Neuron,

Vol. 4, 507-524,

April,

1990, Copyright

0 1990 by Cell Press

Ion Channel Expression by White Matter The O-2A Clial Progenitor Cell Barbara A. BarresF+§ Waiter J. Koroshetz,+ Kenton J. Swartz,*+ Linda 1. Y. Chun,+* and David P. Corey*+5 *Program in Neuroscience Harvard Medical School Boston, Massachusetts 02115 +Department of Neurology *Department of Neurosurgery Massachusetts General Hospital Boston, Massachusetts 02114 SNeuroscience Group Howard Hughes Medical Institute Boston, Massachusetts 02114

Summary We describe electrophysiological properties of the O-2A glial progenitor cell in a new serum-free culture system. O-2A progenitors have many properties characteristic of neurons: they have glutamate-activated ion channels, express the neuronal form of the sodium channel, fire single regenerative potentials, and synthesize the neurotransmitter GABA by an alternative synthetic pathway. Nearly identical properties were observed in acutely isolated O-2A progenitors, indicating that this phenotype is not an artifact of culture. The O-2A did not express a simple subset of channel types found in its descendant cells, the type-2 astrocyte and oligodendrocyte, studied in the same culture system. During development, these electrophysiological properties may contribute to 0-2A function in vivo. Introduction Although it has a simple structure, the function of white matter is not fully understood. The optic nerve is a good preparation for studies of white matter glia because it consists only of retinal ganglion cell axons and three glial cell types: type-l astrocytes, type-2 astrocytes, and oligodendrocytes (Raff et al., 1983a; Miller and Raff, 1984; Miller et al., 1989b). The distinct morphological and antigenic characteristics of each of these cell types (reviewed in Miller et al., 1989b) suggest a diversity of function, as do their differing electrophysiological properties (Barres et al., 1988a, 1989, 1990). The first paper in this series described some electrophysiological properties of type-2 astrocytes and oligodendrocytes in culture (Barres et al., 1988a). This work raised several questions. First, oligodendrocytes and type-2 astrocytes are descended from a common bipotential glial precursor cell, the O-2A progenitor (Raff et al., 198313, 1984; Temple and Raff, 1985). To what extent is the O-2A phenotype characteristic of glia, or does it reflect a transitional state? How do its

Glia:

electrophysiological properties compare with those of its descendants, and do these suggest any specific functions? Second, do the properties of glial cells in vivo correspond with the properties of those in vitro, where they are deprived of normal neuronal contacts and are exposed to unknown serum factors? Bevan et al. (1987) previously found that optic nerve O-2A progenitors in culture express several voltagedependent ionic currents. The channel types they observed were a subset of those found in type-2 astrocytes, but differed from those found in oligodendrocytes (Bevan and Raff, 1985; Barres et al., 1988a). However, each of these three cell types was characterized under different culture conditions, so it was not clear whether these differences reflected a true developmental program of channel expression. More recently, the developmental switch in ion channel phenotype in at least one part of the lineage-from the O-2A progenitor to the oligodendrocyte-has been confirmed for mouse brain glia in culture (Sontheimer et al., 1989). Still unanswered is whether the ion channel phenotype of O-2A lineage cells in culture reflects that in vivo. Thus we have recharacterized ionic currents in O-2A progenitors, type-2 astrocytes, and oligodendrocytes in the new in vitro preparation developed by Lillien and Raff (1990). Under these conditions, O-2A progenitors divide and differentiate into oligodendrocytes and type-2 astrocytes in the same sequence as occurs in vivo. In the present work, we report three findings. First, O-2A progenitor cells in this culture preparation have a “neuronal” ion channel phenotype that is neither a subset nor a superset of both descendant cells. Nearly identical properties are found in O-2A progenitors acutely isolated from optic nerve, including the presence of glutamate-activated ion channels, suggesting that similar properties occur in vivo. Second, these cells synthesize the neurotransmitter, y-aminobutyric acid (CABA). Third, type-2 astrocytes and oligodendrocytes have properties in serum-free culture conditions that are similar, but not identical, to those we previously reported in serum-containing medium. Results Voltage-Dependent Sodium Currents in O-2A Progenitors We studied whole-cell currents in O-2A progenitors, both in culture and in suspension prior to culture, using a series of ion isolation solutions designed to reveal the presence of the main ion channel types (Figure 1; see Experimental Procedures for cell identification and solutions). We observed both inward and outward current components in all cells examined. A sodium current was present in 100% of O-2A progenitors, both in culture and in suspension (Table 1;

Neuron 508

Figure

1. O-2A

Progenitor

Cells

Nomarski (A, B, and C) and fluorescence (D, E, and F) micrographs of O-2A progenitors culture (B and E) and in tissue prints (C and F). Cellswere labeled immunohistochemlcally by rhodamine-conjugated goat anti-mouse immunoglobulin. Bar, 20 Frn (A, B, D, and

Figure 2). Current density was calculated by dividing the peak inward current by the cell capacitance, which is a measure of surface area. In culture the sodium current density in O-2A progenitors was about half of that observed in the acutely isolated cells (Table 1). We have previously reported that at least two forms of sodium channels exist in optic nerve glia: a “glial” form found in type-l astrocytes and most other glial cell types and a “neuronal”form found in retinal ganglion cells and some other neurons; this latter form is also the predominant form in type-2 astrocytes (Barres et al., 1989). The glial form has high tetrodotoxin sensitivity, like the neuronal form, but has a more negative voltage dependence and slower kinetics. Using the same whole-cell isolation solutions, we studied the sodium channels in O-2A progenitors in culture to determine which form was expressed. The voltage dependence and kinetics of the O-2A channel were nearly identical to those of the neuronal form of the channel (Table 2). Calcium currents were never present in O-2A progenitors in culture or in suspension. At least 20 cells were studied in each set of isolation solutions. Regenerative Potentials in O-2A Progenitors Because of the presence of sodium currents in O-2A progenitors, we wondered whether these cells were excitable. With the NaCl bath and KCI pipette solutions (see Experimental Procedures) and the wholecell, current-clamp recording configuration, injection of depolarizing current induced a single regenerative potential in all O-2A progenitor cells in culture and in about half of acutely isolated O-2A progenitors (Fig-

in culture (A and D), in suspension with the monoclonal antibodyA2B5, E); 15 wrn (C and F).

prior to followed

ure 3). Typically, about 100 pA of current was required; these potentials overshot zero, but rarely repolarized back to the resting potential with sustained current injection. Thus this behavior was intermediate between that shown previously for type-2 astrocytes in serum-containing culture (which require at least 1000 pA of injected current, fire a single spike, and repolarize little; Barres et al., 1988a) and that of retinal ganglion cells (which require about 20 pA of injected current, fire singly or repetitively, and repolarize completely; Barres et al., 1988b), under identical recording conditions. Voltage-Dependent Outward Currents in 0-2A Progenitors Two components of outward current were present in all O-2A progenitors. Both currents were carried by potassium ions, since no outward current was observed with chloride isolation solutions. In addition, both components were entirely abolished when barium (5 mM) or 4-aminopyridine (5 mM) were included in the bath. Using voltage ramp and step protocols identical to those used for type-2 astrocytes and oligodendrocytes (Barres et al., 1988a), we observed a sustained current (Ko) and a current that decayed more rapidly (KA) (Figure 2). Both components were observed in all cultured and acutely isolated O-2A progenitor cells. The sustained component was isolated by using prepulses to -45 mV, which inactivated all current carried by the decaying component. Current-voltage relations of the sustained component, Kn, indicated that it was activated by steps to -30 mV or greater, that it activated more slowly, and that it did not decay rap-

“Neuronal” 509

Table

Properties

1. Average

of O-2A

Currents

Progenitor

in O-2A

Cells

Lineage

O-2A Serum-Free Capacitance

Type Suspension

2A

Serum-Free

Oligodendrocyte KS”

Serum-Free

*

12.6 + 4.3

3.6 k 1.6

110-380 15 k IO

80-370 31 & 12

360-2,100 0.7 * 0.22

190-910 0.93 + 0.67

380-2,300 0.43 k 0.16

0.24

+ 0.16

Range (PA) Density (nS/pF)

130-850 0.25 + 0.14

300-1,260 1.34 * 0.59

700-5,600 0.63 + 0.38

0.31

f

KIK Range, Density

70-170 0.17 + 0.13

20-80 0.19 k 0.08

3,400-5,600 0.85 k 0.19

l,OOO-5,300 0.66 & 0.55

Na+ Range Density

(pF)

Cells

(PA) (pA/pF)

26 to 72h 230 14

f &

1,200 4.7

75

30

410 f 3,100 17 * IO

85

f

FCS” 45

0

160 * 70 0

KU Range (PA) Density (nS/pF)

180-3,400 0.10 * 0.01

100-800 0.06 & 0.05

K

- 120 mV (PA) (pA/pF)

Car Range (PA) Density (pA/pF)

0

CaL Range (PA) Density (pA/pF)

0

Conductance WKD ~TKR

0

0

O-400 0.20

0.006

k

0

O.loC

300-3,300 0.24 k 0.19

1,200-10,000 0.57 f 0.38

20-85 1.7 * 0.9

O-220 f 1.8

0

0

2.4

O-50 0.51 f 0.55

O-470" ? 4.4

0

0

3.7

0.06 0.44

0 0.1

Ratios 1.4 12

0.35 5.6

1.5 1.2

1.3 0.8

All values represent the averages of IO-15 cells and are expressed as mean f SD. Each current component was found in 100% of cell unless otherwise noted. Serum-Free: cultures grown in serum-free medium; FCS: culture containing fetal calf serum; Suspension: acutely isolated cells in tissue prints were used. acutely isolated cells in suspension, except K,,, for which a Values taken for comparison from Table 2 of Barres et al. (1988a), except KIR. h Average values taken from youngest to oldest cultures. ( This current component was found in only 23% of cells. d This current component was found in only 60% of cells.

