted in the absence of external calcium, decreased gradually, and then recovered partially after replenishment of extracellular calcium. These findings substantiate the direct role of intracellular calcium in mediating the serotonin response, and indicate that serotonin-induced release of calcium from intracellular stores is sufficient for the activation of conductance in the C6 glioma cell line. 8 1992 Wiley-Liss, Ine.

have been described in glial cells (Hansson, 1988). Among them is a serotonin receptor that stimulates One of the crucial functions of glial cells in the central phosphatidylinositide (PI) turnover and induces memnervous system (CNS) is to regulate the extracellular brane hyperpolarization in astrocytes (Hansson et al., potassium ion concentration ([K'],) (Barres et al., 1987; Hosli et. al., 1987; Walz and Schlue, 1982). 1990; Walz, 1989). If not regulated, potassium efflux C6 glioma cells, a common model for glial cells from activated neurons would readily increase [K 'I,. (Benda et al., 1968) are relatively homogeneous, small Accumulation of [K+I, would tend to alter neuronal ex- sized, and nonbranching, and express a large and concitability and synaptic transmission (Sykova, 1983). In sistent electrogenic response to serotonin. These feathe proposed mechanisms of [K+], regulation, potas- tures make them an ideal model system for the physiosium flux, possibly accompanied by chloride flux logical analysis of neurotransmitter signal transthrough channels in the membranes of adjacent glial duction. cells, is suggested as the immediate regulator of LK+lo Serotonin induces hyperpolarization and elevation of (Barres et al., 1988; Newman et al., 1984). ICa2'l, in C6 cells (Ogura and Amano, 1984; Ogura Various potassium channels-voltage-dependent et. al., 1986; Sugito et al., 1984). It has been suggested and calcium-dependent-have been implicated in that these responses depend on influx of extracellular mechanisms of [K'], regulation by glia. In addition, calcium. Serotonin activates PI turnover and inositolvoltage-dependent chloride channels and calcium chan- phosphate accumulation in these cells (Ananth et al., nels have been included in the proposed models (Barres et al., 1990; Walz, 1989; Quandt and MacVicar, 1986). Modulation of glial potassium conductance by neuroReceived October 17,1991;accepted January 14,1992. transmitters may be part of neuron-glia interaction. Address reprint requests to Dr. David Manor at the address given above Several functional receptors for neurotransmitters INTRODUCTION

0 1992 Wiley-Lm, Inc

SEROTONIN-EVOKED CONDUCTANCE IN GLIAL CELLS

1987). Since 1,4,5-inositol-trisphosphate UP3),a product of PI metabolism, initiates release of calcium from intracellular stores (Berridge and Irvine, 1989; Rana and Hokin, 1990), we reevaluated the relative roles of both sources of calcium (internal vs. external) in the response to serotonin in C6 cells. Using the patch-clamp technique we controlled the internal milieu (Hamil et al., 1981), in particular [Ca"Ii. In a separate set of experiments we used Fluo-3 imaging of [Ca"], (Kao et al., 1989) in a confocal laser scanning microscope to monitor calcium changes associated with the response to serotonin in C6 cells. We demonstrate that intracellular calcium stores are sufficient, at least initially, for producing a response to serotonin in C6 cells, which consists of activation of a large potassium conductance accompanied by a small increase of chloride conductance.

119

with KOH to pH 7.2, BAPTA (tetrapotassium salt) 0.2, CaC1, 0.02. Nucleotidetrisphosphate (NTP)-regenerating components including ATP 1,GTP 0.1, and creatine phosphate (TRIS salt) 20 (Stelzer et al., 1988) were routinely added to the solution to avoid wash-out of the response (creatine phosphokinase was added in some experiments, but as it appeared not to be essential, it was omitted from later experiments). When different free calcium concentrations were required, the free calcium concentrations were calculated using the BAPTAcalcium KDvalue of 110 nM.

