Electrophysiological Properties of Human Astrocytic Tumor Cells In
Situ: Enigma of Spiking Glial Cells
ANGÉLIQUE BORDEY AND HARALD SONTHEIMER
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35924
INTRODUCTION
Unlike most neurons, glial cells retain the ability to divide
postnatally (Gensert and Goldman 1996; Korr 1986). Glial
proliferation accompanies reactive gliosis, the brain’s response to injury (Reier 1986) and the malignant transformation of glial cells. Glial-derived tumors are collectively termed
gliomas and include astrocytic, oligodendroglial, ependymal,
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and glial-neuronal tumors (Kleihues et al. 1993a,b). The majority of gliomas fall into the astrocytic lineage (astrocytomas) and presumably derive from astrocytes. They still share
some common features with astrocytes, including variable
expression of glial fibrillary acidic protein (GFAP), S100,
and growth factor receptors (Bigner et al. 1981; Collins 1995;
Kleihues et al. 1993a,b). This is particularly true for lowgrade astrocytomas, the most prevalent primary brain tumor
in children. Factors that induce the transition from glia to
glioma are not well understood. However, it is evident that
this transition is accompanied by a number of genetic alterations, gene deletions, and gene amplifications (Collins 1995;
Westermark et al. 1995) that are also typical of cancer cells
outside the nervous system.
Attempts to understand mechanisms that underlie cell
growth and differentiation have largely focused on studying
cancers of the body. Several of these studies suggest that
alterations in ion channel complement are associated with
and possibly are essential to allow cell proliferation to occur
(Sontheimer 1995). For example, in lymphocytes entry into
the cell cycle is associated with the up-regulation of Ca 2/ activated K / channels (Cahalan et al. 1991; DeCoursey et
al. 1984) and blockade of these channels retards cell proliferation. Pharmacological block of K / channels also inhibits
proliferation of brown fat cells (Pappone and Ortiz-Miranda
1993), melanoma cells (Nilius and Wohlrab 1992), human
breast cancer cells (Woodfork et al. 1995), lung small cancer
cells (Pancrazio et al. 1993), retinal glial cells (Puro et
al. 1989), Schwann cells (Wilson and Chiu 1993), O2-A
progenitor cells (Gallo et al. 1996), and astrocytes (Pappas
et al. 1994). Mechanisms by which ion channel complement
affects cell proliferation are largely unknown. Some recent
studies have begun to shed light on interactions between
tumor suppressor genes or oncogenes and ion channel function. For example, transfection of fibroblasts with ras21 or
exposure of fibroblasts to epidermal growth factor or platelet-derived growth factor leads to the induction of a novel
Ca 2/ -activated K / channel that is essential for cell cycle
progression in these cells (Huang and Rane 1994). In Drosophila, the tumor suppressor gene dlg interacts with ‘‘Atype’’ K / channels leading to channel clustering (Tejedor
et al. 1997).
Despite our expanding knowledge of ion channel expression in normal rat or mouse glial cells, little is known about
properties of glial-derived tumor cells. The few studies that
have investigated glial cell-derived tumors have largely focused on studying several well-established human cell lines.
These transformed glial cells possess a plethora of ion channels (Brismar 1995; Matute et al. 1992). However, culturing
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Bordey, Angélique and Harald Sontheimer. Electrophysiological properties of human astrocytic tumor cells in situ: enigma of
spiking glial cells. J. Neurophysiol. 79: 2782–2793, 1998. To
better understand physiological changes that accompany the neoplastic transition of astrocytes to become astrocytoma cells, we
studied biopsies of low-grade, pilocytic astrocytomas. This group
of tumors is most prevalent in children and the tumor cells maintain
most antigenic features typical of astrocytes. Astrocytoma cells
were studied with the use of whole cell patch-clamp recordings
in acute biopsy slices from 4-mo- to 14-yr-old pediatric patients.