A

02A Progmtors Cultured Na

B.

KD

KA

02A Progmtors: Acutely Isolated

Figure

2. Voltage-Sensitive

Currents

in O-2A

Progenitors

Whole-cell ionic currents elicited in O-2A progenitors in culture (top row) or after acute isolation prior to culture (middle row) by a series of voltage command steps (bottom row) are demonstrated. Currents are plotted as average current densities (see Table I), so that amplitudes of currents in each column can be directly compared. The acutely isolated cells were either in cell suspensions (Na, KD, KJ or in tissue prints (KIR). The capacitive transients have been blanked. Bath and pipette solutions differed for each type of ionic current (see Experimental Procedures).

NeLlKHl 510

Table

2. Properties

of Whole-Cell

Sodium

Currents

in O-2A

Lineage

O-2A

Activation (mV) (mV) Half-inactivation Equiv. charge of inactivation Tauh (ms) -30 mV -20 mV -10 mV 0 mV

Culture

Suspension

TypeZA Culture

Type lAa Suspension

Suspension

-32 + 2.5 -5% f 3.1 4.4 + 0.3

-2% f 2.2 -57 k 3.6 3.9 & 0.4

-31 f 3.3 -54 + 2.7 4.1 * 0.3

-41 5 1.7 -80 f 5.8 4.4 * 0.5

-37 * 2.9 -5.5 * 4.0 4.0 t 0.3

2.1 1.4 1.0 0.7

f f k &

0.3 0.1 0.2 0.2

2.1 1.3 0.8 0.6

+ f * k

0.4 0.2 0.2 0.1

All values are expressed as mean t SD and represent the averages type-2 and type-l astrocytes. RGC: retinal ganglion cells. a Data taken for comparison from Table 2 in Barres et al. (1989).

idly. To isolate KA from KD, currents elicited with prepulses to -45 mV were digitally subtracted from those elicited with prepulses to -100 mV. KA was activated at lower potentials (to -45 mV or greater), it activated comparatively rapidly, and it then decayed rapidly. Steady-state inactivation curves showed that half of KA was inactivated by prepulses to -70 of- 4 mV. Whereas KI, was the predominant potassium current in O-2A cells in culture, in acutely isolated O-2A progenitors, KA always predominated (Table 1). Determination of current densities revealed that this difference could be attributed to a 4- to 5-fold lower expression of KA in O-2A progenitors in culture (Table 1).

20 )

mV

50ms

Figure 3. Regenerative Culture

Potential

Cells

in an O-2A

Progenitor

Cell

in

A single regenerative potential overshooting zero was elicited in response to a depolarizing current injection. This was recorded in the current clamp configuration with an injection of 100 pA of current. Bath solution: 140 mM NaC!, 5 mM KCI, 2 mM CaCl>, 3 mM dextrose, 5 mM HEPES (pH 7.4). Pipette solution: 140 mM KCI, Ca2+ buffered to 10m6 M, 1 mM MgCI*, 5 mM HEPES (pH 74).

2.0 1.3 1.0 0.7

i i + i

0.3 0.3 0.2 0.1

of at least 10 ceils.

4.4 2.3 1.7 1.3 Temperature

f -t * k

0.5 0.3 0.2 0.2

1.8 1.3 0.7 0.5

was 23OC. Type

2 * * *

0.2 0.2 0.1 0.1

2A and Type

IA:

Calcium Sensitivity of Potassium Currents in O-2A Progenitors The scorpion toxin charybdotoxin (CTX; 30 nM), a specific blocker of some calcium-activated potassium channels (Miller, C., et al., 1985a), blocked more than two-thirds of the sustained current component, KD, in both cultured and acutely isolated O-2A progenitors when average whole-cell current components in different ceils were compared (Table 3). The residual sustained component had voltage dependence and kinetics identical to those of KO, but was CTX-insensitive: it was not blocked further by raising the CTX concentration to 100 nM. The effect of CTX could also be seen in the same cell before and after exposure to the toxin (30 nM). In both of 2 cells, K,, was blocked by approximately 60% and KA was unaffected. Apamin (100 nM), another blocker of some calcium-dependent potassium currents, had no effect on either potassium current, in culture or in suspension. To determine whether these potassium currents were actually calcium-sensitive, we studied whole-ceil outward current in O-2A progenitors with pipette solutions containing calcium buffered to either IO+ M or 1O-9 M with EGTA (10 mM). As expected, K. was strikingly calcium-sensitive. In acutely isolated progenitors, the percentage decrease in current in low calcium closely matched that caused by CTX (Table 3). Surprisingly, lowering calcium to 10e9 M also decreased KA by about SO%, suggesting that at least a portion of this current is also calcium-sensitive, although it could not be blocked by either apamin or CTX. On the other hand, in O-2A progenitors in culture, we observed no decrease in either K,, or KA when the pipette calcium concentration was decreased to 10m9 M (Table 3). This was surprising, since KI, in cultured O-2A progenitors was clearly blocked by CTX. It is possible that calcium was not effectively buffered to 10e9 M in the larger, process-bearing O-2A progenitors in culture. Alternatively, K,, in cultured O-2A progenitors may be calcium-insensitive. Potassium It is difficult detection

Inward Rectification to design isolation of inwardly rectifying

in O-2A Progenitors solutions that permit potassium currents

“Neuronal”

Properties

of O-2A

Progenitor

Cells

511

Table

3. Calcium

Dependence

of Outward

Currents

in O-2A Progenitors

KO

KA

Solution

Culture

Suspension

Culture

Suspension

Ca2+ 10m6 M Ca2+ 10m6 M, CTX CaZ+ 1O-9 M

0.7 2 0.22 0.1 f 0.12 0.79 + 0.37

0.9 f 0.67 0.27 f 0.22 0.21 * 0.17

0.25 f 0.14 0.23 f 0.13 0.35 f 0.25

1.34 * 0.59 0.10 f 0.60 0.59 + 0.30

All values

are conductance

densities

expressed

in nS/pF

(mean

when large outward currents are present. For instance, intracellular cesium blocks outwardly rectifying potassium currents, but also partially blocks inwardly rectifying potassium current (e.g., Barres et al., 1988a). Consequently, we isolated the inwardly rectifying potassium currents by first recording wholecell currents with a bath solution containing 20 mM potassium and a pipette solution containing 140 mM potassium. The current remaining after cesium (5 mM) was added to the bath was then subtracted from the original whole-cell current. Extracellular cesium blocked the inwardly rectifying component, but not current from the outwardly rectifying channels or from seal leakage. Thus the subtracted current consisted only of inwardly rectifying potassium current (Figure 2; Figure 4). Figure 4 demonstrates that these inward currents, even in high bath potassium, appear linear and might otherwise have been inadvertently subtracted away as leakage current. Inwardly rectifying potassium current, KIR, was detected in 100% of O-2A progenitors in culture. It was not observed in acutely isolated O-2A progenitors in suspension. Since all other current components found in O-2A progenitors in culture were also found in cells in suspension, we considered two possible artifactual reasons why KIR was not detected in the acutely isolated cells. First, it was possible that the enzymatic digestion used to isolate the cells specifically digested these channels. Second, the channels might be located in the processes of O-2A progenitors; these processes were always shorn off during the dissociation procedure. To approach this issue experimentally, we employed an enzymatic “tissue print”dissociation to obtain acutely isolated O-2A progenitors still bearing processes (see Experimental Procedures). 0-2A progenitors in tissue prints were identified by their small size and, in all cases, by A2B5 labeling prior to recording (Figures IC and IF). All of these cells, when bearing one or more processes, were found to have an inwardly rectifying potassium current (Table 1). When normalized for surface area, the conductance of KIR was similar to that found in cultured O-2A cells (Table 1). Thus O-2A progenitors in vivo appear to have the inward-rectifier channels, which appear to be localized to processes. The localization of inward-rectifier channels to processes has previously been reported for other glia (Brew et al., 1986; Barres et al., 1988a; Newman, 1989).

f

SD) and

A.

represent

the

averages

25 ms ti

Oligodendrocyte

/

C.

PA

-

-320

--+1600

i

25ms -

Type-2

+

Astrocyte

-

4. Inwardly

PA

1. -600

I Figure

ceils.

