ion channel expression by white matter glia: i. type ...corey.med.harvard.edu/pdfs/1988 barres corey...

GLIA 1:10-30 (1988)

Ion Channel Expression by White Matter Glia: I. Type 2 Astrocytes and Oligodendrocytes

BARBARA A. BAEtRES,'9Z93 LINDA L.Y. CHUN,'s3 AND DAVID P. COREY',2,3 'Department of Neurology and 'Neuroscience Group, Howard Hughes Medical Institute, Massachusetts General Hospital, Wellman 414; and 3Program in Neuroscience, Haruard Medical School, Boston, Massachusetts 02114

KEY WORDS Optic nerve, Patch-clamping, Potassium regulation, Sodium channel, Chloride channel

ABSTRACT White matter is a compact structure consisting primarily of neuronal axons and glial cells. As in other parts of the nervous system, the function of glial cells in white matter is poorly understood. We have explored the electrophysiological properties of two types of glial cells found predominantly in white matter: type 2 astrocytes and oligodendrocytes. Whole-cells and single-channel patch-clamp techniques were used to study these cell types in postnatal rat optic nerve cultures prepared according to the procedures of Raff et al. (Nature, 303:390-396, 1983b). Type 2 astrocytes in culture exhibit a "neuronal" channel phenotype, expressing at least six distinct ion channel types. With whole-cell recording we observed three inward currents: a voltage-sensitive sodium current qualitatively similar to that found in neurons and both transient and sustained calcium currents. In addition, type 2 astrocytes had two components of outward current: a delayed potassium current which activated at 0 mV and an inactivating calcium- dependent potassium current which activated at -30 mV. Type 2 astrocytes in culture could be induced to fire single regenerative potentials in response to injections of depolarizing current. Single-channel recording demonstrated the presence of an outwardly rectifying chloride channel in both type 2 astrocytes and oligodendrocytes, but this channel could only be observed in excised patches. Oligodendrocytes expressed only one other current: an inwardly rectifying potassium current that is mediated by 30- and 120-pS channels. Because these channels preferentially conduct potassium from outside to inside the cell, and because they are open at the resting potential of the cell, they would be appropriate for removing potassium from the extracellular space; thus it is proposed that oligodendrocytes, besides myelinating axons, play an important role in potassium regulation in white matter. The conductances present in oligodendrocytes suggest a "modulated Boyle and Conway mechanism" of potassium accumulation.

INTRODUCTION sible that the glial cells in the CNS white matter con- tinue to play functional roles in the adult. An efficient Central axon tracts have a strikingly different struc- system for clearance of potassium released by axOnS

ture than do peripheral nerves. While peripheral nerves might be needed, or perhaps glial cells of the white

Schwann cell, whose function is to myelinate, central way. Consistent with this possibility, glial mem- not branes contain functional ion channels (Barres et al.,

are composed Of Primarily One glial the matter could modulate or integrate neuronal signals in system (CNS) white matter is

only ofoligodendrocYtes but process-bearing astrocytes et a1.9 1983a). If the function Of axon tracts is

Of at 'east two types of 1985; Bevan et al., 1985; Kettenman et al., 1984a; and Raff, 1984, Raff MacVicar, 1984; Shrager et al., 1985), and, at least in

to culture, Some glial cell types are capable offiring action conduct impulses rapidly and accurately from neurons potentials (Barres, Chun, and Corey, unpublished re- to target then why do differences be- sults). Type 2 astrocytes in the optic nerve have recently tween the CNS and peripheral nervous system (PNS) exist?

one possibility is that the extra glial cell types simply play a transient role during development. It is also POS-

0 1988 Alan R. Liss, Inc.

Received July 28, 1987; accepted September 10, 1987.

'Address reprint requests to B. Barres at this address

ION CHANNEL EXPRESSION BY WHITE MATTER GLIA 11

been demonstrated to extend an axon-like process into close association with nodes of Ranvier (ffrench-Con- stant and Raff, 1986). Could these cells be releasing substances that affect transmission at the node? Pre- vious investigators have demonstrated that glial cells can release neurotransmitters (Dennis and Miledi, 1974; Minchen and Iverson, 1974; Shain et al., 19861, and there is also evidence that glial cells associated with axon tracts express neurotransmitter receptors (Gallo et al., 1986; Ventimiglia et al., 1987; Villegas, 1975).

Further chacterization of electrophysiological properties of glial membranes may be an important step to- ward understanding the function of particular glial cell types in white matter. Voltage-sensitive ion channels have recently been described in cortical astrocytes, the predominant glial cell type in grey matter (Bevan et al., 1985; MacVicar, 1984). But a different complement of glial cell types is present in white matter, primarily type 1 astrocytes, type 2 astrocytes, and oligodendrocytes (Miller and Raff, 1984; Miller et al., 1985; Raff et al., 1983a), and these might be expected to have a different complement of ion channels. (While type 1 and type 2 are specific terms for white matter astrocytes, protoplasmic and fibrous can have other meanings that are not universally accepted; see Miller et al., 1986). Type I astrocytes resemble cortical astrocytes in some properties (Raff et al., 1983a) but differ in others, including surface antigens (Barres et al., 19861, and may represent a distinct glial class. Type 2 astrocytes have recently been described as a biochemically and developmentally distinct astrocytic component of white matter (Miller and RafY, 1984; Raff et al., 1983a). Oligodendrocytes exist in greater numbers in white than in grey matter and are thought to function mainly to myelinate axons. Other possible functions have not been explored.

The ability to study the electrophysiological properties of these cells has been facilitated by the development of culture conditions that preferentially allow the survival and development of each of these three glial cell types (Raff et al., 1983a,b). The three cell types observed in such cultures correspond to the major glial cell types existing in white matter in vivo (Miller and Raff, 1984; Miller et al., 1985). In such cultures prepared from postnatal rat optic nerves, each cell type has both a charac- teristic morphology and antigenic phenotype permitting unique identification of cells studied (Bartlett et al., 1981; Raff et al., 1978, 1979, 1983a,b). Dispersion of cells in culture permits access to clean membrane surfaces for patch clamping and rapid and reliable exchange of solutions.

In this paper, we report the characterization of the ion channel phenotype of type 2 astrocytes and oligodendrocytes in cultures derived from rat optic nerve. This characterization leads to some new hypotheses for glial cell function.

MATERIALS AND METHODS Preparation of Cell Suspensions

Optic nerves from postnatal day 7 Long-Evans rats were dissected from just posterior to the optic foramen through the anterior one-third of the optic chiasm. (Al- though the optic nerve itself does not contain neuronal

cell bodies, the posterior chiasm may contain neuronal cell bodies originating from the suprachiasmatic nucleus; Card et al., 1981). This tissue was then dissociated enzymatically to make a suspension of single cells, essentially as described by Huettner and Baughman (1986; see also Bader et al., 1978; Lam, 1972). Briefly, the tissue was minced and incubated at 37°C for 75 minutes in a papain solution (30 units/ml; Worthington) equili- brated with 95% 02 and 5% C02. This solution also contained Earle’s Balanced Salts and calcium, magnesium, ethylenediamine tetraacetic acid (EDTA), sodium bicarbonate, glucose, and L-cysteine as described (Huett- ner and Baughman, 1986). The tissue was then tritur- ated sequentially with 21- and 23-gauge needles in a solution containing ovomucoid (2 mg/ml; Calbiochem- Behring) and bovine serum albumin (BSA) (1 mg/ml; Sigma) to yield a suspension of single cells. The cells were resuspended in Dulbecco’s Modified Eagle’s Me- dium (DMEM).

Preparation of Cultures

Cultures of each glial cell type were prepared according to the procedures of Raff and coworkers (Raff et al., 1983a,b). Briefly, cultures of type 2 astrocytes and oligodendrocytes were prepared from cell suspensions of optic nerves from P7 rats. Development of type 2 astrocytes was promoted by culture in DMEM containing 10% fetal bovine serum (FBS), penicillin (100 units/ml), strepto- mycin (100 ug/ml), and 1-glutamine (2 mM). Develop- ment of oligodendrocytes was promoted by culture in a modified Bottenstein-Sat0 serum-free medium (Raff et al., 1983b), containing 0.75% FBS. The cells were plated at a density of 12,000 cells/cm2 on 13-mm-diameter round glass coverslips that had been precoated with poly-l- lysine (PLL) and were cultured at 37°C in a humidified atmosphere of 5% C02 and 95% air.

Immunohistochemical Labeling of Cells in Culture

Cells in culture were labeled by incubation with monoclonal antibodies; antibodies to galactocerebroside (GC) were generously provided by B. Ranscht (Ranscht et al., 1982), and antibodies to A2B5 (Eisenbarth et al., 1979) were obtained from American Type Culture Collection. They were also labeled with a rabbit antiserum to glial fibrillary acidic protein (GFAP Accurate Scientific). For labeling of surface antigens, cells were incubated in the primary antibody for 30 minutes, washed, and incubated in a 1:lOO dilution of the secondary antibody. The secondary antibodies (Cappel) were fluorescein- or rhodamine-coupled F(ab‘)2 fragment goat antimouse IgG (F(ab’)z fragment specific). For GFAP labeling, the cells were fixed and permeabilized in methanol at -20°C for 10 minutes prior to incubation in the GFAP antiserum, and a rhodamine-coupled F(ab’)2 fragment goat antirabbit IgG was used to detect the primary antibody. A modified L15 medium (Hawrot and Patterson, 1979) containing BSA (0.1%) was used in all antibody incubations and washes, to decrease nonspecific binding. Controls with the primary antibody omitted and replaced with fresh hybridoma growth medium, a control monoclonal antibody, or preimmune rabbit serum revealed negligi- ble background.