Recordings from 53 cells in six cases of low-grade astrocytomas
were compared to either noncancerous peritumoral astrocytes or
astrocytes obtained from other surgeries. Astrocytoma cells almost
exclusively displayed slowly activating, sustained, tetraethylammonium (TEA)-sensitive outward potassium currents (delayed
rectifying potassium currents; IDR ) and transient, tetrodotoxin
(TTX)-sensitive sodium currents (INa ). By contrast, comparison
glial cells from peritumoral regions or other surgeries showed IDR
and INa , but in addition these cells also expressed transient ‘‘A’’type K / currents and inwardly rectifying K / currents (IIR ), both
of which were absent in astrocytoma cells. IIR constituted the predominant conductance in comparison astrocytes and was responsible for a high-resting K / conductance in these cells. Voltageactivated Na / currents were observed in 37 of 53 astrocytoma
cells. Na / current densities in astrocytoma cells, on average, were
three- to fivefold larger than in comparison astrocytes. Astrocytoma
cells expressing INa could be induced to generate slow action potential-like responses (spikes) by current injections. The threshold for
generating such spikes was 034 mV (from a holding potential of
070 mV). The spike amplitude and time width were 52.5 mV and
12 ms, respectively. No spikes could be elicited in comparison
astrocytes, although some of them expressed Na / currents of similar size. Comparison of astrocytes to astrocytoma cells suggests
that the apparent lack of IIR , which leads to high-input resistance
( ú500 MV ), allows glioma cells to be sufficiently depolarized to
generate Na / spikes, whereas the high resting K / conductance in
astrocytes prevents their depolarization and thus generation of
spikes. Consistent with this notion, Na / spikes could be induced
in spinal cord astrocytes in culture when IIR was experimentally
blocked by 10 mM Ba 2/ , suggesting that the absence of IIR in
astrocytoma cells is primarily responsible for the unusual spiking
behavior seen in these glial tumor cells. It is unlikely that such
glial spikes ever occur in vivo.
ELECTROPHYSIOLOGY OF HUMAN GLIOMA CELLS
METHODS
Patient selection
Human brain specimens were obtained from six pediatric patients who underwent resections of primary central nervous system
neoplasms. Clinically these cases did not present with seizures.
Parallel recordings were performed in comparison tissue also obtained from three pediatric patients. These tissues were resected
outside the tumor margins of a choroid plexus carcinoma and a
hypothalamic pilocytic astrocytoma, respectively. One additional
comparison biopsy was obtained from the cortex of a patient who
underwent a hemispherectomy to surgically treat Rasmussen’s encephalitis. Histopathological examination showed that none of
these comparison tissues contained neoplastic cells. We refer to
these tissues throughout as comparison tissue, acknowledging the
fact that cells in these tissues may also have altered properties.
tinuously perfused with oxygenated ACSF containing 1.8 mM
CaCl2 at room temperature. The chamber was mounted on the stage
of an upright microscope (Nikon Optiphot2) equipped with a 140
(2-mm working distance) water immersion objective and Nomarski optics.
Whole cell recordings and data analysis
Whole cell patch-clamp recordings were obtained as previously
described for hippocampal slices (Bordey and Sontheimer 1997;
Edwards et al. 1989). Patch pipettes were pulled from thin-walled
borosilicate glass (1.55 mm od; 1.2 mm id; TW150F-40, WPI) on
a PP-83 puller (Narishige, Japan). Pipettes had resistances of 4–
6 MV when filled with the following solution (in mM): 145 KCl,
0.2 CaCl2 , 1.0 MgCl2 , 10 ethylene glycol-bis( b-aminoethyl
ether)-N,N,N *,N *-tetraacetic acid (EGTA), and 10 HEPES (sodium salt), pH adjusted to 7.2 with tris(hydroxymethyl)aminomethane (Tris). To label cells for later morphological and antigenic
identification, 0.1% Lucifer yellow (LY; dilithium salt) was added
to the pipette solution. Voltage-clamp recordings were performed
with an Axopatch-200A amplifier (Axon Instruments). Current
signals were low-pass filtered at 5 kHz and digitized on-line at
25–100 kHz by using a Digidata 1200 digitizing board (Axon
Instruments) interfaced with an IBM-compatible computer system.