Progenitor

02A

B.

of 8-15

Rectifying

PA -2000

Currents

in O-2A

Lineage

Cells

Whole-cell currents elicited by a ramp voltage command in cells in culture: (A) an O-2A progenitor cell, (B) an oligodendrocyte, and (0 a type-2 astrocyte. Currents are shown before (solid traces) and after (dotted traces) addition of cesium (5 mM) to the bath. The ramp voltage was increased from -100 mV to -80 mV over250 ms. Linear leakcurrent was not subtracted from thecurrent response. Bath solution: 140 mM NaCI, 20 mM KCI, 2 mM CaCb, 3 mM dextrose, 5 mM HEPES (pH %4). Pipette solution: 140 mM KCI, Ca2+ buffered to 1O-6 M, 1 mM MgC12, 5 mM HEPES fpH 74).

NellKNl 512

presence of intracellular potassium (Brew and Attwell, 1987; Barbour et al., 1988). Glutamate-activated currents were induced in 100% of O-2A progenitors. These currents were inward at negative potentials, were outward at positive potentials, and reversed near zero, at 0.5 & 2 mV (mean & SD; Figures 5B, 5C, 5E, and SF). Currents were never elicited by control puffs of the solution used to dissolve the glutamate. Currents could be activated by glutamate (200 PM; 14 of 14 cells), kainate (100 PM; IO of 10 cells), or quisqualate (100 uM; 10 of 10 cells). Peak curent activated at -80 mV was as follows: glutamate, 24 + 7 pA (average density: 9 + 3 pA/pF); kainate, 27 + 9 pA (average density: 10 2 3 pA/pF); and quisqualate, 46 + 16 pA (average density: 18 + 6 pA/pF). Rapid desensitization was present when quisqualate was the agonist, but kainate-activated cur-

Glutamate-Activated Currents in O-2A Progenitors Glutamate-activated receptors and glutamate-activated channels of the non-NMDA type have recently been demonstrated in O-2A progenitor ceils in culture (Gallo et al., 1989; Cull-Candy et al., 1989, J. Physiol., abstract). Are these receptors also present in vivo? O-2A progenitors were acutely isolated at P7; they were identified by their characteristic morphology along with the presence of the neuronal form of the sodium current. At this age, O-2A progenitors are the only cells in optic nerve suspensions with the neuronal form of the sodium current (Barres et al., 1989). The avoidance of antibody labeling facilitated the tight gigaohm seals necessary for these experiments. Glutamate (200 PM) was pressure-ejected onto O-2A progenitors; the elicited ionic currents were studied with whole-cell recording. The bath contained the NaCl solution, and the recording pipette contained a CsCl solution (see Experimental Procedures). Use of cesium not only eliminated outward potassium currents, but also ensured that any inward current due to activation of electrogenic glutamate transport would not be induced, since such induction is dependent on the

rents did not desensitize. Current when the glutamate concentration 20 PM. All glutamate-activated current was blocked by the glutamate 6-cyano-7-nitroquinoxaline-2,3-dione

was was

not detected decreased to

in 0-2A progenitors receptor antagonist (CNQX; Honore

A Glu + CNQX

Kainic

8 PA L

Acid 13pA

:OpA

125 ms

l0ms

Quisqualic

Acid

25 ms

E

D

+30

T -60

ii

PA

/ 5 PA 250 ms

Figure

5. Glutamate-Activated

i -60

Ionic

Currents

in O-2A

Progenitors

Whole-cell current responses of acutely isolated O-2A progenitor cells to 200 uM glutamate (A and D), 100 uM kainate (B and E) and 100 ficM quisqualate (C and F). (A) Current response at a holding potential of -80 mV to glutamate (bottom trace) or glutamate and 20 PM CNQX (top trace). (B and C) Family of current responses to kainate or quisqualate elicited by test steps from -80 to +60 mV in 20 mV increments. (D) Whole-cell current response to glutamate in this cell shows individual single-channel openings. The largest events are 50 pS and can last tens of milliseconds (see text). (E and F) Peak current-voltage relation plotted from the current responses in (B) and CC). In each panel, the duration of drug exposure is indicated by a horizontal bar beneath the current traces. Bath solution: 140 mM NaCI, 5 mM KCI, 2 mM CaCI,, 3 mM dextrose, 5 mM HEPES (pH i’4). Pipette solution: 140 mM CsCI, Ca*+ buffered to lOA M, 1 mM MgCb, 5 mM HEPES (pH 74).

“Neuronal”

Properties

of O-2A

Progemtor

Cells

513

et al., 1988; 20 PM; 4 of 4 cells; Figure 5A). These currents were always associated with a marked increase in current noise (Figure 5). As reported by Cull-Candy et al. (1989, J. Physiol., abstract; also see Usowicz et al., 1989), the amplitude of the noise was largest when the agonist was glutamate or quisqualate and was smaller when kainate was used (Figure 5). This increased noise suggested that the ion channels activated by quisqualate were of larger conductance than those activated by kainate. Although detailed single-channel studies were not performed, single channel openings of 50 pS conductance, lasting up to tens of milliseconds, were sometimes observed in our whole-cell recordings when either glutamate or quisqualate (but not kainate) was the agonist (Figure 5D). Similar channels were observed in O-2A progenitors and type-2 astrocytes in culture (Cull-Candy et al., 1989, J. Physiol., abstact; Usowicz et al., 1989). Whole-cell current-voltage relations of glutamate current were always linear over the range studied, in contrast to the outwardly rectifying current-voltage relations reported for other cell types (Figures 5E and 5F; see Discussion). No responses were seen to NMDA (100 vM; 3 of 3 cells), even in the absence of magnesium and the presence of glycine (500 nM; johnson and Ascher, 1987). The absence of NMDA responses could indicate that O-2A progenitors do not normally have this class of glutamate-activated receptors or that they were selectively destroyed by the papain used to isolate these cells. Because NMDA-activated channels are present on rat retinal ganglion cells (Aizemann et al., 1988), we sought to determine whether acutely isolated P7 retinal ganglion cells, exposedto the same amount and of papain for the same duration, had NMDA responses. NMDA responses were not observed (3 of 3 cells), although glutamate-activated currents were present in all cells. Thus if NMDA receptors were present in vivo, they probably would have been digested during enzymatic isolation. Although the average glutamate currents were larger in retinal ganglion cells than in O-2A progenitors, the average current density was close to that in O-2A progenitors, about 7 + 2 pA/pF. Glutamate-activated channels have not been detected in cultured oligodendrocytes (Cull-Candy et al., 1989, j. Physiol., abstract); similarly, we failed to detect glutamate-activated currents in acutely isolated oligodendrocytes (9 of 9 cells). Ionic Currents in Oligodendrocytes in Serum-Free Culture We have previously characterized ionic currents in oligodendrocytes in cultures that contained a small amount of fetal calf serum (0.75%). In cultures completely lacking serum, oligodendrocytes also develop from O-2A progenitors (Raff et al., 1983b; Lillien and Raff, 1990); they were found in large numbers in our cultures (Figure 6). We reexamined these oligodendrocytes using whole-cell recording. Inwardly rectifying

TYPE-2 ASTROCYTE

02A PROGENITOR Nap CaT

K~ -

CaL

OLIGODENDROCYTE

KIR

K~

N%

KA K~/~~

KIR

Figure O-2A

6. Developmental

Program

of Channel

Expression

KD

in the

Lineage

Nomarski micrographs of 0.2A progenitors, oligodendrocytes and type-2 astrocytes in culture. The components of whole-cell current observed are listed beneath each ceil. NaN, neuronal form of the sodium current; CaT, transient calcium current; CaL, sustained calcium current; Kn, sustained potassium current not blocked by CTX; KDarx, sustained potassium current blocked by CTX; KA, decaying potassium current; KIR, inwardly rectifying potassium current.

potassium currents were again isolated by subtracting the current remaining after extracellular cesium blockade from the control current. We again found that all oligodendrocytes expressed KIR (Table 1; Figure 4B). These outward currents were also isolated from KIR by omitting potassium from the bath (see Experimental Procedures). After blockade of these inward currents with extracellular cesium (5 mM), a smaller, outward potassium current, KD, was also present in 100% of oligodendrocytes cultured without serum (Table 1; Figure 4B). Since we had not observed an outward potassium current in oligodendrocytes in serum-containing cultures (Barres et al., 1988a), we used the new isolation procedure to record from oligodendrocytes in serumcontaining cultures. We then detected a small outward current, KD, in all oligodendrocytes studied (Table 1). In serum-free cultures older than 14 days, a small additional component of outward current was ob-