12 BARRES ET AL.

Fig. 1. Type 2 astrocytes and oligodendrocyte in culture. Cultures were prepared from P7 optic nerve suspensions. 8: Type 2 astrocytes cultured for 4 days were labeled immunohistochemically with a rabbit antiserum to glial fibrillary acidic protein (GFAP), followed by fluores-

cein-coupled goat antirabbit Ig. B: An oligodendrocyte cultured for 4 days was labeled immunohistochemically with a monoclonal antibody to galactocerebroside (a), followed by fluorescein-coupled goat antimouse Ig. Scale bars = 10 pm.

Immunohistochemical Verification of Cell Types Electrophysiological Recording

Cells in culture, labeled by indirect immunofluores- cence as described above, were identified by viewing with a Zeiss microscope equipped with differential inter- ference contrast (DIC) optics and epifluorescent illumination.

Type 2 astrocytes and oligodendrocytes in culture can be reliably identified by morphology alone (Fig. 1A,B; Raff et al., 1983b). The main contaminants of each culture are non-process-bearing cells, both meningeal cells and type 1 astrocytes. We verified that in type 2 astrocyte cultures greater than 90% of process-bearing cells were type 2 astrocytes and were labeled intracellularly by antibodies to GFAP and on the surface by antibodies to A2B5. Similarly, in oligodendrocyte cultures, greater than 95% of process-bearing cells were oligodendrocytes, and were labeled on the surface by antibodies to GC. However, the accuracy of our morphological identification of these cells was significantly greater than 90% for type 2 astrocytes and greater than 95% for oligodendrocytes because the process-bearing morphology of each cell type is distinct (Fig. 1).

Gigohm-seal recording

A piece of glass coverslip with cultured cells was placed in the recording chamber, which contained the appropriate bath solution (volume 500-750 pl). Standard procedures for preparing pipettes, seal formation, and whole- cell recording were utilized (Corey et al., 1984; Hamill et al., 1981). Micropipettes were drawn from hard boro- silicate capillary glass (Drummond), coated with Syl- gard to reduce their capacitance, and fire-polished to a bubble number of 4.0-4.5 (corresponding to an internal tip diameter of about 1.2 pm; Corey and Stevens, 1983). Pipette capacitance and series resistance were electron- ically compensated. All experiments were done at room temperature, approximately 24 "C.

Data acquisition and analysis

Voltage stimuli were generated, and responses were recorded with a PDP 11/73 computer (INDEC). A Yale


Mark V patch clamp was used. Analog signals were filtered with an eight-pole, low-pass Bessel filter before being digitized and recorded by the computer. In each experiment, linear capacitive and leakage currents were measured and subtracted before storage of data. The BCLAMP program set was used for acquisition and analysis of whole-cell data.

Microtube perfusion system

In some experiments, the behavior of channels in inside-out patches was studied while the internal side of the patch was perfused with a series of different solutions by using a microtube perfusion system, as described by Yellen (1982). The microtube design we used was provided by David Friel; it consisted of a series of 1- pl microcapillary tubes (Drummond) epoxied together in a linear array. Each tube is about 64 mm long, 60 pm in internal diameter, and is connected through narrow tub- ing to a 10-ml syringe barrel containing its respective perfusion solution. The microtubes were lowered into the bath prior to recording with all tubes clamped shut. After obtaining an inside-out patch, the patch was con- secutively moved in front of each microcapillary.

Solutions and Current Isolation

The solutions for the bath and those for the pipette (which replace diffusible constituents of cytoplasm) were designed in each case to isolate current carried by a specific ion through a specific channel type. These solutions are listed in Table 1 and are further discussed below. All ion concentrations are in mM unless otherwise stated.

Sodium current isolation (solutions A and C, Table la)

The bath solution contained cadmium (100 pM) to block voltage-sensitive calcium current. We controlled for the possibility of a cadmium-sensitive sodium current by recording current in the absence of cadmium. In no case was sodium current observed that was cadmium- sensitive .

Calcium current isolation (solutions B and C, Table la)

We used sodium as the major bath cation because we reasoned that it would support sodiudcalcium exchange, and thereby help prevent buildup of calcium in the cell prior to recording. All sodium current was blocked by tetrodotoxin (Tl’X; 10 pM). In some early experiments, the bath solution (B2) contained 120 tetra- methylammonium methanesulfonate (TMA-CH3S03), 10 CaC12,3 dextrose, 10 pM TTX, 5 Hepes, pH 7.4, and the pipette solution (C2) was 120 tetraethylammonium methanesulfonate (TEA-CH~SOZ), 10 EGTA, Ca buffered to lo-’ M, 1 MgC12, and 5 Hepes, pH 7.4. Tetra- ethylammonium hydroxide was obtained from Kodak and from Aldrich; methanesulfonic acid was obtained from Aldrich.

TABLE l a Sodium and calcium isolation solutions*

Na OUT (A)

140 120

Ca OUT (B) Na and Ca IN (C)

135 Ba(OH12 10

Mg++ 1 c1- 7b CH3SO3 140 120 130 Hepes 5 5 5 D-glucose 3 3 EGTA 10 TTX 10 pM Adiusted to

Ca+ + 2 1 nMa Cd++ 100 pM

i H 7.4 with NaOH NaOH CsOH Phenol red 0.001% 0.001% 0.001%

*All values given in rnM unless otherwise noted. All solutions were designed and measured to have junction potentials near 0 mV. In early experiments, other calcium isolation solutions were used; in the text they will be referred to as solutions B2 and C2. Bath: 10 CaCI2, 120 TMA-CH3S03H, 5 dextrose, 5 Hepes 10 pM TTX, pH 7.4. Pipette: 120 TEA-CH:,S03H, 10 EGTA, Ca buffered to M, 1 MgCI,, 5 Hepes, pH 7.4. “Final fee calcium concentration calculated from a dissociation constant for EGTA of bC1- is used to eliminate need for an agar hridge.

M, at 1 mM Mg++ (Caldwell, 1970).

TABLE 1 b. Potassium isolation solutions*

K OUT (D) K IN (E) K,, OUT (F) K,, IN (G)

Na ’ 140 120 K+ 120 20 140 CSCl Ca.+ + 2 ME++ 1

I p M 2 1 1

1 nM 1

Cl= 6 2 146 2 Aspartate- 120 140 CH3SOs 140 Hepes 5 5 5 5 D-glucose 3 3 EGTA 10 10 Adjusted to

pH 7.4 with NaOH KOH NaOH CsOH Phenol red 0.001% 0.001% 0.001% 0.001%

*In early experiments, other solutions were used to isolate inwardly rectifying potassium current (Ki,). In the text they will be referred to as solutions F2 and G2. Bath (F2): 120 TMA, 20 K, 146 C1, 1 M g + + , 2 Cat ’, 3 dextrose, 5 Hepes, pH adjusted to 7.4 with TMA-OH. Pipette solution (G2): 120 Cs, 120 CH:$303-, 1 Mg ‘ ’ , 2 C1, Ca buffered to 1 nM with EGTA, 5 Hepes, pH adjusted to 7.4 with CsOH.

Potassium current isolation

Solutions D and E were designed to reveal potassium current but not chloride current. The use of a high concentration of calcium in the pipette ensured that possible calcium-dependent currents would be activated. In some cases (see “Results”) evidence for calcium dependence was obtained by blockade of a potassium current by charybdotoxin (30 nM; Miller et al., 19851, a specific blocker of large-conductance calcium-dependent potassium channels. Charybdotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, was generously provided by C. Miller (Smith et al., 1986).

Components of potassium current that were predominantly inward were initially recorded in solutions F2 and G2, but in later experiments solutions F and G were

14

v -70

BARRES ET AL.

-40

1000 pA hms Fig. 2. Voltage-sensitive currents in a type 2 astrocyte. Whole-cell

ionic currents elicited by a series of voltage command steps are demonstrated. The command steps are illustrated below: the test steps ranged from -40 to 45 mV, following a prepulse potential of -70 mV. The capacitive transients have been blanked. Bath solution in mM: 140 NaC1, 5 KCl, 2 CaC12, 1 MgCl,, 3 dextrose, 5 Hepes, pH 7.4. Pipette solution: 140 K-Aspartate, 2 EGTA, Ca buffered to M, 1 MgC12, 5 Hepes, pH 7.4.

utilized. The high bath concentration of potassium en- hanced the size of inward potassium currents.

Chloride current isolation (solutions not listed in Table 1)

To look for chloride current in the whole-cell mode we used a bath solution that contained 140 NaC1,5 KC1, 2 CaC12, 1 MgC12, 3 dextrose, 5 Hepes, pH 7.4 and a pipette solution containing 140 CsC1, Ca buffered to

M, 1 MgC12,2 EGTA, and 5 Hepes, pH 7.4. Whole- cell chloride current was never observed in our experiments.