Data acquisition, storage, and analysis were done with the use
of pClamp version 6 (Axon Instruments). For all measurements,
capacitance and series resistance compensation (60–80%) were
used to minimize voltage errors. Settings were determined by compensating the transients of a small (5 mV) 10-ms hyperpolarizing
voltage step; the capacitance reading of the amplifier was used as
value for the whole cell capacitance. After compensation, series
resistances ranged from 6 to 10 MV. Membrane resistance (Rm )
was determined from the average response to 50 hyperpolarizing
(10 mV) current pulses (20 ms). The resulting membrane current
charging curves were fitted either to a single or double exponential.
Unless indicated otherwise, capacitive and leak conductances
were subtracted on-line by a modified P/5 protocol. This P/5
protocol consisted of five hyperpolarized subpulses of 01/5 amplitude executed before each episode, summed and added to the current trace of interest. Subpulses holding potential was 080 mV.
Peak currents were determined by using Clampfit (Axon Instruments) and statistical values (mean { SE in the text, {SD in the
table; n Å number of cells tested) were evaluated with a statistical
graphing and curve-fitting program (Origin, MicroCal). Chemicals
were purchased from Sigma unless otherwise noted.
Steady-state parameters for activation and inactivation of
sodium current
To establish steady-state activation or inactivation curves, the
peak current I was measured at each potential and the corresponding conductance G was calculated by using the following equation
Slice preparation
G Å I/(V 0 Vi )
Methods for preparing slices are described in detail in Ullrich
et al. (1997). Human tissue was obtained during surgery and collected in the operating room where it was immediately placed in
ice-cold (47C) calcium-free artificial cerebrospinal fluid (ACSF–
Ca-free) containing (in mM) 116 NaCl, 4.5 KCl, 0.8 MgCl2 , 26.2
NaHCO3 , 11.1 glucose, and 5 N-2-hydroxyethylpiperazine-N *-2ethanesulfonic acid (HEPES), oxygenated with 95% O2-5% CO2 .
Because of its fragile and mixed consistency, tissue was embedded
in agar and then glued (cyanoacrylate glue) to the stage of a
Vibratome. Slices of 150–300 mm were cut in cold oxygenated
ACSF–Ca-free and transferred to a beaker filled with ACSF–Cafree at room temperature. After a recovery period of ¢2 h in
ACSF–Ca-free, slices were placed in a flow-through chamber con-
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(1)
where V is the membrane command potential and Vi is the predicted
equilibrium (Nernst) potential for the ion (i) under consideration.
At 207C, calculated equilibrium potentials for potassium and sodium were 087.7 and 67.0 mV, respectively.
The measured peak amplitudes and the calculated peak conductance were then normalized and plotted as a function of the membrane potential during the test pulse. The resulting inactivation and
activation curves were fit to the Boltzmann equation
Gi /Gi ( max ) Å 1/(1 / exp[(V1 / 2 0 V )k])
(2)
where Gi ( max ) is the maximum ionic conductance at peak current,
V1 / 2 is the voltage where Gi is one-half of Gi ( max ) , and k, the slope
factor, determines the voltage-dependent relation of Gi .
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cells and passing them on for generations may alter their
phenotypes as has been demonstrated in other cells, including
glial cells (Bigner et al. 1981; Kennedy et al. 1987; Noble et
al. 1995). We recently reported that ion channel complement
of astrocytes in situ differs from the channel complement
of cultured astrocytes (Bordey and Sontheimer 1997). Our
understanding of possible biophysical changes underlying malignant transformation of glial cells is further hampered by
the fact that properties of ‘‘normal’’ human glial cells are
largely unknown.
We set out to investigate properties of astrocyte-derived
tumors as compared with noncancerous control astrocytes in
patient biopsies. We focused on a comparison between the
lowest grade astrocyte tumors, the pilocytic astrocytomas, because unlike higher grade gliomas, these astrocytoma cells
maintain several biochemical and morphological features with
astrocytes and may thus be most relevant in understanding
changes that underlie the transition from glia to glioma. Our
data suggest that astrocytoma cells lose expression of inwardly
rectifying K / channels, which are abundantly expressed in
astrocytes. These channels are believed to be instrumental in
aiding K / buffering in the brain. They also endow astrocytes
with a relatively negative and stable resting potential. Because
of the absence of IIR in conjunction with the prominent expression of INa in astrocytoma cells, these cells could be induced
to fire slow, action potential-like responses not seen in normal
astrocytes. The latter, however, could be induced to exhibit
spikes when IIR was pharmacologically inhibited suggesting
that the lack of IIR in astrocytoma cells is principally responsible for the unusual spiking behavior.