served in all oligodendrocytes studied: an inactivating outward potassium current, KA, whose density appeared to increase with increasing age of the cultures (0.11 I 0.07 nS/pF). In these older cultures, Ko was still present, but at a slightly larger density in all cells (0.22 k 0.17 nS/pF). (It is not clear how old oligodendrocytes actually are in older cultures, since new ones are continually generated from O-2A progenitors still present in older cultures; Lillen and Raff, 1990.) Neither KA nor Kn was blocked by CTX (30 nM). Ionic Currents in Type-2 Astrocytes in Serum-Free Culture We have previously reported that type-2 astrocytes in serum-containing cultures express six components of whole-cell current (Barres et al., 1988a). Because serum might affect the expression (or function) of ion channels, we studied type-2 astrocytes that developed from O-2A progenitors in the serum-free cultures (Figure 6). Like type-2 astrocytes in fetal calf serum, these cells expressed sodium currents at a similar density (Table 1). Whereas sodium currents in type-2 astrocytes in fetal calf serum are composed of two forms, about 85% neuronal and 15% glial (Barres et al., 1989), the type-2 astrocytes that developed in serum-free medium expressed only the neuronal form (Table 2). Expression of the glial form of the sodium current so far coincides with expression of the surface antigen RAN-2: type-2 astrocytes express RAN-2 in our serum-containing cultures, but not in our serum-free cultures. (If a more sensitive detection method is used, RAN-2 does appear in serum-free cultures older than 21 days [Lillien and Raff, 19901.) Both transient, Cat, and sustained, Cat, components of calcium current were found in type-2 astrocytes that developed in serum-free culture, although the density of Cat was considerably lower than that found in serum-containing cultures (Table 1). As in the serum-containing cultures, Cat was found in only about two-thirds of the cells (Table I). We again observed two components of outward current, KA and Ko, at densities near those observed in serum-containing cultures. Whereas we previously reported that about half of the outward potassium current could be blocked by CTX (30 nM) in type-2 astrocytes in serum, none of the outward current in type-2 astrocytes in serum-free medium was blocked (7 of 7 cells). The properties of KA in type-2 astrocytes appeared identical to those found in 0-2A progenitors in terms of kinetics of decay and in steady-state inactivation. Half of the peak KA current was inactivated by prepulses of -71 + 3 mV. All type-2 astrocytes that developed in serum-free medium had a large inwardly rectifying potassium conductance (Table 1; Figure 4C): its density was 2- to 3-fold greater than that found in oligodendrocytes. This current had not previously been detected in type-2 astrocytes that developed in serum-containing medium (Barres et al., 1988a). We again measured in-

wardly rectifying currents in type-2 astrocytes grown in the serum-containing medium, but this time using the cesium subtraction isolation procedure described above. Inwardly rectifying potassium current was observed in 100% of type-2 astrocytes cultured in serum at a density similar to that measured in serum-free medium (Table 1). GABA During

a search

for

unique

antigenic

surface

markers,

we noticed that O-2A progenitors and type-2 astrocytes, but not type-l astrocytes or oligodendrocytes, could be brightly labeled with the monoclonal antibody VC1.l (Figures 7A and 7B). Since this antibody also labels a subset of GABAergic neurons in rat brain and retina (Arimatsu et al., 1987; Naegele et al., 1988; Penn, 1989, Harvard undergraduate thesis), we tested whether GABA was present in O-2A progenitors. PO/P7 cocultures were immunohistochemically labeled with a polyclonal antiserum to GABA 1 and 3 weeks after plating (see Experimental Procedures). All optic nerve glial cell types, including O-2A progenitor cells, were present in these cultures. In addition, because the cultures were prepared from optic nerve along with part of the optic chiasm, a few neurons were also present (Card et al., 1981), typically about 3-6 on a coverslip containing thousands of glial cells. These were easily identified by their characteristic axon-bearing morphology, by the presence of action potentials, and by bright labeling with anti-tau antibodies. We found that all O-2A progenitor cells had GABAlike immunoreactivity and were labeled strongly throughout their somata and processes (Figures 7C and 7D; Figure 8C). Dark labeling of bead-like varicosities along their processes was prominent. Neurons were usually darkly stained and had dark varicosities. Other O-2A lineage cells were labeled: some type-2 astrocytes were labeled as darkly as O-2A progenitors, and oligodendrocytes were labeled to a lesser extent, with labeling localized mainly to the somata (data not shown). Type-l astrocytes and meningeal cells were generally labeled very lightly or not at all. Control experiments with primary antibody omitted or preadsorbed with GABA (coupled to BSA; see Experimental Procedures) produced no labeling of any cells (Figure 7E). The immunohistochemical results suggest the O-2A progenitors may be synthesizing GABA, since the medium contained no serum and the cultures contained extremely few neurons. To verify that the few neurons present were not the source of the GABA-like immunoreactivity in O-2A progenitors, cultures were prepared using optic nerve tissue specifically excluding the optic chiasm to ensure that no neurons would be present (see Experimental Procedures). The elimination of GABAergic neurons in these cultures was verified by immunohistochemical staining with the GABA antiserum: the rare cells with neuronal morphology that stained darkly with the antibody were completely absent with the more stringent dissec-

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Cells

GABA Figure

Standard 8. Neurochemical

Putrescine+

PutrescineVerification

of GABA

in 0-2A

Cultures

HPLC with electrochemical detection was used to detect the presence of GABA in HC104 (0.1 M) extracts of cultured optic nerve cells. Chromatograms illustrate the analysis of authentic GABA, 50 pmol injection (A), an extract of cells grown in the absence of putrescine for 72 hr (B), or an extract of cells grown in the presence of putrescine (C). The retention time of GABA was 171 min (A). A peak (indicated by the arrow) that eluted at the CABA retention time is seen in (C) but not in (B). If any CABA was in (B), it was present at concentrations below the sensitivity ofthisassayforGABA(1 pmol per injection).GABA-likeimmunoreactivity (see Figure 7) in cultured O-2A progenitors grown with putrescine (inset C) is diminished in cultures grown without putrescine (inset B).

Figure Z lmmunohistochemical lmmunoreactivity in O-2A

Demonstration Progenitors

of

CABA-like

(A and B) O-2A progenitors in culture (Nomarski [A]; fluorescence [B]) are labeled by the monoclonal antibody VC1.l. An oligodendrocyte in the center of the field is not labeled. (C and D) Anti-GABA immunohistochemical label of O-2A progenitors in culture. In (C) O-2A progenitors are labeled, as well as a process of a neuron (upper left), shown for comparison. In (D) O-2A progenitorsare still labeled with theCABAantibody in a neuronfree culture. (E) CABA labeling is eliminated by preadsorption of the primary antibody with GABA-BSA. (F) CABA labeling of acutely isolated 0.2A lineage cells in PlO tissue prints. Type-l astrocytes, the process-bearing cells with large somata, are not labeled, whereas the smaller cells bearing fewer or no processes (O-2A progenitors and oligodendrocytes) are labeled. Immunohistochemical protocol was indirect immunofluorescence (B) or indirect immunoperoxidase (C, D, E, and F). Bar, 15 nm (A and B); 20 pm (C, D, E, and F).

tion. O-2A progenitors in these neuron-free optic nerve cultures continued to label with antibodies to GABA (Figure 7D). Because of the possibility that the GABA antibody detected a molecule other than GABA, we verified the presence of GABA in our neuron-free c:ultures by HPLC measurements. A peak eluting at the GABA retention time of I%1 min was present (Figures 8A and 8C; see Experimental Procedures for the chromatographic conditions). The peak corresponding to GABA also eluted with authentic GABA under a second chromatographic scheme in which the mobile phase contained 37% methanol (pH 5.12). Finally, addition of authentic GABA to the samples of cultured cells produced a single clean peak at 17.1 min, which was equal to the concentration of the endogenous peak plus the quantity of authentic GABA added (data not shown). To detect a synthetic enzyme, we labeled the cultures with antibodies to glutamic acid decarboxylase (GAD; see Experimental Procedures; Wu et al., 1986; Chang and Cottlieb, 1988). While almost all of the contaminating neurons in the optic nerve cultures were labeled darkly with the GAD antibodies, we were unable to detect GAD in O-2A progenitors, type2 astrocytes, or any of the other glial cell types in these cultures.

This result suggested that GABA was synthesized via an alternative pathway that did not involve GAD. An alternative synthetic pathway occurring in nonneural tissues and the brain has been previously described; it utilizes putrescine as a pecursor of GABA rather than glutamate (Seiler and Al-Therib, 1974; DeMello et al., 1976; Seiler et al., 1979). Putrescine is a component of our serum-free medium. To test this possibility, we omitted the putrescine from the medium for 48 hr, beginning on the fifth day of culture, and then immunohistochemically labeled the cultures for GABA. Although the occasional neurons present in these cultures continued to be darkly labeled, labeling in the O-2A progenitors (and other O-2A lineage cells) was greatly diminished (Figure 8B). However, in sister cultures fed with medium containing putrescine, O-2A progenitors continued to label darkly in concurrently developed control slides (Figure 8C). Quantitation of the amount of GABA using HPLC showed that the cells cultured with putrescine contained CABA at a concentration of 214 f 28 pmol per 35-mm dish (n = 2; Figure 8C). In contrast, the cells cultured without putrescine contained GABA at a concentration below the sensitivity of our assay, less than 20 pmol per 35-mm dish (n = 2). Is this GABA synthesis simply an artifact of putrestine-containing tissue culture conditions? Preliminary data suggest that this is not the case: in P7 optic nerve tissue prints labeled with the GABA antibody, many of the O-2A lineage cells present at this age, O-2A progenitors and oligodendrocytes, were labeled. Cells not of the O-2A lineage were not labeled (Figure 7F).