Intracellular Calcium Imaging

Cells were plated on fire-cleaned 12 mm glass coverslips 1or 2 days prior to the experiment. The cells were preincubated at room temperature for 1 h in external solution containing 10 pM Fluo-3-AM and 0.5 mg/ml MATERIALS AND METHODS pluronic acid F-127 (Kao et al., 1989). A confocal argon laser scanning microscope (CLSM, C6 cells were cultured in DMEM supplemented with Leitz, Germany) was used for calcium imaging. A cover10% heat-inactivated horse serum (Whitaker-Azmitia slip was mounted, inverted, on a 150 pl perfusion chamand Azmitia, 1986). We used two methods to study the ber under a 50x water-immersion objective, which enserotonin effects: patch-clamp, to measure membrane abled us to view three to ten cells in a field. Fluo-3 was conductance and to manipulate the intracellular mi- excited at 488 nm, through 90% neutral density filter to lieu; and intracellular calcium imaging of intact cells to reduce photodynamic damage, and emission was monimonitor change in [Ca2+Iilevel. tored at 512 nm. The cells were continuously perfused with external solution, supplemented with 5 mM glucose, using gravitational force on the out-flowing fluid Patch-Clamp Experiments column. Lowering the draining tubing outlet created a larger gravitational force that maintained a flow of 3 Cells were plated at low density in 35 mm Petri mumin during the switch to drug-containing external dishes (Nunc) 24-48 h before use. Experiments were solution, and the field was scanned at a rate of 3 conducted with cells that did not have apparent connec- framesh, yielding images of 128 x 128 pixels. At the tions with neighboring cells for better control of mem- end of the scanning procedure, the cells were washed brane potential and intracellular milieu. quickly and thoroughly with drug-free solution. The Thin-wall borosilicate glass pipettes (1.5 mm outer images were saved on an optical disk for later analysis. diameter) were pulled on a vertical puller, and fireRelative responses to serotonin are normalized by the polished to yield a tip diameter of approximately 2 pm. maximum response that could be induced in a particuCells were voltage-clamped in a "whole-cell"configu- lar cell (patch-clamp experiments) or group of cells (calration using Axopatch-1B amplifier and a software- cium imaging experiments), usually using 1-10 pM of hardware control (pCLAMPprogram, TL1 and TM-100 serotonin. Labmaster A/D and D/A peripherals; Axon Instruments). The membrane potential (corrected for the measured Data Analysis junction potentials between the internal and external solutions) was usually clamped to -60 mV. Membrane The dependence of reversal potential, E,,,, on extercurrents were recorded on a pen-recorder or were digi- nal KCl was fitted with the Goldman equation (Goldtized and stored for further analysis. man, 1943): Serotonin (100 pM in external solution) was usually applied by diffusion from a 2 pm wide pipette placed PNa 10-20 pm away from the cell. In experiments in which [K'], "a+], - [Clkli RT PK PK timing of application or exact concentrations were cru- Ere, = -In (1) F P N a Pa cial, serotonin was pressure-ejected from a micro-pi[Kt]i __ [Na+]i -[Clk], pette with less than 1 pm tip situated near the cell. PK PK Standard solutions contained (in mM): external solution: NaCl 140, KC1 2.5, CaCl, 2, MgCl, 2, HEPES 10, where [K+], [Cl-1, and "a+] are the activities of K', titrated with NaOH to pH 7.4; internal solution: potas- Cl-, and Na+,respectively (function of their concentrasium gluconate or KCll40, MgC1,2, HEPES 10 titrated tions; see Robinson and Stokes, 1965),P,, PNa,and P,,

+ +

+ +

120

MANOR ET AL.

are the permeability of potassium, sodium, and chloride, respectively, and R, T, and F have their usual thermodynamic meanings. All ion concentrations were held constant except that of K', which w a s varied by partial substitution of Na+ in the external solution. We determined the parameters (C,) in the Hill equation by fitting equation 2 to obtained dose-response relationships:

1

+(3c3 -

where y is the response, x the agonist concentration, c1 the maximum response, c2 the half-effective concentration, and cg the Hill coefficient. K, values for serotonergic antagonists were calculated from the equation (Mahan, 1975):

(3)

0

NTP regenerating

10

20

time after 1 st serotonin application

(min.)

where EC,,,,,, and EC5,, are the half-effective concentrations of serotonin in the presence or absence of a n antagonist, respectively, and [I] is the antagonist concentration. Chemicals were purchased from Sigma (serotonin, ATP, GTP, creatine phosphate), Molecular Probes (Fluo-3 AM), Calbiochem (BAPTA), and Research Biochemicals Inc. (serotonergic agonists and antagonists). RESULTS Serotonin-Induced Current Application of serotonin (1-100 FM) onto more than 100 C6 cells caused a typical electrogenic response consisting of a rapid (1-5 s) and marked increase in membrane conductance (by 2 0 4 0 nS, Fig. lA,B). At a holding potential of -60 mV we measured a large outward current of 500-1,200 PA. Usually the current inactivated during continuous application of serotonin, but occasionally it reactivated repeatedly. Therefore, we preferred short applications of serotonin (5-10 s), which yielded more uniform responses lasting for 15-60 s (decay to half maximum in 9.8 2 0.9 s in a sample of ten cells). Repeated applications of serotonin, when using a standard non-supplemented intracellular solution, resulted in a progressively smaller response, which tended to disappear within a few minutes after breaking the patch to attain the whole-cell configuration (Fig. lA,C). Reproducible responses to serotonin could be achieved with the addition of NTP-regenerating components to the intracellular solution (Fig. 1B,C). Consequently, in all patch-clamp experiments described below, these components were routinely included in the internal solution.

Fig. 1. An NTP-regenerating system is required for the maintenance of electrogenic responses to serotonin in C6 cells. A Current trace demonstrating outward membrane current in response to repetitive applications of serotonin (100 p M , horizontal bars). Cells were clamped at a holding potential of -53 mV with superimposed 1 s, -25 mV hyperpolarizing pulses, to demonstrate increase of membrane conductance. Diminution of the serotonin-induced membrane current with a non-supplemented standard internal solution in the pipette occurs several minutes after attaining the "whole-cell'' configuration. B: Diminution is markedly reduced by addition of NTP-regenerating components to the pipette solution. C: Time-course of change of the amplitude of the serotonin-induced current without and with NTPregenerating system (each point represents an average peak current i S E M of three to ten measurements).

Ions Carrying the Serotonin-Induced Current The peak amplitude of the serotonin-induced current was linearly related to membrane potential. Figure 2A,B shows serotonin-induced currents at different membranes potentials with 140 mM KC1 in the pipette and 20 mM KC1 outside the cell. The current reversed a t -32 mV, which is above the calculated Nernst potential for potassium in these conditions (-47 mV). Ramp voltage commands were applied during the responses to serotonin to find the dependence of E,,, on the extracellular KC1 concentration (Fig. 2C). The results could be fitted by the Goldman equation (Eq. 11, yielding a K' to C1- permeability ratio of about 60, with K' to Na' permeability ratio larger than 1,000 (Fig. 3D). The effect of the presumed chloride conductance could be prevented by replacing KC1 in the internal solution with potassium gluconate. In these conditions the reversal potential of the response to serotonin closely followed the Nernst potential for K' (Fig. 2D). The peak outward current was reduced by replacement of internal potassium by cesium (Fig. 3A). The response to serotonin was reduced by 75% after the

SEROTONIN-EVOKED CONDUCTANCE IN GLIAL CELLS

121

*

B

-

- 1 00

11.5 1.0 s). The time-course of the change in calcium level (Fig. 4E) was similar to that of the serotonininduced current (compare with Fig. lA,B). Dose Dependence of the Responses to Serotonin