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A. BORDEY AND H. SONTHEIMER
RESULTS
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Sodium current, INa
Transient, fast, inward currents were observed in 37 of 53
(70%) glioma cells. These currents were almost completely
blocked by 100 nM TTX (Fig. 3A; n Å 10 cells, 3 cases).
TTX-block was partially reversible and did not significantly
alter the resting potential of the cell. Currents were activated
with voltage steps more positive than 050 mV that peaked
within õ3 ms and inactivated rapidly (Fig. 3A, left panel).
Current amplitudes were voltage-dependent, peaked at Ç0
mV, and decreased with more depolarized voltage steps. IV curves were generated either in the presence of TEA (10
mM) to block K / currents (Fig. 3B, ● ) or used the TTXsensitive current component after subtraction (Fig. 3 B, s ).
This analysis included 14 cells from five cases (every biopsy
except giant cell astrocytoma) and mean values were plotted
in Fig. 3B. The extrapolated current reversal potential was
64.7 { 1.7 (SE) mV (n Å 14), which is in good agreement
with the predicted equilibrium potential for Na / ions (ENa Å
/68 mV) under the imposed ionic gradients suggesting that
these fast, transient, inward currents were carried by Na /
ions. Steady-state activation and inactivation curves (Fig.
3D) were constructed according to Eqs. 1 and 2 (see METHODS ) and again included 14 cells from the same five cases.
Boltzmann fits of the activation curves gave values of
020.0 { 2.5 mV for V1 / 2 and 6.9 { 0.7 mV 01 for k (n Å
7). Sodium currents demonstrated typical steady-state inacti-
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Whole cell recordings were obtained from acute tissue
slices of nine patient biopsies and included 53 glioma cells
and 9 nonneoplastic astrocytes for comparison. Although
comparison tissue was also derived from patients, these tissues did not contain neoplastic cells as confirmed by histopathological examinations. Hence, despite the possibility
that cells in these tissues may also have altered properties,
they represent the best possible comparison tissue for purposes of this study. All tissues were histopathologically characterized and classified with the use of criteria set forth
by the World Health Organization (WHO) (Kleihues et al.
1993a,b). They included four cases of pilocytic astrocytomas (WHO grade I) and one desmoplastic and one giant
cell astrocytoma, also considered to be WHO grade I tumors.
These tumors were resected from children between 4-mo
and 14-yr old (mean age 5 yr). Comparison tissue (3 cases)
was cortical tissue associated with a choroid plexus carcinoma and a hypothalamic pilocytic astrocytoma and cortical
tissue from a patient with Rasmussen’s disease (3-yr-old
patient).
Tumor tissue consisted of densely packed, round tumor
cells (Fig. 1A; black arrows point out round cells) and their
processes. Cell processes stained positive for GFAP (Fig.
1B, arrows), whereas most glioma cell bodies were only
weakly GFAP positive (Fig. 1B) as this was previously
reported (Bilzer et al. 1991; Bressler and Edwards 1997;
Kharbanda et al. 1993). Tumor tissue could be readily distinguished from surrounding normal peritumoral tissue microscopically (Fig. 1C). The tumor tissue was fragile and
showed thinner sections after cutting. Figure 2 shows two
representative cells, one from a pilocytic astrocytoma cell
from the hypothalamus and one from a nonneoplastic comparison astrocyte from peritumoral issue from a choroid
plexus carcinoma. These cells were each filled with LY during whole cell recording to allow morphological and antigenic characterization. The comparison astrocyte was GFAP
positive and its morphology resembled that of stellate astrocytes in rat hippocampus in situ (Bordey and Sontheimer
1997) with 2–3 major processes and many smaller processes
of Ç50–60 mm (Fig. 2A). The pilocytic astrocytoma cells
had a round cell body and extended only 2–3 major processes (Fig. 2B). This cell, like many others, showed some
LY coupling to adjacent cells. Typically, dye spread to less
than five adjacent cells.