Discussion O-2A Ion Channel Phenotype The O-2A glial progenitor cell possesses a complex set of physiological properties. First, it has at least five types of ionic currents active under whole-cell recording conditions. These most likely reflect the presence of specific ion channel types: a sodium channel, an inwardly rectifying potassium channel, a delayed potassium channel, a calcium-dependent sustained potassium channel, and an inactivating A-type potassium channel, of which at least 50% is calcium-dependent. Second, acutely isolated O-2A progenitors possess non-NMDA glutamate receptors that directly gate ion channels. Third, our observations strongly suggest that O-2A progenitors are able to synthesize GABA, and it appears that they do so by an alternative synthetic path not used by most neurons. The O-2A ion channel phenotype is neuronal in character: both glutamate-activated ion channels and voltage-dependent sodium channels are expressed at a density similar to that found in neurons. The sodium channels have properties identical to those found in neurons such as retinal ganglion cells, but distinct from those in type-l astrocytes. In addition, O-2A pro-

genitors fire tion of small

single regenerative potentials with amounts of depolarizing current.

injec-

Comparison of O-2A Properties In Vitro and In Vivo The serum-free culture conditions of Lillien and Raff (1990) promote the sequential development of cells from O-2A progenitors in an order mimicking that occurring in vivo. To assess whether the electrophysiological properties of O-2A progenitors in these cultures are like those occurring in vivo, we studied acutely isolated O-2A progenitors prepared by enzymatic digestion and trituration. Four of the five basic types of voltage-dependent currents present in the cells in culture were also present in these acutely isolated cells. Moreover, we confirmed that the nonNMDA, glutamate-activated ionic currents present in O-2A progenitors in vitro (Cull-Candy et al., 1989, J. Physiol., abstract) are also present in the acutely isolated O-2A cells. Acutely isoiated O-2A cells are still not perfect models of O-2A progenitors in vivo, since they lack processes and have been exposed to enzymes. Thus a channel type preferentially localized to a process might be absent or present at lower density in the acutely isolated cells. Such was the case with the inwardly rectifying potassium current. To demonstrate this current, we used a new tissue print preparation that yields cells still bearing processes. (This procedure and its use in characterization of optic nerve glia will be further described in a later paper in this series.) O-2A progenitors isolated by tissue printing did express the inwardly rectifying potassium current. Fortunately, extracellular papain appeared not to digest any of the basic types of voltage-dependent ion channels. We observed one prominent difference between acutely isolated cells and cultured cells: KA in culture was only a minor component of the total potassium current. Its density was about 5 times lower than that in acutely isolated cells. This difference cannot be attributed to loss of processes in the acutely isolated cells (nor to a digestive effect of enzymesj and must reflect a decreased expression of KA in O-2A progenitors in cu!ture. Possibly a cell type present in vivo, such as neurons, can up-regulate expression of KA in O-2A progenitors. Since the ion channel phenotype of O-2A progenitors in serum-free culture is overall similar to that of acutely isolated cells (suspensions or prints), it probably reflects the phenotype occurring in vivo. Glutamate-Activated Channels in O-2A Progenitors Glutamate receptors and glutamate-activated ion channels were recently described in type-2 astrocytes and O-2A progenitors in culture, both from optic nerve and cerebellum (Gallo et al., 1989; Cull-Candy et al., 1989, J. Physiol., abstract). Using both whole-cell and single-channel recording, Cull-Candy et al. found that these cells express glutamate-activated ion channels

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that are activated by both kainate and quisqualate, but not by NMDA. These channels were mapped to both processes and soma. We have found that acutely isolated O-2A progenitors have non-NMDA, glutamategated ion channels, which indicates that these channels are found on O-2A progenitors in vivo as well as in vitro. The low density at which these channels occurred in both retinal ganglion cells and O-2A progenitors may reflect the loss of extra-somatic channels in processes during the dissociation procedure. While we did not study glutamate channels with single-channel recording, our whole-cell data suggest that the microscopic properties of the channels underlying these currents may be similar to those studied in type-2 astrocytes by Usowicz et al. (1989): the increase in current noise elicited by kainate is smaller than that induced by quisqualate (and glutamate), suggesting that each of these agonists activates channels of different elemental conductances. Single-channel events could be observed in some of our wholecell recordings; quisqualate and glutamate (but not kainate) activated a large and long-duration conductance state of 50 pS. In neurons, this 50 pS conductance is generally activated only by NMDA and not by non-NMDA agonists; thus it could not be activated in neurons lacking NMDA receptors (Llano et al., 1988). In addition, the 50 pS channel activated by non-NMDA agonists in type-2 astrocytes appeared to differ from the 50 pS conductance activated by NMDA in neurons since it could not be blocked by extracellular magnesium (Usowicz et al., 1989). The acutely isolated O-2A progenitors lacked NMDA receptors; however, papain has previously been reported to digest NMDA receptors (Akaike et al., 1988). Indeed, NMDA receptors were not found on retinal ganglion cells acutely isolated by the same enzymatic procedure used to isolate the O-2A progenitors. Thus our results do not rule out the presence of NMDAactivated ion channels in O-2A progenitors in vivo. However, their absence in culture suggests that this is unlikely. We noticed one other difference between glutamate-activated ion channels in O-2A progenitors and non-NMDA channels described previously: our wholecell current-voltage relations are not rectifying, while others, including those expressed by type-2 astrocytes (e.g., Usowicz et al., 1989; Ascher and Nowak, 1988), show outward rectification even though the open single channels do not rectify. Although a definitive explanation of this difference is not at hand, one possibility is that an incompletely blocked inwardly rectifying conductance could decrease the space constant along the processes at negative potentials. In cells with long processes, this would decrease the amplitude of extra-somatic glutamate-activated current that is recorded by the electrode at the soma at negative potentials. In a type-2 astrocyte, for instance, such a conductance would be significant in the presence of a typical bath solution containing 5 mM potassium

and would be incompletely blocked by cesium in the pipette. The acutely isolated O-2A progenitors lack both extensive processes and inwardly rectifying conductances and thus would not be subject to this sort of artifact. Developmental O-2A Lineage

Program

of Channel

Expression

in the

In the serum-free cultures of Lillien and Raff (1990), O-2A progenitors divide and differentiate first into oligodendrocytes and then into type-2 astrocytes in the same sequence that occurs in vivo. We have examined the electrophysiological properties of cells in the O-2A lineage as they develop under these conditions. We found that each cell in the O-2A lineage has a distinct ion channel phenotype and that these are highly homogeneous among cells of a given type. These phenotypes are sufficiently characteristic that each cell type can be identified as accurately by its ion channel phenotype as by its antigenic phenotype. For instance, each cell has a characteristic ratio of outward to inward potassium conductance: in O-2A progenitors it is much greater than 1, in type-2 astrocytes it is near 1, and in oligodendrocytes it is less than 1 (Table 1). There is a close correspondence between development of cell type based on antigenic and morphological phenotype and development of channel expression. When the O-2A progenitor decides to become an oligodendrocyte, multiple changes occur: sodium channels, the A-type potassium channel, and the CTXsensitive potassium current disappear, while the potassium inward rectifier and the component of delayed potassium current that is not CTX-sensitive persist. On the other hand, if the O-2A progenitor decides to become a type-2 astrocyte, then sodium currents and the A-type potassium current persist, the inward rectifier and delayed potassium current persist but are upregulated, the CTX-sensitive component of delayed potassium current is lost, and new expression of Car and CaL occurs (Figure 6). The developmental expression of glutamate-activated channels in cells of the O-2A lineage also follows a similar program: Cull-Candy et al. (1989, J. Physiol., abstract) found that, in culture, O-2A progenitors and type-2 astrocytes make glutamate-activated channels, whereas oligodendrocytes do not. Similarly we found that acutely isolated O-2A progenitors express glutamate-activated channels, but acutely isolated oligodendrocytes from postnatal optic nerve do not. (Glutamate responses in acutely isolated type-2 astrocytes have not yet been examined.) Interestingly, three neuronal characteristics-the expression of glutamate-activated channels, the neuronal form of the sodium channel, and KA-occur together in O-2A progenitors and type-2 astrocytes, but are lost in oligodendrocytes. Our experiments have not provided any evidence for heterogeneity among O-2A progenitors. Thus our

work rules out the possibility that there are two antigenitally similar populations of O-2A progenitors that are electrophysiologically distinct: one determined to become type-2 astrocytes and the other, oligodendrocytes. Comparison of O-2A Progenitors from Rat Optic Nerve and Mouse Brain The developmental program of channel expression in the O-2A lineage from rat optic nerve differs in several respects from that reported for mouse brain (Sontheimer et al., 1989). First, Sontheimer and co-workers reported that mouse O-2A progenitors lack KIR. However, some inward current does seem to be present in the mouse O-2A progenitor, and inward current is increased when potassium is added to the bath (see Figure 6 of Sontheimer et al., 1989). Second, voltage-dependent outward currents were reported to be absent from mouse oligodendrocytes. However, the addition of 4-aminopyridine reduces total outward current by several nanoamperes in those experiments, and the blockable current is voltage-dependent (see Figure 7 of Sontheimer et al., 1989). Thus, these disparate interpretations probably do not reflect true differences between species. Serum Effects on Ion Channel Expression To study the function of glial cells in vitro, tant to assess whether functional properties tive to culture conditions. Thus we have

it is imporare sensiasked how

the electrophysiological properties of type-2 astrocytes and oligodendrocytes that developed in serumfree medium compare with those we previously described in serum-containing medium (Barres et al., 1988a). Oligodendrocytes in both types of cultures are remarkably similar. They both express KIR and Kn at similar densities. The delayed potassium current, Kn, is small compared with KIR and was missed in our earlier study. It can be revealed if it is isolated from KIR by extracellular cesium blockade or by the use of zero extracellular potassium. One difference is that in serum-free cultures, oligodendrocytes developed an additional component of current, KA, after 1 or 2 weeks in culture; this current is never seen in oligodendrocytes in serum-containing culture. However, in these serum-containing cultures, oligodendrocytes always died after about 5 days of culture. They might have expressed KA had they survived longer (we will never know!). Type-2 astrocytes were generally similar in the two culture conditions, although there were several differences. First, sodium currents were present at similar density in both, but although these currents were composed of both neuronal and glial forms of the sodium channel with serum-containing medium (Barres et al., 1989), we detected only the neuronal form in type-2 astrocytes in serum-free medium. Second, calcium currents of the L-type (Ca,) were found at severalfold lower density in serum-free type-2 astrocytes.