Single chloride-selective channels were observed with excised inside-out patches. Pipette solutions (external to the patch) contained either 130 KC1 or 130 TMA-C1 and 0.5 CaC12, 1 MgC12, 5 Hepes, pH 7.4. Microtube perfusion solutions (internal to the patch) contained either 130 TMA-C1, 130 K-gluconate, or 130 NaC1, and 10 EGTA, 1 MgC12, calcium buffered to lo-’ M, and 5 Hepes, pH 7.4.

RESULTS Type 2 Astrocytes Express Inward and Outward

Voltage-Sensitive Current Components

Whole-cell currents in type 2 astrocytes in culture were recorded using a saline solution in the bath chamber (140 NaCl, 5 KC1, 2 CaC12, 1 MgC12, 3 dextrose, 5 Hepes, pH 7.4), and a potassium aspartate solution in the pipette (140 K-aspartate, 2 EGTA, Ca buffered to

M, 1 MgC12, 5 Hepes, pH 7.4). Type 2 astrocytes in our cultures rarely live longer than 9 days, and most

experiments were performed on cells 3-6 days after plating. We observed both inward and outward voltage-sensitive current components in all cells examined (Fig. 2). Solutions designed to isolate specific currents were used to dissect these components further.

Type 2 Astrocytes Express Voltage-Sensitive Sodium Currents

All type 2 astrocytes in culture show sodium currents when studied with isolation solutions A and C (Table la). Figure 3A shows a family of current responses elicited by test steps that ranged from -30 to 0 mV and that were preceded by a prepulse potential of - 100 mV. Qualitatively these currents closely resemble neuronal sodium currents: the current activated rapidly and then decayed rapidly, and the kinetics of activation and current decay became more rapid at more depolarized potentials. The peak current-voltage [IW)] relationship shows that the current was activated at potentials positive to -40 mV, peaked at -10 mV, and reversed positive to 40 mV (Fig. 3B). TTX (10 pM) completely blocked this current, but a dose-response relationship was not measured.

Whole-cell sodium current ranged from 410 to 3,100 pA per cell, and averaged 1,400 f 950 pA per cell or 17 f 10 picoamperes per picofarad (pNpF) of membrane capacitance (Table 2a). Sodium currents were observed in all of the cells studied.

When steps to a constant test potential were made from increasingly depolarized prepulse potentials (Fig. 3C), the peak inward current was diminished, illustrat- ing a voltage-dependent inactivation. The voltage sensitivity of the inactivation process was examined by plotting the peak currents (as percent of maximum peak current) against the voltage of the prepulse potential to yield a steady-state inactivation (or “h-infinity”) curve (Fig. 3D). The resultant plot was fit by eye with a sigmoidal curve. On average, the point a t which one-half of the channels were inactivated occurred at -52 i 7 mV (7 cells). The curve was steeply voltage-dependent with a slope corresponding to 4.5 k 0.5 equivalent charges per channel. These values are extremely close to those reported for sodium channels in many neuronal cell types. We observed that the h-infinity curves of many cells were not perfectly sigmoidal but could be fitted well instead by the sum of two sigmoids. This result could be accounted for by the presence of two types of sodium channels, by the presence of one type of channel in two spatially disparate locations, or by an inactivation mechanism that involves two conformational tran- sitions. Further experiments were needed to decide among these possibilities (Barres, Chun, and Corey, unpublished results).

Type 2 Astrocytes Express Two Distinct Types of Voltage-Sensitive Calcium Channels

We next chose isolation solutions that would not support the sodium current but would reveal even small calcium currents (solutions B and C, or B2 and C2, Table la). Figure 4A shows inward current elicited in response to a ramp voltage command that changed from - 100 to


TABLE 2a. Average peak inward currents in type 2 astrocytes and oligodendrocytes

Type 2 astrocytes Oligodendrocytes, Sodium Calcium (TI Calcium (L) potassium (IR)

Peak current per cell (PA): Average -1,400 k 950 Low -410 High -3,100

Average capacitance (pF) 80 k 30 Current density (pA/pF)

Average 17 k 10 Low 6.0 High 33

% cells expressing 100

Total No. of cells 7 current

-4,600 k 3,000b -130 + 70a -87C -goc - 1,200 -220 470 - 10,000

-210 k 190a

160 _+ 70 70 + 30 70 k 30

2.4 F 1.8a 3.7 +_ 4.4" 5.1 2.5 Lo= 0.8' 2.3 5.1 11.1 10 86 71 100

7 7 11 Solutions (Table 1) A, C Bd, C Bd, C F, G "Peak current averages in positive cells only were 150 f 50 (CaT) and 300 f 140 pA (Car,). Average current densities in positive cells only were 2.8 f 1.6 (Ca,r) and 5.2 "Currents for oligodendrocytes are given as values at -100 niV, where maximum conductance was observed. Corresponding to an average conductance a t ~ 100 mV of 92 f 61 nS. "The lowest value found among cells expressing this current. dThe calcium in solution B was replaced with an equal amount of barium.

4.3 pNpF (Ca,,).

TABLE 2b. Potassium currents in type 2 astrocytes

K (total) K (Ca) K (d) Conductance

Averagekell 48 F 23 AverageIpF 0.55 k 0.36 Low 0.18 High 1.21 No. of cells 7

0 mV 1,470 + 280 370 + 90 75 mV 3,300 k 470 4,300 +_ 1,400 No. of cells 3 3 Solutions D, E D, E

Average current (PAP

aSolutions not listed in Table 1. They were, bath: 140 NaCl, 5 KCI, 2 CaCI,, 3 dextrose, 5 Hepes, pH 7.4. Pipette: I40 KCI, 10 EGTA, Ca buffered to M. 1 MgC12, 5 Hepes, pH 7.4. The chord conductance was calculated as I/W ~ V,,), where V,, was -85 mV. The conductance was plotted vs. voltage, and the plateau conductance was determined. bCurrentS were separated by measuring whole-cell currents before and after block with charybdotoxin (see text). Chord conductances were not determined because the bath solution did not contain potassium.

100 mV over 500 ms. Because a time-dependent inactivation could occur as the voltage is changing, such curves represent only an approximation of the actual I(V) relationship.

Most cells exhibited I(V) relationships that contained two peaks. Such curves suggested the existence of either two channel types or one type of channel that was lo- cated in spatially disparate regions of the cell. This latter possibility needed to be considered seriously because of the extremely long processes formed by type 2 astrocytes in culture.

Several experiments indicate that these two peaks in fact represent two distinct types of calcium channel. First, about 30% of the cells expressed only the peak occurring at hyperpolarized potentials (Table 2 and Fig. 5). Second, families of current responses suggested that the currents were composed of two components because one component appeared to inactivate more rapidly than. the other (Fig. 4B). The rapidly inactivating component

activated at about -60 mV, corresponding to the first peak, while the slowly inactivating component typically activated at about -30 mV, corresponding to the second peak. As has been observed for other calcium currents during whole-cell recording, the I(V) relation of both components shifted 20-30 mV in the hyperpolarizing direction during the first 10 minutes of recording (Corey et al., 1984); Figure 4A shows the I(V) relation before the shift.

Current responses were studied in three cells that contained predominantly the rapidly inactivating component of current (Fig. 5A). Steady-state inactivation curves were obtained as described for sodium currents above (Fig. 5B). The current could be half-inactivated by prepulses to -63 & 5 mV. This curve was also steeply voltage-dependent with a slope of 3.9 0.4 equivalent charges per channel. Because the component of current that activated positive to -30 mV did not display this strong voltage-dependent inactivation, the relative amplitude of the two peaks could be shifted in favor of the second peak by depolarizing prepulses (data not shown), providing additional evidence that the two peaks of calcium current represent distinct channel types.

Further evidence that these two components represent distinct molecular entities was obtained by comparing the selectivity of divalent cations between the two components. On average the amplitude of current in the first peak was identical in solutions containing calcium (10 mM) or barium (10 mM): 2.4 _+ 1.0 pNpF (calcium) vs. 2.4 f 1.8 pA/pF (barium) (Table 2a). In contrast, average amplitudes of the second peak were 1.9 _+ 2.2 pA/pF (calcium) vs. 3.7 f 4.4 pA/pF (barium) (Table 2a). It was difficult to hold seals stably for long enough to allow perfusion of new bath solutions. In only one cell were we able to determine the I(V) relationship first in a calcium-containing bath solution, and then again after replacement of calcium with barium. In this cell, which contained only the first peak of current, inward currents were diminished by about 50% in the barium-containing solution, a result somewhat different from the average behavior of the first peak but still contrasting with the barium enhancement of the second peak.