The recordings from these two cells (Fig. 2, A and B,
right panels) are representative examples for each tissue
group, and the traces show raw data without leak subtraction
elicited by 50-ms voltage steps to potentials ranging from
0160 to 60 mV (10-mV increments). These recordings show
prominent expression of outward potassium currents in both
glioma and comparison astrocytes. However, comparison
cells always expressed large inward K / currents in addition
to outward K / currents. Inward K / currents were absent in
glioma cells. This difference in rectification is more apparent
in the corresponding current-voltage curves (I-V ) plotted in
Fig. 2C. Current amplitudes were determined 4 ms ( ●, j )
and 35 ms ( s, h ) after the beginning of the recording and
plotted as a function of applied potentials. I-V curves of
astrocytoma cells ( j, h ) were always outwardly rectifying,
whereas I-V curves of comparison astrocytes ( ●, s ) were
either inwardly rectifying or linear at potentials near the
resting potential. We previously reported expression of outwardly rectifying glioma-associated chloride currents
(GCC) that are activated by very positive voltages [e.g.,
ú45 mV (Ullrich et al. 1997)]. To eliminate possible contamination of current traces with GCC, we restricted our
analysis of peak currents to potentials õ30 mV, where Cl 0
currents were not activated. A more detailed dissection of
inward and outward currents is discussed below and shown
in Figs. 3–4.
Data from all recorded cells are summarized in Table 1,
which in addition to densities of current and conductance of
the different current subtypes also gives mean values ( {SD)
of resting membrane potential (Vr ), membrane capacitance
(Cm ), and resting membrane resistance (Rm , measured at
080 mV). For consistent comparison, all biophysical parameters (e.g., Cm , Vr , and Rm ) were determined in the first 3
min of whole cell recordings. Conductance densities were
used as measures that allow us to compare the relative expression of current components independent of cell size.
Conductance of outward currents was determined at 30 mV
and conductance of IIR at 0150 mV. Kinetic and pharmacological analysis of the outward and inward currents in tumor
cells were performed in cells having series resistances õ10
MV and with the use of leak-subtraction protocols.
Astrocytoma cells had significantly more depolarized
membrane potentials than comparison astrocytes whose potential was similar to that reported previously for rat astrocytes (Bordey and Sontheimer 1997) (P õ 0.001). However, mean membrane capacitance values were similar, suggesting that cells were of approximately equal size. This is
consistent with our morphological observations (Fig. 2).
ELECTROPHYSIOLOGY OF HUMAN GLIOMA CELLS
2785
vation (Fig. 3, C and D). The Boltzmann fits of the inactivation curves gave an e-fold change per 8.0 { 0.4 mV 01 (n Å
18) and a mean V1 / 2 of 059.2 { 1.9 mV (n Å 18). Parameters for the activation and inactivation curves in comparison
astrocytes were similar with values for V1 / 2 of 021.6 and
057.4 mV and 8.7 and 7.5 mV 01 for k, respectively (data
not shown).
To compare the relative Na / channel expression in astrocytoma cells to comparison astrocytes, we determined sodium conductance density, GNa (sodium conductance divided
by the membrane capacitance), for each cell and pooled data
from each patient. Astrocytoma cells in the four pilocytic and
the desmoplastic tumors consistently expressed conductance
densities that were approximately three- to fivefold higher
than in comparison cells (Table 1).
Outward potassium currents
To study outward currents, depolarizing voltage steps
were applied from a holding potential of 070 mV. This
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protocol activated sodium currents followed by sustained
outward currents. The outward currents recorded in 38 of
53 (72%) tumor cells showed delayed and slow inactivation
(Fig. 4A). The I-V relationship of outward currents derived
from mean data of eight cells from two pilocytic astrocytomas suggested an activation threshold of approximately 050
mV (Fig. 4B) as compared with approximately 030 mV for
two comparison astrocytes. The voltage-dependence of the
sustained current showed kinetics reminiscent of delayed
rectifying potassium currents (IDR ) described in numerous
other preparations (Rudy 1988). Bath application of 10 mM
TEA reduced IDR by 32% (n Å 4; Fig. 4C). Tail current
analysis yielded a reversal potential of 085 mV closed to
the predicted Nernst reversal potential for K / (EK Å 088
mV; Fig. 4, D and E).