Third, a large component type-2 astrocytes in serum we found no CTX-sensitive 2 astrocytes in serum-free

of the outward current in is CTX-sensitive, whereas potassium current in typemedium. inwardly rectify-

ing currents have now been observed in type-2 astrocytes, in both serum-containing and serum-free medium. Moreover, these currents are present at a density that is Z- to 3-fold greater than that in oligodendrocytes. Thus some differences in ion channel expression in type-2 astrocytes occur depending on whether they develop in serum-free or serum-containing medium. A question to be taken up in a later paper in this series involves the extent to which these in vitro phenotypes match those in vivo.

GABA Synthesis by O-2A Progenitors Our observations provide evidence that O-2A progenitors synthesize GABA and that they accomplish this by an alternative synthetic pathway different from that generally used by neurons. First, O-2A progenitors and type-2 astrocytes were labeled by the monoclonai antibody VCl.l, a marker of a subset of GABAergic neurons. Second, a polyclonal antibody to GABA !abeled O-2A progenitors darkly (and type-2 astrocytes identified by morphology only; their astrocyte identification must still be confirmed with a glial fibrillary acidic protein double immunohistochemical label). HPLC analysis confirmed the presence of GABA in totally neuron-free and serum-free cultures. In O-2A progenitors, we were unable to detect immunohistochemically CAD, which converts glutamate to GABA, but we could detect it in GABAergic neurons. In certain nonneural tissues, GABA is synthesized instead from putrescine (H2N-CH2-CH2-CH2CH2-NH*), although the exact pathway has not been established (Seiler and Al-Therib, 1974; Kremzner et al., 1975; Seiler et al., 1979; Caron et al., 1988). In support of this possibility, when we eliminated putrestine from our serum-free medium for 48 hr, GABA immunohistochemical labeling was markedly decreased in O-2A progenitors, while neurons were still darkly labeled. Similarly, the CABA peak measured by HPLC analysis of cell extracts was eliminated by removal of putrescine from the medium. Although these experiments were performed on cultured cells, GABA may be synthesized by O-2A progenitors in vivo. GABA-like immunoreactivity was detected in acutely isolated O-2A progenitors. In addition, putrescine is normally found in the brain (Noto et al., 1987). Brain homogenates have been previously demonstrated, using radioactive precursor experiments, to synthesize GABA from putrescine, although the cell types responsible were not established (Seiler and Al-Therib, 1974; Seiler et al., 1979); glioma cells are also able to synthesize GABA by this alternative pathway. (Sobue and Nakajima, 1977). The putrescine pathway has been shown to account for 20% of CABA synthesis in 6-day-old chick embryo retina, where it is

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probably synthesized by neuroblasts at a time prior to synaptogenesis (DeMello et al., 1976). Spontaneous release of GABA formed by this pathway in the adult brain, as well as enhanced release by potassium depolarization, has also been reported (Noto et al., 1987). The capacity of O-2A progenitors and possibly type2 astrocytes to synthesize GABA raises the question of whether they secrete it. Electrical activity or glutamate-induced depolarization of 0-2A progenitors could mediate release of the synthesized GABA. In fact, release of [3H]GABA, preloaded into O-2A progenitors and type-2 astrocytes, is induced by non-NMDA glutamate analogs (Gallo et al., 1986,1989). Such secretion might even be mediated by the same mechanism that carried the GABA into these cells. Recent evidence suggests that electrogenic carriers might mediate secretion, at least under certain experimental conditions (Schwartz, 1987; O’Malley and Masland, 1989). Targets for GABA may well be present in optic nerve: GABA receptors have been found on astrocytes and axons (Bormann and Kettenmann, 1988; Bhisitkul et al., 1987). Thus an important next step will be to determine whether O-2A progenitors and type-2 astrocytes synthesize and release GABA in vivo. Possible Functions of Channels in O-2A Progenitors The unique electrophysiological phenotype of each cell in the O-2A lineage suggests that the active membrane properties of the O-2A progenitor may contribute to O-2A function during development. The channels expressed at different stages are not simple subsets or supersets of one another, and the pattern of expression changes rapidly and in concert with antigenic changes marking differentiation. Since the apparent function of the O-2A cell is simply that of a progenitor, the channels may be involved in transduction of developmental signals. Although many aspects of development are thought to be intrinsically determined, extrinsic signals may regulate mitosis, celltype specification, and morphogenesis. For instance, neurotransmitters can alter the rate and direction of growth cones (Mattson et al., 1988) or increase glial cell division (Ashkenazi et al., 1989). O-2A progenitors sense environmental signals that mediate proliferation, differentiation, and perhaps migration (Hughes and Raff, 1987; Small et al., 1987; Noble et al., 1988; Lillien et al., 1988). It has been previously proposed that dilution of a membrane molecule could underlie the intrinsic clock in O-2A progenitors that is able to count divisions (Raff et al., 1985; Temple and Raff, 1986). Dilution of a channel molecule might potentially fulfill such a role. However, we did not observe large differences in channel densities among O-2A progenitors. Glutamate receptors on O-2A progenitors may obviously serve as the receptor for some extrinsic signal. Yet it is not clear what the source of the glutamate would be, since release of neurotransmitter into white matter is not generally thought to occur. Some evi-

dence suggests the possibility of impulse-mediated nonsynaptic glutamate release from axons, including optic nerve axons (reviewed in Barres, 1989, and Barres et al., 1990), and retinal ganglion cells have been shown to fire spontaneously during this period of early postnatal development. Another potential source of glutamate is dying retinal ganglion cell axons, since about 50% of retinal ganglion cells die between birth and PI0 (Perry et al., 1983), a time when O-2A progenitors appear to be migrating into the optic nerve (Small et al., 1987). Nor is it clear what purpose a glutamate signal to O-2A progenitors would have; a possibility is an effect on migration or process outgrowth (e.g., Mattson et al., 1988). In this respect, the ability of O-2A progenitors to fire regenerative potentials is especially intriguing. Although it is not clear they do so in vivo, the density of sodium channels is similar to that of neurons, and it offers a way to amplify the response to a glutamate signal. Sodium channels in either axons or glia play some critical role, since grossly abnormal development of optic nerve glia is observed when sodium channels are blocked by intracranial infusion of tetrodotoxin (Friedman and Shatz, 1990). Of the several types of potassium currents present in O-2A progenitors and their descendants, only KIR is expected to be active at their resting potential. Thus it is likely that KIR underlies the resting potential in each cell type of the O-2A lineage (see Barres et al., 1990, for further discussion). In the first paper of this series, possible functions of ion channels in type-2 astrocytes and oligodendrocytes were discussed (Barres et al., 1988a). We proposed a potassium regulatory mechanism, a modulated Boyle and Conway hypothesis of local potassium accumulation in oligodendrocytes. According to this hypothesis, neuronal activity could trigger the activation of otherwise inactive voltage-dependent chloride channels, allowing movement of potassium, chloride, and water into oligodendrocytes. Because these same chloride channels were found in type-2 astrocytes (Barres et al., 1988a), it appears that type-2 astrocytes may be equally capable of potassium accumulation by this mechanism. Type-2 astrocytes processes may be closely apposed to nodes of Ranvier (ffrench-constant and Raff, 1986; Miller et al., 1989a) and may therefore act as local potassium accumulators at nodes of Ranvier. In addition, the /igad-gated chloride conductance activated in cultured astrocytes by GABAp, receptors (Bormann and Kettenmann, 1988) would function equally well in the modulated Boyle and Conway mechanism. Glutamate may be the humoral factor postulated to be axonally released; it would depolarize the type-2 astrocytes (Usowicz et al., 1989), possibly triggering GABA secretion, which would in turn activate its associated chloride channel both on oligodendrocytes and on the type-2 astrocyte itself. The presence of GABAA receptors on astrocytes in vivo

NWtOtl 520

has not yet been demonstrated, although they are found on acutely isolated Miiller cells (Malchow et al., 1989). In summary, O-2A glial progenitor cells have neuronal electrophysiological properties and may be dynamically sensing and responding to their environment. They may nevertheless accomplish these tasks by mechanisms that differ from those of neurons: O-2A progenitors have glutamate receptors that appear to differ from those of neurons (Usowicz et al., 1989), and they appear to synthesize GABA by a pathway not used by most neurons. If O-2A progenitors do secrete GABA, it is likely they do so by a nonvesicular mechanism: synapses, synaptic vesicles, and synaptic vesicle-associated antigens have not been observed, and voltage-dependent calcium channels are also absent. Experimental