16 BARRES ET AL.

t - i

-\ 4;;-

I A

, -1 0

/ 0

-30 V

-100

500 pA

t - i

-\ 4;;-

C I

, -1 0

/

” -1 0

1

-100

1 5002p~s

Fig. 3. Voltage-sensitive sodium current in type 2 astrocyte. Whole- cell currents were recorded with isolation solutions A and C (Table la). A Ionic currents elicited by voltage steps to -30, -20, -10, and 0 mV from a prepulse potential of -100 mV. B: Peak I(V) relationship for the same cell. C: Peak sodium current was diminished when the voltage was stepped to a constant potential of -10 mV from a series of

We were also able to separate the two current components pharmacologically. While both components of the current were completely abolished by high concentrations of cobalt (1 mM) or cadmium (1 mM), we observed that lower concentrations of cadmium (100 pM) selec- tively blocked the second component while only slightly diminishing the first component (Fig. 6). The effects of dihydropyridine calcium channel antagonists were not studied.

Together, these data strongly indicate that these two components of calcium current are analogous to two types of calcium channels described in dorsal root gan. glia (DRG) by Nowycky et al. (1985). The first component has a rapid voltage-dependent inactivation and acti-

-60 6

-2000 -

0.8 l . O I .. Q)

0.6 1 (II

.- r E l

2 a I ‘\ \ i

0.4 4 +-

o.2 i ‘\ a

0.0 -1 00 -80 -60 -40 -20

Prepulse potential (mV)

prepulse potentials ranging from -100 to -30 mV. D: Steady-state inactivation curve for a typical cell demonstrating the voltage dependence of inactivation. The percent of maximum peak inward current was plotted versus the prepulse potential and fit by eye to a sigmoidal curve. One-half of the channels were inactivated at a prepulse potential of -52 mV in this cell; the average for all cells was -52 5 7 mV.

vates at relatively hyperpolarized potentials (usually about -60 mV, Fig. 6A). Thus, it is analogous to the “T” or transient calcium current of Tsien’s classification. In type 2 astrocytes, peak T currents ranged from 87 to 220 pA per cell, averaging 130 + 70 pA per cell (in 10 mM Ba++; Table 2). They were observed in 86% of cells.

The second component of the current activates a t more depolarized potentials (about -30 mV; Fig. 4B) and inactivates slowly. This current is analogous to the “L” or long-lasting calcium current. Like the “L” channel in dorsal root ganglion neurons, the inactivation of this channel was not particularly voltage-sensitive, but rather appeared to be calcium-dependent because it decayed more slowly when barium was substituted for


v -1 00

A

~

-60

+400 I I: A I

50 rns *

100

-1 00

B I

Fig. 4. Voltage-sensitive calcium current in a type 2 astrocyte. Whole- cell current was recorded with solutions B2 and C2 (Table la). A: Current response obtained immediately after establishing the whole- cell configuration. The ramp voltage command is illustrated below: the voltage was increased from -100 to f l O O mV over 500 ms. Linear leak current was subtracted from the current response. B: Family of calcium current responses obtained from a similar cell. Voltage com- mands were taken from a prepulse potential of -100 mV to test steps ranging from -60 to 0 mV.

calcium (data not shown) and because rapid repetition intervals diminished its amplitude. In type 2 astrocytes, peak L currents ranged from 90 to 470 pA per cell, and averaged 210 k 190 pA per cell (in 10 mM Baf+ ; Table

I l W P A p m s

Prepulse potential (mv)

Fig. 5 . Whole-cell calcium current in type 2 astrocyte. A Isolation of calcium current in a cell that expressed only the transient component of calcium current. Command steps from - 75 to - 15 mV from a prepulse potential of - 110 mV. Solutions B2 and C2 were used (Table la). B: Steady-state inactivation curve for three cells demonstrating voltage-dependent inactivation of this component of calcium current. One-half of the channels were inactivated with a prepulse potential of -63 mV (average).

2a). They were observed in 71% of cells. We never observed a component of calcium current analogous to the "N" or intermediate calcium current described in dorsal root ganglia.

The differential pharmacological sensitivity is largely consistent with the sensitivity of T and L channels in other cell types (Nowycky et al., 1985). The differential permeability to divalent cations is also consistent: the L channel has generally been found to be more permeable to barium than calcium as we found, while T channels in most other cell types have been reported to have equal permeability to calcium and barium.

In summary, type 2 astrocytes express two types of calcium channels. Generally cells contain both types of channel, but we have observed cells that express T chan-

BARRES ET AL. 18

A

Jf +zoo /

J’

+loo ,J’ Control J/

i i

B

Fig. 6 . Evidence that the two components of calcium current in a type 2 astrocyte are derived from distinct channel types. Whole-cell currents were recorded in solutions B and C (Table 1). A Leak-subtracted ramp current. B: Leak-subtracted ramp current after perfusion of the recording chamber with a cadmium solution (100 pM).

nels but not L, and (rarely) cells that express L channels but not T.

Type 2 Astrocytes Express at Least Two Outward Currents

Depolarization activated outward current in all type 2 astrocytes (Fig. 2). Initially, whole-cell currents were recorded with a bath solution containing 140 NaC1, 5 KC1,2 CaC12, 1 MgC12, 3 dextrose, 5 Hepes, pH 7.4 and a pi ette solution containing 140 KC1, Ca buffered to

current could be abolished by addition of barium to the bath solution suggesting that almost all of the current was carried by potassium. Outward currents recorded in the presence of these isolation solutions must be carried by potassium because current carried by chloride would have reversed at 0 mV and would have generated tail currents, which were never observed (e.g., Fig. 2). In

10- .p M, 1 MgC12, 2 EGTA, 5 Hepes, pH 7.4. Outward

addition, isolation solutions that allowed only chloride current to be observed did not reveal any chloride current (see below). Total outward current ranged from 1,910 to 9,560 pA per cell at test steps to +45 mV. Conductance-voltage relationships showed that peak potassium conductance ranged from 14.7 to 73.5 ns, averaging 48 f 23 nS per cell (Table 2b).

With potassium isolation solutions (solutions D and E), the shape of the I(V) relationship, elicited with a ramp voltage command, suggests a t least two distinct components: one activating at - 30 mV and one activating at 0 mV (Fig. 7A). Current responses to a series of voltage steps (Fig. 7B) also reveal two outward current components: there appears to be a component of current that activates rapidly and inactivates and a component that activates more slowly and does not inactivate. All cells examined showed both current components.

The inactivation of the faster component appeared from prepulse experiments to be voltage-dependent (Fig. 8). When a series of test steps was preceded by a prepulse to -40 mV, the current activated more slowly and showed less of an inactivating component.

In single-channel recording experiments we observed the presence of two types of potassium channels supporting our whole-cell observations that the outward current is composed of primarily two channel types. These channels had conductances of about 20 pS and 100 pS in cell- attached patches when the pipette contained 5 mM KC1 (data not shown).

Although the isolation solutions used were designed to record outward potassium currents, the external solution contained normal amounts of calcium and sodium. Therefore, the currents shown represent the sum of inward calcium and sodium currents and outward potassium currents. It can be seen in Figure 7D that block of the rapidly activating component of outward current revealed the inward sodium current, but this current is wholly inactivated in 2-3 ms. Similarly, calcium current with 2 mM calcium in the bath is very small relative to the much larger outward currents. (Calcium was included to ensure the activation of any calcium-dependent potassium channels that might be present.)

When the scorpion toxin charybdotoxin (30 nM), a specific blocker of some calcium-activated potassium channels (Miller et al., 1985), was added to the bath solution, the I(V) relationship revealed a decrease in outward current, primarily the more negatively activating component (Fig. 7C). Figure 7D demonstrates that the current component blocked was the rapidly activating, inactivating component. Block by charybdotoxin suggests that this component might be calcium-sensitive. In support of this, we observed that in the presence of cadmium (500 pM) in the bath to prevent calcium entry and a pipette solution containing calcium buffered to lo-’ M to reduce intracellular calcium, the inactivating current component was significantly diminished. In addition, under these conditions the charybdotoxin-sensitive component was diminished (in both of two cells; data not shown). Inactivating potassium currents (“A- currents”) have not previously been reported to be blocked by charybdotoxin. However, it is possible that the apparent voltage-dependent inactivation of this current is actually an indirect effect since a component of the calcium current is through channels with a voltage-


A

+ 45

v -30 -110

-50

25 rns

C /------

D

12.5 rns H

Fig. 7. Voltage-sensitive potassium current in a type 2 astrocyte. Wholecell currents were recorded with solutions D and E (Table lb). A Current elicited by a ramp voltage step from - 100 to 100 mV over 250 ms. A linear leakage current was subtracted from the ramp current. B: A current family obtained with steps from a prepulse of - 110

dependent inactivation (T-channels), the decrease in potassium current with depolarizing prepulses might re- flect inactivation of calcium channels. Consistent with this idea, in some cells the time course of the potassium current decay paralleled the time course of the “T” calcium current decay. However, in other cells the charybdotoxin-blockable potassium current contained both an inactivating and a sustained component, perhaps reflecting activation by both T and L calcium currents. In one cell, charybdotoxin-blockable current was mainly a sustained current, and we observed after addition of barium to the bath solution that this cell contained an L but not a T current. Thus it seems possible that the postassium current blocked by charybdotoxin is a con- ventional Ca+ +-activated Kf current, the time course and voltage dependence of which derive from the calcium currents (see “Discussion” below).