If currents were activated after applying a conditioning
200-ms prepulse to 0110 mV (a protocol used to activate
transient A-type K / currents), no additional transient out-
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FIG . 1. Human astrocytoma biopsies. A: high power photograph ( 1400) of a live section of a pilocytic astrocytoma.
Cells are densely packed and tend to have round cell bodies
(arrows point to representative tumor cells). B: glial fibrillary
acidic protein (GFAP) staining of the same slice as in A;
primarily processes traversing the slice stain positive for
GFAP (white arrows). Cell bodies are only weakly stained.
C: low power photograph ( 1100) of same slice as in A shows
tumor margins delineated by a dashed white line.
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A. BORDEY AND H. SONTHEIMER
ward potassium current could be activated in 89% of all
studied cells (data not shown). In the remaining six pilocytic
astrocytoma cells, which were all from one tumor case (posterior fossa), transient A-currents with conductance densities
of 391.6 { 148.7 pS/pF could be identified and example
recordings are shown in Fig. 5. These currents could be
isolated by activation from a prepulse potential of 0110 mV
(Fig. 5A) and subsequent subtraction of currents activated
from a prepulse potential of 070 mV (Fig. 5B) yielding a
transient current component that activates at potentials
greater than 060 mV (Fig. 5C). This is more readily visible
if peak currents are plotted as a function of applied potential
(Fig. 5D). Steady-state inactivation of the transient current
was analyzed by varying the prepulse potential from 0180
to 020 mV. The subtracted transient current (from protocols
with and without prepulse; Fig. 5E) was plotted as a function
of applied prepulse potential in Fig. 5D ( s ) for five tumor
cells and showed a V1 / 2 (for inactivation) of 084.4 { 1.7
(n Å 5). This value was more hyperpolarized than a V1 / 2
of 060.3 mV in a comparison astrocyte (data not shown).
Moreover, at the resting membrane potential of pilocytic
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astrocytoma cells ( 043.4 { 4.7; n Å 6), this transient outward potassium current displayed no overlap between
steady-state activation and inactivation curves (Fig. 5 D),
suggesting that currents were not available for activation at
the cells resting potential.
Whole cell recordings were also obtained in which the
membrane was stepped from a prepulse potential of 0 mV
to more negative potentials ranging from 0180 to 0 mV
(data not shown). This protocol was applied to facilitate
isolation of IIR (Ransom and Sontheimer 1995). This type
of current was found only in 2 of 11 cells of one pilocytic
astrocytoma and was absent in 96% of all studied cells (see
Table for conductance values). The inward current showed
a marked current inactivation at potentials more negative
than 0130 mV. In addition, the I-V curve showed inward
rectification typical of cesium- and barium-sensitive inwardly rectifying K / channels.
Spikes induced by current injections in astrocytoma cells
Current injection into some tumor cells induced transient
overshooting responses reminiscent of slow action potentials
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FIG . 2. Astrocytoma cells are electrophysiologically
distinct from comparison astrocytes. Left panel: characteristic morphology of Lucifer yellow–filled cells; right
panel: representative whole cell currents after stepping
the cell membrane from 0160 to / 80 mV from a holding
potential of 080 mV of the corresponding recorded cell.
(A: comparison nontumorous astrocyte; B: pilocytic
astrocytoma cell; C: corresponding current-voltage relationships of the displayed traces from A and B.) Current
amplitudes were determined 4 ms ( j, ● ) and 35 ms ( h,
s ) after the beginning of the recording and plotted as a
function of applied potentials. I-V curve of the pilocytic
astrocytoma cell ( j, h ) was outwardly rectifying, whereas
I-V curve of the comparison astrocyte ( ●, s ) was linear.