___I

100 ms

-

/ I000PA

A

__I

rn looms n n -50

-90-50-10 E M

-50

(mv)

Fig. 2. Serotonin-activated current is carried mainly by potassium. Internal solution usually contained KC1140 mM. NaCl in the external solution was replaced part for part by the indicated concentrations of KCI. A The direction of current (lower series of superimposed traces) induced by serotonin (puff of 1 kM, horizontal bar) depends on membrane potential (lower traces, voltage jumps to -80, -60, -40, and -20 mV from a holding potential of -50 mV). B: Peak current to membrane potential (EM)relationship is linear and current reverses at -32 mV (calculated Nernst potential for potassium, -42 mV). C: Ramp voltage commands (1 s, ranging between peak values of -90 mV and -10 mV, upper trace) were applied at various stages during serotonin-induced responses, eliciting ramp-shaped currents (lower panels). Each series of superimposed traces belongs to one serotonin application. Larger transmembrane currents correspond to a phase of increased conductance. Due to linearity of voltage commands with time, time base can be converted to membrane-potential base (bottom scale). The cross-over points (arrowheads) correspond to reversal potential of the currents. Note the shifted reversal potential of the currents in different external KCl concentrations. D: The reversal potential dependence on KC1 could be fitted (squares, continuous line, R = 0.99)with the Goldman equation (Eq. 2), yielding a permeability ratio between K ' and C1- of 56, with a permeability ratio between K ' and Na' larger than 1,000 (each point represents four to five cells). The reversal potential of the response to serotonin, after replacement of KCI in the internal solution by K' -gluconate (triangles, each point represents one to four measurements), closely follows the Nernst potential of K+ (dashed line).

addition of 20 mM tetraethylammonium (TEA) to the external solution (Fig. 3B). These experiments suggest that the serotonin-induced electrogenic response in C6 cells is mediated mainly by a potassium current, with a possible minor contribution of a chloride current. Serotonin-Induced [Ca2+IiElevation Imaging of Fluo-3-loaded cells with the CLSM revealed a transient rise of intracellular calcium concentration during a 1 min superfusion of C6 cells with serotonin (Fig. 4, decay to half-maximum response in

The dependence of the serotonin-induced outward current on serotonin concentration was assayed in patch-clamp experiments by random puff-applications of varying concentrations of serotonin (up to 10 pM). The half-effective dose of serotonin (ED50)was about 9 nM and maximum response was achieved with concentrations above 50 nM. The dependence of [Ca2+Iielevation on serotonin concentration, measured with the CLSM, yielded an ED,, of 35 nM and a maximum response at about 1 p.M serotonin (Fig. 5 ) . Membrane Conductance Directly Activated by [Ca2+li In order to explore the dependence of the current on [Ca2+I1, we manipulated intracellular calcium concentration using patch-clamp in "whole-cell"configuration. Outward current could be induced upon breaking into cells with pipettes containing high levels of calcium (Fig. 6 and Table 1). In these conditions the peak amplitude of the initial current was directly related to lCa2'l, (Fig. 6C). E, of [Ca2' ],-induced current shifted in different external K+ concentrations according to the Goldman equation (Eq. 11, yielding a K' to C1- permeability ratio of 17 and K' to Na' permeability ratio larger than 1,000 (Fig. 6D). These findings indicate that elevation of [Ca"], activates a membrane conductance, which resembles the serotonin-induced conductance, although it has a 3 to 4 times larger chloride component. [Ca2+liDependence of the Response to Serotonin Clamping [Ca2+lia t a constant level could prevent the subsequent electrogenic response to serotonin. Applying intracellular solutions containing different calcium-BAPTA ratios (Table l ) demonstrated that the fraction of cells responding to serotonin was inversely linearly dependent (R = -0.94) on free BAPTA concentration, which determines the calcium buffering capacity (Fig. 7). Only the (presumed) saturation of the response by high concentration of calcium inside the cell abolished this relationship (see Figs. 6B and 7, arrow). No such correlation was found between cell responsiveness to serotonin and total BAPTA, total calcium, or even free calcium (Table 1). This finding indicates that preventing the elevation of [Ca2+li,abolishes the increase of serotonin-induced, calcium-dependent potassium conductance.