In summary, our observations are consistent with two types of potassium currents: a delayed potassium current which activates positive to 0 mV but is not calcium- sensitive, and an inactivating charybdotoxin-sensitive

mV to steps from -30 to 45 mV. C: Ramp current in the same cell described in A and B before and after addition of charybdotoxin (30 nM) to the bath solution. D: Current responses elicited by the same voltage command steps in the same ceIl described in B, after charybdotoxin.

current activating positive to -30 mV which is calcium- activated. We determined the average amount of each type of current by measuring total current and charybdotoxin-blockable current in three cells (Table Zb). As expected, the relative amount of peak current contrib- uted by each of these two channel types varied with the test potential: a t 0 mV the charybdotoxin-blockable current formed 80% of the outward current, but at 75 mV the more slowly activating component formed 57% of the outward current.

In some systems, TEA preferentially blocks calcium- activated potassium channels, but in this case TEA seemed to diminish all components partially. Similarly, we did not achieve better separation with the blocker 4- aminopyridine (4-AP).

Other Whole-Cell Currents

There does ngt appear to be a significant component of whole-cell chloride current in type 2 astrocytes. Re-

20 BARRES ET AL.

I

A

i Chloride Channels Are Observed in Excised Patches

I

V -40

- 31

ride channels were often present in excised, inside-out

1 1 + 60

V 0

1 30rnV -110 1 1 25ms

B

7 / I-

Fig. 9. Action potential in a type 2 astrocyte. A single regenerative potential overshooting zero was elicited in response to a depolarizing current injection. This was recorded in the current clamp configuration with a prepulse current of -350 pA and an injection of 1,500 pA of current. Bath solution: 140 NaCl, 5 KCl, 2 CaC12, 3 dextrose, 5 Hepes, pH 7.4. Pipette solution: 140 KCl, Ca buffered to M, 1 MgC12, 5 Hepes, pH 7.4.

Fig. 8. Two components of outward current in a type 2 astrocyte. Whole-cell currents were recorded with solutions D and E (Table lb). A Whole-cell potassium current elicited by steps to 0, 20, 40, and 60 mV from a prepulse potential of -110 mV. B: Current response obtained in the same cell when the prepulse potential was to -40 mV. C: Current inactivated by the depolarizing prepulse to -40 mV, derived by subtracting currents in I3 from those shown in A.

cording in isolation solutions that would allow chloride to carry current revealed no outward current after several minutes of cell dialysis to remove intracellular potassium. We looked for but did not observe inwardly rectifying potassium channels.

Type 2 Astrocytes in Culture Are Excitable

Because inward and outward current components were present, we wondered whether type 2 astrocytes could be induced to fire an action potential with current injection. Injection of depolarizing current induced a single regenerative potential in many type 2 astrocytes in our cultures (four out of eight cells). Figure 9 illustrates a regenerative action potential which overshoots zero mV, recorded from a cell in whole-cell current-clamp configuration. The solutions used for these experiments ap- proximated physiological ion concentrations (see legend to Fig. 9). Similarly cortical motor neurons studied under the same conditions also fired a single action poten-


rnV + 50

50 rns ti

0 . 5 - 7 -150

I -1600

Fig, 10. Inwardly rectifying current in oligodendrocytes. The current elicited by a ramp voltage command ranging from -150 to +70 mV over 500 ms with isolation solutions F2 and G2 (Table lb). A large inward current was observed at potentials more positive than -120 mV in this cell. The current-voltage relation shows marked inward rectification.

tial, but on average required only 10% as much depolarizing current to exceed threshold. Motor neurons have about ten times the sodium channel density as type 2 astrocytes (Barres, Chun, and Corey, unpublished).

Voltage-Sensitive Current in Oligodendrocytes

Because oligodendrocytes rarely lived more than 5 days in culture, most experiments were performed on cells between 3 and 5 days after plating. While we looked for sodium, calcium, and chloride currents, we observed only voltage-sensitive potassium currents in whole-cell configuration.

When a pipette solution containing 120 C s C H ~ S O ~ H (solution F2) and a bath solution containing 120-TM-A- C1 and 20 KC1 (solution G2) were used we observed the I(V) relation shown in Figure 10. In this figure, the stimulus was a ramp voltage from - 150 to 70 mV over 500 ms. Large inward currents were activated at potentials positive to -120 mV suggesting that the channels were voltage-dependent but normally active at physiological potentials. Several lines of evidence suggest that this inward current was carried by potassium: when extracellular potassium was increased, inward current was increased (Fig. 11). If potassium was omitted from the bath solution and replaced with sodium, no inward current was observed. In ten cells studied with 20 mM K+ in the bath and 140 mM Kf in the pipette (solutions F and G), the current reversed on average at -40 mV, near the potassium equilibrium potential (- 50 mV), demonstrating a selectivity of the channel for potassium. (These traces were not corrected for a small leakage current, which would make the apparent reversal closer to 0 mV than the actual reversal). This current was completely blocked by extracellular barium (10 mM, lower concentrations not tested), but not by TEA (20 mM) or 4-AP (1 mM).

In Figures 10 and 11, with cesium in the cell, outward current was smaller than inward current; such rectification might arise from a preferential selectivity for potassium over cesium. However, when the cesium in

T +looo

Fig. 11. Increase in wholecell current in an oligodendrocyte with increase in extracellular potassium. Currents recorded with solutions F2 and G2 (Table lb) were increased when bath potassium was increased from 20 to 40 mM, and the reversal potential was shifted to a more positive potential (the middle trace represents current at a value of bath potassium somewhere between 20 and 40 mM).

T t4000 1

T ‘\X

iPA 25 ms - - -8000

Control

Fig. 12. Voltage-dependent block of potassium current by extracellular cesium in an oligodendrocyte. Whole-cell current elicited by a ramp voltage from + 100 to - 150 mV using solutions F and G (Table lb) is shown as the control trace, and responses to extracellular cesium of 0.1, 0.5, and 5 mM are superimposed. Cesium block is more pronounced at negative potentials but also diminishes the outward current at positive potentials.

the pipette was replaced with potassium, inward rectification was still observed (Fig. 13A). Inward current a t - 100 mV ranged from - 1,240 to - 10,000 pA per cell, averaging -4,600 i 3,000 pA per cell or 30 f 20 pA/pF (Table 2a, solutions F and G). Inwardly rectifying potassium current was observed in all cells studied.

As is true of all previously reported inwardly rectifying potassium channels, we found that the current was decreased by small amounts of extracellular cesium in a manner that suggests voltage-dependent block (Fig. 12, solutions F and G). At -90 mV inward current was diminished by 50% with 100 pM cesium and was completely blocked by 5 mM. The block increased at more negative potentials, as if the blocking ion was being driven into the pore. Because small amounts of extracellular cesium produced a potent block, we wondered whether cesium could be leaking from the pipette, prior to seal formation, into the bath solution around the cell when cesium was the intracellular cation in whole-cell experiments. Since the pipette solution G2 contained 120 mM cesium, leakage sufficient to produce a local

22 BARRES ET AL.

A T +*OO0

- I - -4000

B I

I

V

t 60 + 25

-175

Fig. 13. Whole-cell current in oligodendrocyte. A Current elicited by ramp voltage from +60 to -250 mV using solutions F and G (Table lb). The IW) relationship is strongly inwardly rectifying. B: Currents elicited by test steps to - 175 to +25 mV from prepulses of + 60 mV. Sustained inward current was seen except at very negative test steps where the current decayed. Leak subtraction was performed with leak steps obtained from 60 to 85 mV. In both A and B, the current has not been corrected for the uncompensated series resistance of the pipette.

concentration of 0.1% of the pipette solution could significantly diminish the whole-cell currents measured. In fact, currents recorded with solutions F and G, which lacked cesium, were many times larger than with solution G2: peak inward currents averaged 30 pNpF com- pared to 5 pNpF (Table 2a). We did not rule out the additional possibility that cesium may block the channel internally.

Is the apparent voltage dependence of activation simply voltage-dependent block by extracellular cesium leaked from the pipette? Figure 13A shows current responses obtained with solutions F and G, which do not contain cesium, to a ramp voltage swept from 60 mV to -250 mV. As we previously observed with cesium-containing isolation solutions, the current was inwardly rectifying; however, inward current was seen even below an apparent potential of -250 mV (this result was unaltered by the direction of the ramp). Nevertheless,

-60 mV

-+Lf-d4>.7--

&% A 7

7swu“

T

-80 mV

-1 00 mV ----z----

-120 mV

Gs ms

-- Fig. 14. Single-channel records of an inwardly rectifying potassium

channel in an oligodendrocyte from a cell-attached patch with pipette solution: 130 KC1, 0.5 Ca, 1 MgClz, 5 Hepes, pH 7.4. Potentials are given relative to the resting potential. At negative potentials, openings were rare and brief; a t depolarized potentials the channel was almost continuously open. Channel openings are indicated by downward current deflections. Single-channel amplitudes increased with hyperpolarization.

these currents showed evidence of decay at extreme hyperpolarizing voltage. Current responses to a series of voltage steps from a prepulse of 60 mV to hyperpolarized potentials demonstrated the time course of this decay (Fig. 13B). Because a t very negative potentials inward current was large, even a small amount of uncompensated series resistance could produce a significant voltage drop across the pipette. This problem would tend to cause a shift of the negative region of the IW) relationship in a hyperpolarizing direction, and impaired our ability to study the voltage dependence of the channel at hyperpolarized potentials using whole-cell recording.