Procedures

Preparation of Cell Suspensions Optic nerves from PO, P4, or P7 Long-Evans rats were dissected from just posterior of the optic foramen through the anterior optic chiasm. Although 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). Occasional neurons were present in our cultures when any chiasm tissue was taken. For certain experiments in which totally neuron-free cultures were required (see text), the optic nerve was taken without the chiasm. This tissue was then dissociated enzymatically to make a suspension of single cells, essentially as described by Huettner and Baughman (1986). Briefly, the tissue was minced and incubated at 37OC for 75 min in a papain solution (30 U/ml; Worthington) equilibrated with 95% OS, 5% CO,. This solution also contained Earle’s balanced salts, calcium, magnesium, EDTA, sodium bicarbonate, glucose, and L-cysteine as described (Huettner and Baughman, 1986). The tissue was then triturated sequentially with 21 and 23 gauge needles in a solution containing ovomucoid (2 mgiml; Calbiothem-Behring) and bovine serum albumin (BSA; 1 mgiml; Sigma) to yield a suspension of single cells. The cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM). Preparation of Tissue Prints Use of the tissue printing method will be described in more detail elsewhere (unpublished data; see also Cassab and Varner, 1987, for a similar technique). In brief, cleaned glass coverslips were coated with nitrocellulose by dipping them into a nitrocellulose solution (1% in amyl acetate; Ladd) and then air-dried overnight. An enzymatically treated optic nerve (as above, except incubated in the papain solution in a HEPES-buffered solution at room temperature for 30-45 min) was transferred to a 35.mm petri dish containing a low calcium saline solution. The optic nerve was gently pressed onto a small square of prehydrated nitrocellulose paper (0.45 pm pore size; Schleicher & Schuell) and sliced once longitudinally down the middle with a razor blade. The nerve was then gently opened up with forceps so that the inside of the nerve faced upward. This piece of nitrocellulose paper was inverted, gently touched to the surface of the glass coverslip, and removed. A thin layer of cells remained tightly adhered to the coverslip. On several coverslips, the viability of the adherent cells was established by trypan blue exclusion; most, but not all, cells excluded trypan blue. (Cells that did not exclude trypan blue appeared damaged with differential interference contrast optics.) Coverslips used for physiology were immunohistochemically labeled (as below, except that coverslips were first incubated in 5% BSA for 5 min and antibody incubations were shortened to 5-10

min) and then transferred fied essential medium mained in this solution

to a petri dish containing Eagle’s modicontaining 0.1% BSA (Sigma). They refor 5 min to 2 hr before recording.

Preparation of Cultures Type-2 Asfrocyfes and Oligodendrocytes in Serum-Containing Medium Cultures of each glial cell type were prepared according to the procedures of Raff and co-workers (Raff et al., 1983a, 1983b; Miller and Raff, 1984). 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 12.5% fetal bovine serum, 100 U/ml penicillin, 100 &ml streptomycin, and 2 nM L-glutamine. Development of oligodendrocytes was promoted by culture in a modified Bottenstein-Sato medium (Raff et al., 198313) containing 0.75% fetal bovine serum. The cells were plated at a density of 15,000 cells per cm* on round (13 mm diameter) glass coverslips that had been precoated with poly-Llysine and were cultured at 37°C in a humidified atmosphere of 5% CO*, 95% air.

O-2A Progenitors, Type-2 Asfrocyfes, and Oligodendrocytes in Serum-Free Medium We used the recently described protocol of Lillien and Raff (1990). In previous work they have shown that each glial cell type arises in the nerve with a precise time course: oligodendrocytes first develop around birth, whereas type-2 astrocytes only begin toappear in the second postnatal week (Miller, R. H., et al., 1985, 1989a; reviewed in Raff, 1989). In their new serum-free culture system, O-2A progenitors divide and differentiate into both oligodendrocytes and type-2 astrocytes, and this same developmental sequence of appearance is replicated. In brief, cocultures of optic nerve suspensions prepared from both PO and P7 animals were plated into a modified Bottenstein-Sato serumfree medium containing human platelet-derived growth factor (PDCF; 3 rig/ml; R&D) at a density of 20,000-30,000 PO cells and lO,OOO-15,000 P7 cells per round (13 mm diameter) glass coverslip. In experiments in which recordings were made from O-2A progenitors, we plated P4 optic nerve suspensions at 30,000 cells percoverslip and cultured these in the PDCF-containing, serumfree medium described above. (These plating densities are higher than those used by Lillien and Raff [1990]; a possible explanation is that we plated cells directly into the final volume of medium, whereas they allowed cells to adhere in a small volume first.) Coverslips were precoated with poly-Nysine (Lillien et al., 1988). These cultures were fed with fresh PDGF every 2 days and with fresh medium (one-half volume) every 4 days. lmmunohistochemical Labeling of Cells in Culture Cells in culture or in suspensions were labeled by incubation with monoclonal antibody supernatants; antibodies to galactocerebroside 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. For labeling of surface antigens, cells were incubated in the primary antibody for 15 min. washed, and incubated in a I:100 diluton of the secondary antibody. The secondary antibodies (Cappel) were fluoresceinor rhodamine-coupled F(ab’)z fragment goat antimouse IgC (F(ab’)* fragment-specific). Eagle’s medium containing 0.1% BSA 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 or a control monoclonal antibody, revealed negligible background. Cells in culture, labeled by indirect immunofluorescence as described above, were identified by viewing with a Zeiss microscope equipped with differential interference contrast optics and epifluorescent illumination. The verification of cell-type identity, by both morphology and antigenic phenotype, in our cultures has previously been described (Barres et al., 1988a).

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Cells

521

Identification of Cell Types 0-ZA Progenitors Cultured O-2A cells were identified either by labeling with the A2B5 antibody, which binds to a surface antigen, or by their characteristic bi- or tripolar morphology during the first few days of culture (Raff et al., 1985), which allowed unambiguous identification of O-2A progenitors even without the use of A2B5 labeling (Figures IA and ID; Figure 6). Acutely isolated O-2A progenitors in suspension were identified either by labeling with the A2B5 antibody (see Experimental Procedures), a specific surface marker of O-2A progenitor cells at this age (Miller, R. H., et al., 1985), or by their small size (Figures IB and IE). Cells were studied at similar ages: cultured O-2A progenitors were prepared from P4 optic nerve and cultured for 2-4 days prior to recording (or in POIP7cocultures; see Experimental Procedures); acutely isolated O-2A progenitors prior to culture were obtained from P6-P8 optic nerve. At this developmental stage (P6-P8), oligodendrocytes are being generated and are simultaneously present in the nerve, but type-2 astrocytes have not yet developed. Control experiments, in which acutely isolated or cultured cells were labeled with both the A2B5 antibody and the antibodies to galactocerebroside, a glycolipid characteristic of oligodendrocytes (Raff et al., 1978; Ranscht et al., 1982), revealed that cells with the O-2A morphology both in culture and in suspension, which brightly labeled with A2B5, never also labeled with galactocerebroside. However, about 20%-25% of the cultured cells that brightly labeled with galactocerebroside were also lightly labeled with A2B5 (this was not observed in cell suspensions). These doublelabeled cells usually had an early oligodendrocyte morphology, appearing transitional, and were not recorded from. Oligodendrocytes Oligodendrocytes were recognized either by their characteristic morphology (Figure 6) or by galactocerebroside staining. Type-2 Astrocytes Type-2 astrocytes were recognized by their characteristic morphology (Figure 6) as well as by A265 labeling. Control experiments demonstrated that ceils with this morphology were positive for both A2B5 and glial fibrillary acidic protein, as reported by Lillien and Raff (1990). Occasional type-2 astrocytes developed withinafewdaysafter plating in PO/P7cocultures, butwere not found in significant number until l-2 weeks later. Thus most of our experiments were done on type-2 astrocytes in 7- to 21day-old cultures. Electrophysiological Recording Gigaohm 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 used (Hamill et al., 1981; Corey et al., 1984). Micropipettes were drawn from hard borosilicate capillary glass (Drummond), coated with Sylgard 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 electronically compensated. All experiments were done at room temperature, approximately 24OC. Data Acquisition and Analysis Voltage stimuli were generated, and responses were recorded with a PDP II/73 computer (INDEC). A Yale Mark V patch clamp was used. Analog signals were filtered with an 8-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. Solutions and Current Isolation The solutions for the bath and those for the pipette (which place diffusible constituents of cytoplasm) were designed each case to isolate current carried by a specific ion through