MANOR ET AL

122

A "O0:

-a

B

1001

1000.

800.

a .-c Q E m

600400-

200 -

"

n,

140\0

70\70

0\140

[K' ] (mM) '\ [Cs'] (mM)

TEA

OmM

TEA 20mM

Fig. 3. Peak current in response to serotonin is affected by potassium channel blockers. A Outward current is blocked by replacement,of potassium gluconate with cesium sulphate in the pipette (from left to right three, five and three cells). B: Tetraethylammonium (TEA) (20 mM) partially blocks the serotonininduced current (data from five cells, normalized as in Materials and Methods).

Serotonin-Induced Responses in Calcium-Free External Solutions

DISCUSSION

Serotonin induces increased membrane conductance and potassium current in the C6 cell line (Figs. 1, 2). This observation extends earlier reports on serotonininduced hyperpolarization in this cell line (Ogura and Amano, 1984). We also observe (Fig. 41, as has been described before (Sugito et al., 1984), that serotonin induces transient elevation of ICa2+li with a similar time-course as the electrogenic response. The existing experimental evidence correlates the effect of serotonin on membrane conductance with its effect on lCa2+li. However, the immediate causative link between the two responses is still lacking. Our new results substantiate the role of calcium as the intracellular mediator of the response to serotonin in the following ways: 1)increase of membrane conductance can be induced by perfusing the cytoplasm with The Serotonin Receptor in the C6 Cell calcium-containing solutions (Fig. 6); 2) the response to serotonin is not additive with the conductance increase To characterize further the serotonin receptor-the starting point of the serotonin-signal transduction cas- achieved by perfusing the cytoplasm with high [Ca2+li cade-we examined the effects of several serotonin ago- (Fig. 6A,B); and 3) the responsiveness of C6 cells to nists and antagonists on [Ca"'], elevation (Fig. 9). The serotonin is negatively correlated with intracellular 5-HT2 agonist tx-methylserotonin produced a dose-re- calcium buffering capacity (Fig. 7). It should be noted sponse curve similar to that obtained with serotonin. that the effectiveness of the calcium buffer BAPTA beThere was no effect of the 5-HT1 agonist 5-carboximi- comes pronounced only when its free component in the dotryptamine (5-CT)or the 5-HT1, agonist 8-OH-DPAT solution is in the millimolar range. This observation (8-hydroxy-dipropylaminotetralin)in concentrations may indicate that the serotonin-induced current reup to 100 pM. The response to serotonin was dimin- quires a robust calcium flux into an extensively buffished by the nonselective 5-HT antagonist mianserin ered sub-membrane compartment. Indeed, the calciumand the 5-HT2-selectiveantagonist ketanserin. Spiper- buffering capacity of membrane lipids is in the one (10 nM) potently inhibited the response to seroto- millimolar range (Carafoli, 1987). We demonstrated that extracellular calcium influx is nin in a non-competitive manner. This pharmacological profile is suggestive of the 5-HT2 sub-class of the sero- not required, initially, for the response to serotonin tonin receptors, which reportedly utilizes IP, as a sec- (Fig. 8). The discrepancies between our results and those of others, who suggested that the serotonergic ond messenger (Frazer et al., 1990; Peroutka, 1988).