Properties of Single Potassium Channels in Oligodendroc ytes

We therefore recorded from single channels in cell- attached patches. Although large inward currents were observed in whole-cell recordings, we found channels to be rare in patches formed on the soma, and it was difficult to seal to the fine processes. We could only find the channels in patches on the soma if we recorded during a time window from 24 to 36 hours after plating; after this time channels were found only when recording from patches on processes. We suspect that channels were inserted into the membrane shortly after plating and were then transported to processes.

For cell-attached recording the pipette solution contained 130 mM KC1, 0.5 CaC12, 1 MgCl2, 3 dextrose, 5 Hepes, pH 7.4. We observed two kinds of potassium channels in these patches. The redominant channel type had a conductance in 130 K of 30 pS (31 f 1.91, but less frequently, we observed a larger conductance

r:


-80 mV -40

T

rest +40 /

.. t " PA ! -8 2.5 ms

M

Fig. 15. Average ramp current from a channel in a cell-attached patch on an oligodendrocyte. The single-channel currents elicited by multiple voltage ramps were averaged, demonstrating a IN) relationship that was very similar to the wholecell IW) relationship. All voltages are given relative to the resting potential (probably about -60 mV).

channel of 120 pS (120 & 6.3). Six of 45 channels studied under these conditions had the larger conductance (13%). However, both channels had a similar voltage dependence, and both demonstrated the pronounced inward rectification.

Typical single-channel responses from a cell-attached patch containing a 30-pS channel are shown in Figure 14. Responses are shown at potentials from - 120 to +20 mV (relative to the cell's resting potential, which was not measured but is estimated from the open channel reversal to be about -60 mV). At very negative potentials, the channel opened only rarely and these openings were brief. As the potential became more positive the openings became longer, and the amplitudes of the single-channel currents became smaller. Identical behavior was observed for the 120 pS channels (not shown).

When the responses of either type of single channel to voltage ramps were averaged (Fig. 15), the resuking average mimicked the whole-cell ramp response. Such ramp responses only approximate the actual I(V) relationship, because they do not take into account the time dependence of the channel responses. However, as whole- cell and single-channel responses appeared identical, these single channels probably form the molecular constituents of the whole-cell current.

Open-channel I(V) relationships of the potassium channels were derived from analysis of many single- channel ramp responses, by separately averaging open and closed segments from many ramps (Fig. 16A). The closed average response (leakage current) was sub- stracted from the open average response at each potential (Fig. 16B,C). Both small and large conductance channeIs demonstrated marked inward rectification of the open-channel IN) relationship, indicating that the rectification is not a consequence of voltage-dependent gating. Instead the open channel current was large at negative potentials, indicating a voltage-dependent closing at very negative potentials.

Dividing the average single-channel current by the open channel current gives the voltage dependence of the open probability P(V) (Fig. 17). This curve demonstrates that probability of channel opening increases with increasing depolarization and contrasts it with some other inwardly rectifying potassium channels that are thought to be closed a t positive potentials but to

12.5 ms t-i

Fig. 16. Openchannel IN) relationship of potassium channels in an oligodendrocyte. A The openchannel IW) relationship was determined by averaging open and closed current segments from many ramps and subtracting the closed average at each voltage from the open average. Cell-attached patches were studied using a pipette solutions of 130 KCI, 0.5 Ca, 1 MgC12, 5 Hepes, pH 7.4. B: IN) relationship of the 30- pS channel. C: IN) relationship of the 120-pS channel. Both small and large channels demonstrate marked inward rectification. Note that the extracted IN) has been inverted so that negative potentials are shown on the left to match convention.

open with increasing hyperpolarization (for instance inward rectifiers in horizontal cells: Shingai and Quandt, 1986). _ _ - -,.

As with other inward rectifiers, the conductance of the channel was not proportional to the extracellular potassium concentration, but was proportional to the square root of extracellular potassium (data not shown), suggesting the interaction of multiple potassium ions in the pore of the channel (Hagiwara and Takahashi, 1974; Hille and Schwarz, 1978). The possible effect of extracellular potassium on channel activation was not studied.

24 BARRES ET AL.

T 1.0

Fig. 17. Voltage dependence of probability of potassium channel opening in an oligodendrocyte. To determine the open probability of these channels as a function of voltage, PW), the average current at each voltage (I) was divided by the open single-channel current (i) (and fitted with a sigmoidal curve). At very negative potentials, the open probability was zero, but rose to greater than .5 a t the resting potential and above. It was difficult to determine the maximum probability precisely because the strong rectification resulted in very small currents at positive potentials.

Chloride Channels Are Observed in Excised Patches

We unexpectedly observed another channel type that was apparent only in excised patches. Both inward and outward currents were carried by this channel, and the current reversed at zero mV. The solutions in these experiments contained symmetric KC1, so that the channel most likely was either a chloride channel or a potassium channel. In order to determine which possibility was correct we recorded from inside-out patches with pipette (extracellular) solutions containing either 130 KCl or 130 TMA-Cl and 0.5 CaC12, 1 MgC12, 5 Hepes, pH 7.4, and then studied the behavior of the channel in front of a series of microtube perfusion (intracellular) solutions containing 130 TMA-Cl, 130 K-gluconate, or 130 NaC1, and in each case 10 EGTA, 1 MgC12, calcium buffered to lo-’ M, and 5 Hepes, pH 7.4. With either pipette solution (KCl or TMA-C1) the current through this channel reversed at exactly 0 mV regardless of the cation as long as the microtube solution contained 130 mM chloride; however, if the microtube solution contained K-gluconate the reversal potential shifted far negatively, and all inward currents were abolished. Therefore, the channel is purely selective for chloride, Although we did not observe chloride current in whole- cell recordings or chloride channels in cell-attached patches, we observed these chloride channels in 12 of the 35 excised inside-out patches we studied. However, these channels were never observed immediately after excision of the patch, but usually became active 2-10 min after excision.

During either hyperpolarizing or depolarizing voltage steps, the channel rapidly flickered open and closed, only occasionally closing for a prolonged period of tens or hundreds of milliseconds. It was only poorly voltage- sensitive: amplitude histograms demonstrated that during steps to -100 mV the channel was open about 35% of the time, while during steps to + 100 mV it was open about 60% of the time.

A

t I ‘

B

c +* n - , P

I -2 12.5 ms *

Fig. 18. Singlechannel records from a single chloride channel in an inside-out patch. A Channel openings elicited by a ramp voltage command from -70 to +lo0 mV. The pipette solution contained 130 KCl, 0.5 CaCI2, 1 MgC12, 5 Hepes, pH 7.4. The microtube solution contained 130 TMA-Cl, Ca lO-’M buffered with 10 EGTA, 1 MgC12, and 5 Hepes, pH 7.4. The channel opens frequently both at negative and positive potentials, and the current reverses at 0 mV. B: Average ramp current obtained by averaging 100 ramp responses. Average current is outwardly rectifying.

Figure 18A demonstrates the current elicited by a ramp voltage command ranging from - 70 to + 100 mV over 125 ms from an excised inside-out patch. The amplitudes of the channel openings rectify in an outward direction (that is, positive current flow is outward, but the direction of chloride ion movement is from outside to inside). The slope conductance ranged from about 25 pS at -70 mV to 60 pS at +lo0 mV in the presence of symmetrical 130 mM chloride. This rectification could occur either because the open channel is outwardly rectifying or because the channel has substates that are voltage-dependent. Our data support the former possibility because of the poor voltage dependence of channel opening, and because we did not observe such substates in our recordings.

The average ramp current obtained by averaging about 100 ramp traces demonstrates the I(V) relation that would be expected by the contribution of these channels to whole-cell current (Fig. 18B). This relation-


ship continues to be outwardly rectifying and, again, demonstrates the 0 mV reversal of the current.

Because these channels are not normally active in cell- attached patches we investigated the effects of two acti- vators of other chloride channels. First we studied the effect of calcium on the behavior of the channel. In these experiments microtube perfusion solutions containing calcium buffered to 10-5.5 to lo-’ M with EGTA (10 mM), 130 TMA-C1, 1 MgC12, and 5 Hepes were used to expose the inside of excised patches to varying calcium concentrations. No effect of calcium on the opening probability or average ramp currents was observed. In addition we attempted to activate chloride channels in cell- attached patches with GABA (1 pM) in the pipette. These experiments are preliminary, but so far we have not observed activation of chloride channels in 15 of 15 patches.

DISCUSSION

Type 2 Astrocytes

Type 2 astrocytes in culture exhibit a surprisingly complex ion channel phenotype. We have observed three voltage-sensitive inward currents (a sodium current and both transient and sustained calcium currents) and two voltage-sensitive outward currents (a delayed rectifying potassium current and an inactivating, calcium-dependent potassium current).

Bevan and Raff (1985) have also observed the presence of voltage-sensitive potassium channels in type 2 astrocytes in optic nerve cultures. Our results are consistent with their observations. They observed primarily a delayed rectifying potassium current, but if the pipette solution contained cesium (10 mM) they observed an inactivating outward current that could be abolished with depolarizing prepulses of -40 mV. Since this small amount of internal cesium would be expected to block the delayed rectifying current, but not the calcium-dependent potassium current, this essentially isolated the latter current.