rein a

specific channel type. These are described in more detail by Barres et al. (1988a). All solutions contained 5 mM HEPES, were adjusted to pH 7.4, and had junctional potentials less than 1-2 mV. -Glutamate-activated currents. External: 140 mM NaCI, 2 mM CaC&, 5 mM KCI, 0 mM MgCI,. Internal: 140 mM CsCI, 1 mM MgCI,, Ca*+ buffered to IO-’ with 2 mM ECTA. -Sodium currents. External: 140 mM NaCH3S03H 2 mM CaC&, 0.1 mM CdCIZ. Internal: 140 mM CsCH$03H, 1 mM MgCl?, CaZf buffered to 1O-9 M with 10 mM EGTA. -Calcium currents. External: 140 mM BaCH$O,H, 10 PM tetrodotoxin. Internal: 140 mM CsCH$S03H, 1 mM MgCi2, Ca2+ buffered to IOh with 10 mM ECTA. -Chloride currents. External: 140 mM NaCI, 2 mM CaC&, 5 mM KCI (or 0 if KIR is present). Internal: 140 mM CsCI, 1 mM MgC12, Ca*+ buffered to 10m6 with 10 mM ECTA. -Outwardly rectifying potassium currents. External: 140 mM NaCI, 2 mM CaCI,, 1 mM MgClz, 5 mM KCI. Internal: 140 mM KCI, 1 mM MgCI,, Ca’+ buffered to 10mh with 10 mM ECTA. -Inwardly rectifying potassium current. External: 120 mM NaCI, 20 mM KCI, 2 mM CaCI,, 1 mM MgCI?. Internal: 140 mM KCI, 1 mM MgCIZ, Ca 2+ buffered to IO-” with 10 mM ECTA. In addition, two alternative solutions were used to study outward potassium currents when an inward rectifier was present, or to identify an inward rectifier by its elimination. In one, cesium (5 mM) was added to the bath; in the other, potassium was entirely eliminated from the bath. In the latter, elimination of potassium from the bath caused the rapid depletion of intracellular potassium. In that case, we used a new piece of coverslip for each cell recorded and dialyzed cells with the potassiumcontaining internal solution for at least 5 min prior to measuring currents. Current densities (pA/pF) were calculated for inward current components as peak inward currents divided by the cell capacitance. Conductance densities (nS/pF) were calculated for outward potassium currents by dividing the chord conductance by the cell capacitance. Chord conductance was calculated as I/(V - V,,), where V,,, the equilibrium potential for potassium, was -50 when the bath potassium was 20 mM and -85 when the bath potassium was 5 mM. In the case where bath potassium was omitted, a value of V,, of -100 was used to allow an approximation of conductance for comparison with the other calculations, since current clamp measurements showed the resting potential to be about -110 to -100 mV in these cells. V was +60 mV for outwardly potassium currents and -120 mV for inwardly rectifying potassium currents (see Barres et al., 1988a).

Application of Drugs to Whole Cells Drugs were applied by pressure ejection through double-barreled pipettes with tips of 5 pm diameter separated by 50 pm. During application of drugs, the recording chamber was continuously perfused with fresh bath solution at a rate of 1 mllmin. The large tip separation prevented cross-contamination of the barrels. Phenol red was used at a concentration of 0.01% to allow visualization of the ejected solutions. In all experiments using glutamate or its agonists, magnesium was omitted from the bath solution. In experiments using NMDA, glycine (500 nM) was added.

CABA lmmunohistochemistry Cultures on glass coverslips were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 10 min and permeabilized with Triton X-100 (0.4%) for 2 hr. Coverslips were then incubated in BSA (5%) to block nonspecific binding and incubated overnight at 4OC in a rabbit anti-GABA polyclonal antiserum (Incstar) at I:1500 in Triton X-100 (0.1%) with BSA (1%). The primary antibody was detected with horseradish peroxidase-conjugated goat anti-rabbit IgC (1:150; Boehringer-Mannheim). In control experiments, coverslips were labeled with the rabbit anti-GABA antiserum after first preadsorbing it for 24 hr with 70 &ml GABA linked to BSA with glutaraldehyde (Storm-Mathisen et al., 1983).

NCWr0n

522

GAD Immunohistochemistry Cultures on glass coverslips were fixed in paraformaldehyde-lysine-periodate (2%) for 40 min, permeabilized with Triton X-100 (0.4%) for 1 hr, and blocked with BSA (5%) for 1 hr. Coverslips were incubated overnight at 4OC in a rabbit anti-GAD antiserum (courtesy of Dr. J. W. Wu; Wu et al., 1986) at I:1000 in Triton X-100 (0.1%) and BSA (1%). The primary antibody was detected by incubating the coverslips in a goat anti-rabbit immunoglobulin antiserum (1:20; Hyclone) for 1 hr, followed by rabbit peroxidase-antiperoxidase (1:80; Sternberger-Meyer) for 1 hr. Cultures were also labeled with a monoclonal antibody to GAD (courtesy of Dr. D. Cottlieb; Chang and Gottlieb, 1988). Cells were labeled as above, except the duration of fixation was decreased to 20 min, the primary antibody (GAD-6 ascites) was used at l:lOO, and goat anti-mouse immunoglobulin and mouse peroxidase-antiperoxidase were used. In all cases, negative controls were performed by omitting the primary antibody. In these experiments, contaminating CABAergic neurons from the optic chiasm (see text) served as positive controls.

CABA Measurements PO/P7 optic nerve cocultures were prepared as described above, except that only optic nerves were used, taking care to discard all optic chiasm tissue. Cells were plated into 35-mm petri dishes coated with poJy-D-lysine at a density of 140,000 PO cells and 85,000 P7cells per dish and allowed to grow to high density. After 1 week in culture, the cells were collected for measurements. In some dishes, putrescine was omitted from the culture medium 72 hr before sample collection. The presence and concentration of GABA inside the cells were studied with HPLC (see below). The medium was replaced after 7 days of culture with fresh medium (1 ml) containing the CABA-transaminase inhibitor y-vinyl-GABA (100 FM; MerrilDow; Jung et al., 1977) for 1 hr. This medium was then discarded, cells were rinsed three times with PBS, and 0.1 ml of perchloric acid (0.1 M) at 4OC was added. Cells were collected with a scraper into 1.5 ml Eppendorf tubes and immediately frozen at -8O’C. Analytical Measurement of CABA Cells collected into perchloric acid were thawed, sonicated for 4 s, and centrifuged at 12,000 rpm for 5 min at O°C. An aliquot of supernatant was taken and diluted I:4 with borate buffer (0.1 M, pH 9). GABA was measured by HPLC (Ellison et al., 1987; Allison et al., 1984; Simons and Johnson, 1976). This method uses a precolumn derivatization using o-phthaldialdehyde and 2.mercaptoethanol followed by reverse phase HPLC with electrochemical detection. The system consisted of a Waters 712 WISP automated sample injector, a Waters 501 solvent delivery system, a Bioanalytical Systems amperometric eletrochemical detector (LC-4), an Omniscribe BS 117-2 chart recorder, and a Rainin 5 ttrn Microsorb (25 cm) C-18 reverse phase column. A Bioanalytical Systems detector cell containing a glassy carbon working electrode and AgiAgCl reference electrode was used. The isoindole derivative was detected by oxidation at +700 mV with a detector sensitivity of IO nA/V. An isocratic solvent system with a mobile phase of NaH*PO, (0.1 M) and 46% (v/v) methanol (HPLC solvent grade) at pH 6.0 was employed, with a constant flow rate of 1.0 mlimin, resulting in an average running pressure of 2800 psi. For some samples, a different solvent system, in which the mobile phase was modified and contained 37% methanol at pH 5.12, was used. The precoiumn reagent for derivatization of amino acids contained 50 ~1 of 2-mercaptoethanol and 50 mg of o-phthaldialdehyde in a total volume of 10 ml of borate buffer (pH 9). In all cases, 20 Pi of sample or standard was injected with 5 ul of derivitization reagent. The samples were maintained at 5OC during analysis. The sensitivity of the method for GABA was 1 pmol per injection at a signal to noise ratio of 5:l. Cell extracts diluted in borate buffer were reacted with the 2-mercaptoethan01, o-phthaldialdehyde reagent in the sample loop of the automated sampler using a program that enabled the samplerto pick up the reagent, pick up the sample, hold the mixture for 90 s, and then inject it onto the column. A IO-cm piece of low volume

tubing was coiled with an approximate diameter of 1 cm and inserted in line between the injector and the column to promote mixing of reagent and sample by eddy diffusion. Three standard solutions containing 5, 10, and 20 pmoli20 1.11CABA in borate buffer were run in between every sample. The retention times of GABA under these conditions was 17.1 min. Acknowledgments We thank Laura Liilien and Martin Raff for sharing their new culture procedure with us long before publication, Dr. D. Cottlieb for enthusiastic and helpful discussion (even though we just called to pester him for an antibody), and Drs. D. O’Malley and R. Baughmann for advice on the CABA experiments. We also thank the following colleagues for donation of antisera and monoclonal antibodies: Dr. D. Gottlieb for GAD-6, the monoclonal antibody to glutamic acid decarboxylase, Dr. J. Wu for a polyclonal antiserum to glutamic acid decarboxylase, Dr. C. Barnstable for the monoclonal antibody VC1.1, and Dr. B. Ranscht for a monoclonal antibody to galactocerebroside. This work was supported by National Institutes of Health grants F32 NS-07970 (to B. A. B.), NS-22059 (to D. P. C.), and NS21269 and NS-16367 (To L. L. Y. C.), and by the Howard Hughes Medical Institute. Received

September

12, 1989;

revised

December

4, 1989.

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