Serotonin could induce an electrogenic response as well as a rise of [Ca"], when extracellular calcium had been washed away with calcium-free external solution containing 4 mM BAPTA (Fig. 8). Only upon repeated serotonin applications did both responses decrease. After washing the cells with calcium-containing solution the responses recovered partially. These results indicate that the intracellular calcium stores are sufficient to activate the calcium-dependent potassium conductance, although these stores probably are depleted during consecutive applications of serotonin, and extracellular calcium is needed for their reloading.

123

SEROTONIN-EVOKED CONDUCTANCE IN GLIAL CELLS o fluorescence

1 .o

n

current

0.8 0.6

0.4

0.2 0.0

101

loo

101

102

103

to'

serotonin (nM) Fig. 5. Dose-response relationships, fitted with Eq. 1,of the serotonin-induced outward current (EC,, 9.3 nM, Hill coefficient 3.3, data from five cells) and serotonin-induced rise of lCa2' I, (EC,, 35 nM, Hill coefficient 0.8, data from 14 experiments). Inset: Current activation curve was constructed by plotting the fitted values of the relative current against the relative fluorescence induced by the same serotonin concentrations, yielding half-current activation at 0.28 of fluorescence-increase and Hill coefficient of 3.9.

1 i\

close to 4; Figs. 5 , 6). We estimate that half-maximum activation of membrane conductance is achieved at 0.8 [Ca2+Iiof 280 nM (Fig. 6). In this concentration, which is lower than the KDof Fluo-3 (400 nM), the change in [Ca2+lican be assumed to be linearly represented by changes in fluorescence level. Nevertheless, monitoring [Ca2+liwith dual wavelength calcium indicator should yield more accurate estimation of current activation by [Ca2+I,. The direction of serotonin-activated current depends on membrane potential (Fig. 2 ) . Application of the 0.0 patch-clamp technique allows better control of mem0 1 0 20 30 4 0 5 0 brane potential than methods applied before for studytime (s) ing serotonin-induced responses in glial cells, and also allows accurate determination of reversal potentials. Fig. 4. Transient rise of intracellular calcium concentration induced The dependence of the reversal potential of the serotoby serotonin. Fluorescence signal from Fluo-3-loaded cells before (A), nin-induced current on KC1 concentration shows that it at the peak (B), and after (C) serotonin-induced response. The maximum net increase of fluorescence (D) was obtained by digitally sub- is carried mainly by potassium with a small contributracting A from B. Note that the elevation of calcium concentration (as tion of chloride (K' to C1F permeability ratio of 60; Fig. indicated by the net fluorescence increase) is more prominent in the 2D). The role of potassium as the carrier of the current nuclear area, identified by observation in transmitted light. Scale bar = 5 pm. E: Time-course of average net change of fluorescence becomes pronounced by replacing C1- by gluconate in induced by serotonin application (1 )LM, horizontal bar) in the cell the internal solution. In this condition the reversal podelimited by the rectangle. tential of the response to serotonin depends on [K+],, as predicted by the Nernst potential for potassium. The involvement of chloride is more pronounced in the coneffect on C6 cells is mainly dependent on extracellular ductance increase induced by perfusing the cytoplasm calcium (Ogura and Amano, 1984; Ogura et al., 1986) with high [Ca2+Ii(Fig. 6; Kt to C1- permeability ratio of may arise from the tendency of C6 cells to alter their about 17).In both of these situations the sodium compophenotype in different culturing conditions or even nent is very small (K+ to Na+ permeability ratio larger spontaneously (Mangoutra et al., 1989), or may be due than 1,000). The difference between calcium-dependent conducto different experimental conditions and methods. Interestingly, a similar chain of events has been sug- tances activated by serotonin and cytoplasm perfusion gested in the response of C6 cells to bradykinin (Reizer with calcium may be: 1)spatial, i.e., serotonin activates et al., 1990). The activation of membrane conductance mainly potassium channels localized near submembraby [Ca2'Ii shows high cooperativity (Hill coefficient nal intracellular calcium stores, while the perfusing