There is evidence from other preparations that some transient potassium currents are sensitive to internal calcium (e.g., MacDermott and Weight, 1982; Salkoff, 1983; Wei and Salkoff, 1986). However it remains un- clear, in this and other preparations, whether the inactivation of the current is a direct attribute of the channel’s voltage dependence or instead is imparted in- directly by the time course of calcium entry. Our whole- cell data suggest the possibility of an indirect inactivation, but resolution of the question awaits single-channel studies.

It is interesting that a glial cell type may express such a complex ion-channel phenotype and suggests that the channels may be generating electrically meaningful events in the type 2 astrocyte itself. Injection of a depolarizing current did in fact elicit an action potential in one-half of the type 2 astrocytes in culture. It has previously been suggested that glial cells (Schwann cells and astrocytes) produce ion channels for transport to axonal membrane at nodes of Ranvier (Bevan et al., 1985; Shra- ger et al., 1985; Waxman and Ritchie, 1985). However, nodes of Ranvier are not thought to contain most of the channel types observed in type 2 astrocytes (Waxman

and Ritchie, 1985), so this explanation is limited to a few channel types at best.

Oligodendrocytes

All oligodendrocytes examined expressed high densities of an inwardly rectifying potassium current. Single- channel experiments demonstrate that this current de- rives from two distinct channel species, which differ in their single-channel conductance but are similar in all other properties that we examined.

There are several possible reasons why the inward rectification of potassium channels in oligodendrocytes has not previously been observed. In the whole-cell experiments of Bevan and Raff (1985), the use of physiological concentrations of potassium in the bath solution would have tended to linearize the I(V) relationship: inward current would be small because of the low extracellular potassium, and outward current would be small because of the rectification. It would thus look like leakage.

Many of the single-channel properties of the potassium channels have been reported by Kettenman et al. (1982, 1984a), who studied oligodendrocytes from mouse spinal cord. There are two principal differences between our results and theirs. First, Kettenman et al. found a range of channel conductances between 6 and 125 pS, whereas we saw just two: 30 pS and 120 pS. Second, they did not observe the inward rectification in the openchannel I(V) relation, which was quite pronounced in our studies. One explanation is simply the differences in tissue used. However, there are also some differences in the experimental protocol. Kettenman et al. studied excised patches, whereas we used cell-attached membrane patches. This suggested to us that excision might affect the rectification. But when we recorded from channels in the cell-attached mode and then excised those patches, the rectification persisted, so that excision is not the essential difference. Another difference is that we always included Mg+ + in the solution facing the intracellular surface, whereas Kettenman’s bath solution had no added Mg++. Recent studies of some other inward rectifiers have found that binding of Mg+ + to an intracellular site on the channel produces the rectification (Matsuda et al., 1987; Vandenberg, 1987). This is the most attractive explanation for our findings; it suggests, moreover, that in vivo the channels do show inward rectification. Inward rectification would also explain the microelectrode data of Kettenman et al. (1984b), which indicated that input resistance of oligodendrocytes increased with membrane depolarization.

Outwardly Rectifying Chloride Channels

Although our whole-cell recording and cell-attached recordings in oligodendrocytes and type 2 astrocytes did not reveal evidence of chloride current, we observed the presence of chloride channels in excised patches in both of these cell types. The appearance of chloride channels in excised patches, typically with a delay after excision, is now a common observation for many (but not all) chloride channels. For instance the large-conductance (450 pS) chloride channel observed in many cell types including cortical astrocytes (Sonhof and Schachner,

26 BARRES ET AL.

1984) and Schwann cells (Gray et al., 1984) and a 60-pS phosphoprotein found at most neuronal synaptic termi- chloride conductance observed in skeletal muscle (Blatz nals, has not been observed in optic nerve sections (de and Magleby, 1985) are observed only in excised patches. Camilli, personal communication). Similarly, synaptic This suggests that these channels are normally inhib- vesicles have not been observed in astrocytes in ultra- ited by some mechanism or factor that can be alleviated structural studies of the optic nerve. Nevertheless, more with excision; in no case is the nature of this inhibition detailed ultrastructural examination of the perinodal understood. We were unable to activate this channel with either calcium or GABA (although the latter result was expected since the channel has distinctly different properties than GABA-activated chloride channels.

Chloride channels have not been observed in oligodendrocytes or type 2 astrocytes previously, but cortical astrocytes have been demonstrated to express a t least three other types of chloride channels that are distinct from the outwardly rectifying chloride channel we have reported here (Gray and Ritchie, 1986; Nowak et al., 1987; Sonhof and Schachner, 1984).

In contrast to most other rectifying chloride channels, the outward rectification we observed is a property of the open channel conductance and not a voltage-dependent gating. Gating-derived outward rectification has been observed in a t least three types of chloride channels: calcium-activated chloride channels (Evans and Marty, 1986a), GABA- and glycine-activated chloride channels (Bormann et al., 1987), and chloride channels from Torpedo electroplax (Miller, 1982).

However, the oligodendrocyte chloride channel closely resembles a chloride channel found in tracheal epithe- lial cells, in its outward rectification, slight voltage dependence of gating, and conductance. This channel is normally inactive in cell-attached patches but can be activated by certain modulators applied outside the cell (Welsh et al., 1986).

Possible Function of Ion Channels in Type 2 Astrocytes

Our understanding of the function of glial cells in the optic nerve would be aided by a better understanding of the structural organization of this nerve. Despite its apparent simplicity and despite previously published anatomical studies, fundamental questions about glial cell arrangement remain. What is the relationship of one subclass of glial cell to processes of other glial cells, axons, and blood vessels? Which glial cell types commu- nicative via gap junctions?

Recent evidence suggests that perinodal glial processes that appear to originate from type 2 astrocytes are in close association with the nodes (ffrench-Constant and Raff, 1986). If the electrophysiological properties we have observed in the type 2 astrocyte in culture are also found in vivo, these cells may fire action potentials that propagate along their process. Perhaps such a signal might propagate along a chain of type 2 astrocytes, or perhaps an action potential generated in a type 2 astrocyte soma could propagate to perinodal processes and trigger release of neuroactive substances that affect the node of Ranvier itself. In support of this idea, we have observed large voltage-dependent calcium currents in type 2 astrocytes similar to those that have been impli- cated in neurotransmitter release in neurons (Miller, 1987; Perney et al., 1986).

Conversely, several points argue against the idea of signaling by type 2 astrocytes. For instance, relatively large amounts of current were required to depolarize the type 2 astrocytes in culture. In addition, synapsin I, a

processes of type 2 astrocytes is needed, and nonvesicu lar release of neuroactive substances from these processes is always a possibility.

Possible Function of Ion Channels in Oligodendroc ytes

The presence of a high density of inwardly rectifying voltage-sensitive potassium channels in a cell type whose sole function has been considered to be myelination is perhaps surprising. In other cell types, inward rectifiers are thought to provide a significant potassium conductance for maintenance of the resting potential but to turn off with slight depolarization, thereby permitting regenerative (action) potentials. Hence, modulation of inward rectifiers can be used to increase excitability (e.g., Stan- field et al., 1985). But this function for inward rectifiers cannot be applicable in the absence of channel types that might cause regenerative depolarizations.

Because these channels are open at the resting potential and would also allow potassium to enter the cell rapidly, it seems reasonable that inward-rectifying channels in oligodendrocytes may participate in homeostatic regulation of potassium, either as part of a spatial buffering mechanism (Orkand et al., 1966) or as part of a potassium accumulation mechanism (Kettenman et al., 1983). Regulation of potassium in the nervous system has generally been considered to be mediated by active mechanisms such as ion pumps and ion transport- ers; but in Muller cells a role for potassium channels in potassium regulation has been postulated by Newman (1985), and new evidence suggests a passive influx of potassium through channels in astrocytes (Ballanyi et al., 1987).

Oligodendrocytes are logical candidates to participate in a potassium regulation mechanism: axonal potassium channels are directly opposed to oliogdendrocyte membranes in the paranode and internode (Chiu and Ritchie, 1980, 1981; Waxman and Ritchie, 1985). Potassium ex- iting these channels into the limited extracellular space during axonal repolarization could rapidly enter oligodendrocytes. Although earlier reports have suggested that potassium channels are nearly absent at nodes of Ranvier and that the role of internodal potassium channels in repolarization of myelinated nerve fibers is neg- ligible, there is now good evidence for both the presence and function of several kinds of potassium channels in nodes and in internodes (Baker et al., 1987; Dubois, 1981; Kocsis et al., 1986). The paranodal axoglial junctions once thought to isolate the internodal axon from the general extracellular space now appear at best to provide partial isolation (Baker et al., 1987; Barrett and Barrett, 1982). Therefore, it is not surprising that an increase in potassium in the extracellular space occurs during axonal firing in optic nerve (Ransom et al., 1985b).