124

MANOR ET AL

B

D

C

Fig. 6. Conductance induced by [Ca” ’ I,. A: Transient initial outward current, induced upon breaking into a “whole-cell”configuration (arrowhead), with intracellular solution containing 300 nM free calcium (total calcium 1.5 mM, total BAFTA 2 mM), subsequently followed by a response to serotonin application (100 p M , horizontal bar). B: Prolonged initial outward current was similarly induced by 600 nM free calcium in internal solution (total calcium 1.7 mM, total BAF’TA 2 mM) but the cell did not respond to a subsequent application of serotonin. C: The dependence of the initial current amplitude on [Ca”]],. Symbols: averaged amplitudes of currents induced by breaking into cells with pipettes containing 0.1, 0.3, and 0.6 pM calculated ICa”], with 2 mM BAPTA (from Table 1).Line: best fit (R = 0.94) with Eq. 1,

using a Hill coefficient of 3.9 (from current activation curve, Fig, 5, inset), yielding half-activation a t 0.3 pM of ICa2 ’ I and maximum current of 1,139 PA. D: The reversal potential of [Cali ],-inducedcurrent was varied by partial substitution of NaCl by KCl in external solutions. The internal solution contained 140 mM potassium chloride. Membrane current was induced by 600 nM free calcium in internal solution. The reversal potential of the current was determined by 1 s ramp voltage commands. The reversal potential shift was fitted by the Goldman equation (Eq. 1) yielding a permeability ratio of about 17 between K+ and C1-, and a permeability ratio of about 1,000 between K ’ and Na ’ (average data from two cells).

TABLE 1 . Cell responsiveness to serotonin at various combinations of calcium-BAPTA buffera

No. of tested cells 11

I

Total BAPTA (FM)

2,000 2.000

2;ooo

13 11 6

2,000 1,100 1,100 1.000 200 200 200

Total calcium (PM)

Calculated free calcium (PM)

200 1.000 1;500 1,700 550 940 100 20 100 170

0.01 0.1 0.33 0.6 0.1 0.6 0.01 0.01 0.1 0.6

Calculated unbound BAPTA (PM)

1,800

1.ooo

500 300 550 160 900 180 100 30 ~~~

Fraction of cells responding to serotonin 0.18 0.57 0.8 0 0.60 0.80 0.75 0.85 0.91 1

aThe ability of serotonin to induce potassium conductance was tested in the presence of the indicated calcium buffering systems in the pipette solution, in whole-cell configuration.

solution acts more diffusely on both chloride and potassium channels; or 2) temporal, i.e., while the inactivation of the response to serotonin starts after few seconds (Figs. 2, 3), the response to cell perfusion is more prolonged (time scale of minutes, as in Fig. 6), and may thus reveal slowly activating calcium-dependent chloride conductance.

Serotonin induced a n increase in membrane conductance and elevation of [Ca2+Iiwith high efficacy (ED,, of 10 nM and 35 nM, respectively; Fig. 5). Our findings are in accord with binding studies performed on this cell line by Whitaker-Azmitia and Azmitia (1986), who found a high-affinity binding site to serotonin (K, 12 nM). The pharmacological profile of the receptor (acti-

125

SEROTONIN-EVOKED CONDUCTANCE IN GLIAL CELLS

o serotonin o+ketanserin 100 nM a +ketanserin 10 nM ~+mianserin1 nM

1.0 43

0

2

0.0

Lc

1

I

0

1.5

1

0.5

unbound BAPTA

2

(mM)

Io"

Ioo

10'

I o2

Io3

10'

I

o5

agonist (nM) Fig. 7. Intracellular calcium buffering reduces the responsiveness of the cells to serotonin (see Table 1).The fraction of cells in which serotonin induced an outward current correlated inversely (R = -0.94) with the unbound BAPTA concentration in the patch pipette. Arrow, a group of cells perfused with an intracellular solution containing 1.7 mM calcium and 2 mM BAPTA (with calculated free calcium 600 nM) does not fit the line, presumably because of the maintained maximum initial activation of conductance by the high calcium in the pipette (see Fig. 6B). 1.01

I

1 -

[Ca"]

*..l...l. ...I1..I* ..2mM . .... ...-....[Ca"]

I

0.0

-

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-

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".......*...I._..._...1

-

-

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Fig. 9. The effects of serotonergic agonists and antagonists on [Ca''], elevation. Serotonin and a-methylserotonin have similar high efficacies (ED,, of 35 nM and 40 nM, respectively). The non-specific 5-HT antagonist mianserin at 1 nM concentration shifts the apparent ED,, of serotonin to 996 nM (4,calculated by Eq. 3,36 pM). The 5-HT2 antagonists ketanserin 10 nM and 100 nM, and spiperone 10 nM (broken lines) shift the apparent ED,, of serotonin to 121 nM, 624 nM, and 1,476 nM, (calculated K,s for k e t a n s e r i n 4 . 1 and 5.9 nM; 24 nM for spiperone). a-Methylserotonin and the antagonists were tested in two to five experiments each.

to use them a s one; see Mangoutra et al., 1989) . The electrogenic response to serotonin in native glia is smaller and less consistent than that of C6 cells (Hosli et al., 1987; Ogura and Amano, 1984).The ionic conductances ofthese cell types also differ (Picker et al., 1981). Nonetheless, our data may offer the following implications, relevant to the function of glia in the CNS. The existence of high-affinity receptors on glial cells may have physiological significance. While concentration of serotonin in the synaptic gap may be relatively high, only small amounts may escape the efficient uptake mechanism to diffuse away and convey signals to the neighboring glial cells. Indeed, microdialysis experiments indicate that serotonin concentration around stimulated nerve fibers rises only within the nanomolar range (Sharp et al., 1989). Neurotransmitter-activated conductance, like the one described in our experiments, may participate in [K'l, buffering (the "spatial potassium buffering mechanism''; Barres et al., 1988; Newman et al., 1984). The chloride component of the serotonin-induced conductance, which was demonstrated to be activated in low [K'],, would help to "clamp" the membrane potential away from the E,. As a result, potassium efflux from the glia cells would be maintained, relieving these cells from the potassium load.

*ll...-...*.l..."l..."

sequential applications of serotonin Fig. 8. Responses to serotonin persist in calcium-freemedium. Serotonin-induced rise of intracellular calcium concentration as indicated by fluorescence increase in Fluo-3-loaded cells ( 0 ) and serotonin-induced potassium current (o),in the presence or absence of calcium in the external solution (4 mM BAPTA was added to 0 calcium solution). Current responses were induced by 100 pM serotonin applied by perfusion a t 2 4 min intervals (data from six cells). Fluorescence increase was produced by superfusion with 1 pM serotonin a t similar intervals (seven groups of cells).

vation by a-methylserotonin, potent inhibition by mianserin and spiperone, inhibition by ketanserin, and ineffectiveness of 5-CT and 8-OH-DPAT) indicates that the serotonin receptor in C6 belongs to the 5-HT2 subtype (Peroutka, 1988). Interestingly, this receptor subtype, originally classified based on its low affinity to serotonin, was efficiently activated by serotonin in our experiments. This finding may be explained by multiple-affinity states of 5-HT receptors, which probably reflect their interaction with G proteins (Frazer et al., 1990). A possible criticism of the use of C6 cells is that they are not a good model for glial cells (albeit it is accepted

REFERENCES Ananth, U.S., Leli, U., and Hauser, G . (1987) Stimulation of phosphoinositides hydrolysis by serotonin in C6 glioma cells. J . Neurochem., 48:253-261. Barres, B.A., Chun, L.L., and Corey, D.P. (1988) Ion channel expression by white matter glia. Glia, 1:1&30.

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