Oligodendrocytes could be involved in potassium regulation in two ways. According to a spatial buffering mechanism, potassium would enter oligodendrocytes wherever the local potassium reversal potential was


more positive than the resting potential. Potassium would be rapidly shunted by current flow from a proxi- mal region of excess to a more distal region (Orkand et al., 1966) primarily driven by the voltage gradient within the cell (Gardner-Medwin, 1983a,b). The local membrane potential would become more positive because of the influx but would not rise much if it were well coupled to the rest of the cell, since the resting potential would be maintained by the normal potassium in distal regions. Moreover, if the local membrane potential of the oligodendrocyte reached EK, there would be no further potassium entry, subverting the purpose. As first pointed out by Newman (1985), inwardly rectifying potassium channels are well suited for a role in a spatial buffering mechanism. They allow potassium influx into the cell but inhibit its efflux. Also, the depolarization induced by potassium entry tends to increase membrane resistance, thus increasing the electrical space constant, and therefore the distance potassium could be carried.

However, if a spatial buffering mechanism is operat- ing in white matter, the shunt of potassium must occur radially since no long-distance gradient of extracellular potassium would exist along the length of the axon during axonal firing. Interestingly, there is anatomical evidence that the processes of type 1 astrocytes are radially oriented in the optic nerve and terminate on blood vessels (Miller and Raff, 1984; Raff, personal communication). Furthermore, gap junctions have been observed between oligodendrocytes and neighboring perinodal astrocyte processes, although it is not known yet whether these astrocyte processes arise from type 1 or type 2 astrocytes (Massa and Mugnaini, 1982; Waxman and Black, 1984).

Alternatively (or in addition), oligodendrocytes may participate in potassium regulation by a potassium accumulation mechanism. Intracellular potassium accumulation by oligodendrocytes in culture occurs when these cells are placed in a bath solution containing elevated potassium (Kettenman et al., 1983), although the mechanism mediating this elevation is not understood. In our experiments with oligodendrocytes in culture, we have frequently observed that these cells lyse after several minutes when placed in solutions containing elevated potassium especially when greater than 20 mM potassium was present; in contrast, neurons and type 1 and type 2 astrocytes can withstand 140 mM potassium for long periods.

An inwardly rectifying potassium conductance and a chloride conductance also dominate resting conductance is muscle. In muscle, when extracellular potassium is elevated, passive potassium uptake occurs by uptake of potassium, chloride, and water (Boyle and Conway, 1941; Hodgkin and Horowicz, 1959). A driving force €or chloride entry occurs when potassium is elevated because membrane potential becomes positive to Ecl and because chloride is passively distributed, that is, Ecl equals the normal resting potential. Potassium accumulation occurring by this mechanism would be limited by the rate at which chloride ions could be transported into the cell because of the need for electroneutrality.

The chloride conductance in oligodendrocytes is appar- ently inhibited under normal conditions since we only observed channels in excised patches after a few minutes. (Chloride permeability of the oligodendrocytes resting membrane was also not observed by Kettenman et al. 1983). However, a modulatory factor that could

activate these chloride channels would be expected to trigger rapid potassium accumulation in oligodendrocytes under conditions of high potassium. Such a factor could be liberated by axons during firing, by astrocytes, or even by the oligodendrocytes themselves. A similar neurohumoral mechanism has recently been proposed to occur in squid axons (Lieberman et al., 1987).

As suggested by Bevan et al. (1985), the virtue of a local potassium accumulation mechanism is that potassium is not shunted to remote regions, thus facilitating the recovery of potassium by the neuron. The observations of Hodgkin and Horowicz (1959) suggest an advan- tage of potassium inward rectifiers over other types of potassium channels for such a potassium accumulation mechanism. They showed that while the muscle cells instantly depolarize in elevated potassium solutions, when the muscle is returned to a low potassium solution the muscle repolarizes only slowly over minutes to hours. Since Ecl is then greater than Ek, potassium chloride leaves the fibre. However, the exit of potassium chloride is greatly slowed because the recitification slows outward potassium current (Hodgkin and Horowicz, 1959: Fig. 7, p 140). The same effect would be expected to occur in oligodendrocytes, although it might be even more pronounced because the outwardly rectifying chloride channels would retard outward movement of chloride ions. Such a protective mechanism might be crucial since even a small amount of efflux of potassium into the limited periaxonal space could impair axonal conduction.

If potassium accumulation by passive ion flux occurs according to the “modulated Boyle and Conway” mechanism we have suggested above, then several predic- tions follow: 1) water should follow potassium and chloride into cells, resulting in a decrease in extracellular volume, 2) blockers of chloride channels should decrease the accumulation of potassium, and 3) potassium elevation alone should be an insufficient stimulus for triggering rapid potassium accumulation (since elevated extracellular potassium alone does not activate the chloride channels). The experiments of Ransom et al. (1985b) provide strong support for the model. They observed a neural-activity-dependent shrinkage of the extracellular space (ECS) in isolated optic nerves whose magnitude correlated closely with the degree of evoked increase in extracellular potassium, yet they observed no shrinkage of the ECS to iontophoretically applied potassium. More- over, the activity-dependent shrinkage of the ECS was blocked by up to 70% in chloride-free solutions, SITS (1 mM, 4-acetamido-4-isothiocyanostilbene-2,2‘-disulfonic acid), or furosemide (10 mM). While they concluded that anion transport systems were involved, these concentrations of SITS and furosemide are now known to block chloride channels (Evans and Marty, 1986b; Gray and Ritchie, 1986).

According to such a model, why would chloride channels be modulated rather than always open? If chloride was actively pumped from the cell so that Ecl was negative to the resting potential, this could accelerate the rate of K + and C1- influx when the chloride channels were finally opened. The ability to close the channels would also slow the eventual release of K+. On the other hand, C1- entry would dissipate the membrane potential gradient that would drive potassium to distal regions of the cell under the spatial buffer hypothesis.

Several observations lend support to the hypothesis

28 BARRES ET AL.

that oligodendrocytes (and their inwardly rectifying potassium channels) play a role in potassium regulation. First, the elegant experiments of Ballanyi et al. (1987) provide evidence supporting a passive uptake mechanism of potassium through potassium channels. Using ion-sensitive microelectrodes, they showed that the potassium elevation occurring in astrocytes during stimulation of guinea-pig olfactory slices could be greatly reduced by extracellular barium. In addition, the astrocytes depolarized during stimulation from -80 to -60 mV; this suggests that an inwardly rectifying potassium channel might have been blocked, since other types of potassium channels are not activated until more depolarized potentials are reached. Second, Ransom and coworkers have studied clearance of extracellular potassium from rat optic nerves during repetitive depolarization. Nerves depleted of oligodendrocytes by 5-azacytidine treatment exhibited a decreased ability to clear potassium, even though the number of astrocytes in these nerves had increased from hyperplasia (Black et al., 1986; Ransom et al., 1985a; Yamate and Ransom, 1985). Third, in peripheral nerves Wurtz and Ellisman (1986) have observed swelling and vacuolization of the paranodal apparatus at nodes of Ranvier during high- frequency stimulation, and they have suggested that sequestration of extracellular potassium may account for these morphological changes.

Potassium homeostatic mechanisms have generally been studied in grey matter (Gardner-Medwin 1983a,b, Kimelberg, 1979; Sykova, 1983; Walz and Hertz, 1983a,b). These studies suggest that cortical astrocytes may play a central role in shunting or accumulating excess extracellular potassium. However, we have not observed inwardly rectifying potassium channels in astrocytes despite an exhaustive search of type 1 and type 2 astrocytes in optic nerve cultures and of astrocytes in cortical cultures (although they have been observed in Muller cells: Newman, 1985). This suggests (among other interpretations) that potassium homeostatic mechanisms in white matter may be significantly different than in grey matter. In the white matter, the problem may also be more urgent: axons occur in high density, the extracellular space is smaller than that found in grey matter (C. Nicholson, personal communication), neuronal firing rates can be extremely rapid (retinal ganglion cells in the cat discharge at 200 to 800 times per second: Kuffler, 19531, and small increases of extracellular potassium could lower the reliability of axonal conduction. Although we have not studied glia in corpus callosum or other white matter tracts, we suspect that our studies of optic nerve glia will be generally applicable to white matter, since glial cell types with similar morphologic and antigenic properties are observed in cultures prepared from corpus callosum (Raff et al., 1983a).

It should be possible to test directly whether inwardly rectifying potassium channels in oligodendrocytes are involved in potassium homeostatic mechanisms, by specific pharmacological blockade with low concentrations of extracellular cesium. Inwardly rectifying channels are not known to be present either in optic nerve axons or in other optic nerve glial cells (but see Baker et al., 19871, and small concentrations of extracellular cesium will not block other types of potassium channels. We predict that extracellular cesium (1 mM) will acutely

disrupt potassium homeostatic mechanisms in white matter.

ACKNOWLEDGMENTS

We would like to thank Peter Grafe for helpful discus- sions. We are also grateful to Eric Newman and an anonymous reviewer for their helpful critiques of this manuscript and to Martin Raff for advice and encouragement.

This work was supported by National Institutes of Health grants NS-22059 (to D.P.C.), NS-21269 and NS- 16367 (to L.L.Y.C.), and F32 NS-07970 (to B.A.B.), by a grant from the Alfred P. Sloan Foundation (to D.P.C.), and by the Howard Hughes Medical Institute.

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