ELECTROPHYSIOLOGY OF HUMAN GLIOMA CELLS
2787
(Fig. 5, A and B, at different timescale). Only single spikes
could be induced with each current injection; thus we consider these spikes not regenerative. In four cells held at 070
mV, an average of 10 action potentials gave a mean spike
amplitude of 52.5 { 3.5 mV. The threshold was 033.9 {
1.0 mV. The mean rise time (beginning of the pulse to the
peak spike value) was 17.1 { 2.8 ms and the duration of
the spike was 11.8 { 0.6 ms. Because generation of action
potentials depends on the ratio of inward to outward current,
we calculated conductance densities (conductance obtained
by Eq. 1, METHODS, divided by the membrane capacitance)
mediated by potassium channels (GK , corresponding to the
total potassium density of conductance in these cells) and
sodium channels (GNa ) to obtain the ratio GK :GNa . The ratio
GK :GNa in astrocytoma cells was 2.7, a value that is significantly lower (P õ 0.001) than a ratio of 11.8 { 5.6 (n Å
8) in nontumor astrocytes (Table 1).
spikes can be induced, is correct. At 7–10 days in vitro,
these cells express prominent inwardly rectifying potassium
currents in addition to INa (Fig. 7A, left panel) (see also
Ransom and Sontheimer 1995). The potassium current was
blocked by 10 mM barium (Fig. 7A, right panel) as previously reported (Ransom and Sontheimer 1995). Current
injection under current clamp could not elicit a spiking response from these cells (Fig. 7B). Blockade of IIR by 10
mM Ba 2/ transforms these ‘‘passive’’ cells into spiking cells
(Fig. 7B, right panel). Figure 7B illustrated the progressive
transformation due to the Ba 2/ application in current-clamp
recordings during the wash in of the drug. A current injection
of 200 pA was applied every 5 s and the respective potential
values (resting potential and potential in response to the
current injection) were plotted against time (Fig. 7B, right
panel). These data clearly suggest that expression of inwardly rectifying K / channels prevents the spike generation
by current injection.
Experimental spike induction in spinal cord astrocytes in
vitro
DISCUSSION
Spinal cord astrocytes were shown to possess significant
Na / channel densities in vitro, allowing action potential-like
response induced by current injection (Sontheimer and Waxman 1992). This appears largely because of a lack of inhibition by neurons whose presence suppresses Na / channel
expression in these cells (Thio et al. 1993) and is probably
an artifact of cell culture. However, these cells present a
good model system to study whether our hypothesis, namely
that the relative expression of IIR and INa determines whether
Our results suggest that marked differences exist between
astrocytoma cells and comparison astrocytes, most strikingly
the loss of IIR and A-type outward potassium currents (IA ) in
transformed glial cells that appeared to exclusively express
delayed rectifier potassium channels. In addition, astrocytoma cells showed an up-regulation of sodium currents and
were able to generate current-induced spikes that were absent
in comparison tissue. Blocking IIR in normal rat glial cells
in culture allowed these cells to generate current-induced
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FIG . 3. Astrocytoma cells express tetrodotoxin (TTX)-sensitive, voltage-dependent Na / currents. A: whole cell inward
currents in response to 8-ms step depolarizations starting at 070 mV, incremented
by 10 mV from a holding potential of 0110
mV. Currents before exposure to TTX
(left), after 3-min exposure to TTX 100
nM (right). B: mean peak current amplitudes obtained in 14 cells from 5 pilocytic
astrocytomas ( s ) and from traces shown
in A ( ● ) were plotted vs. corresponding
membrane potential. Extrapolated reversal
potential /66.0 mV. C: whole cell Na /
currents after a 200-ms inactivating prepulse potential. Prepulse potentials started
at 0160 mV and were incremented by 10mV steps while the command potential was
kept at 010 mV. D: activation ( s ) and inactivation curves ( ● ). Data from Fig. 2A
plotted as activation curve for peak current.
rrr, best fit according to Eq. 2 with
V1 / 2 Å 020.1 mV, k Å 8.2 mV 01 per e-fold.
Fraction of peak current measured from 18
cells is plotted vs. inactivation voltage.
– – – , best fit to a Boltzmann distribution
with values of –59.5 mV for V1 / 2 and 9.6
mV per e-fold for k.
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A. BORDEY AND H. SONTHEIMER
spikes, suggesting that expression of IIR prevents depolarization and spike generation in normal astrocytes.
Delayed rectifier potassium channels
Astrocytoma cells and comparison astrocytes consistently
expressed IDR . Indeed, these channels were ubiquitously expressed in nonexcitable cells (Rudy 1988) and were implicated in the control of proliferation of various cell types
(Gallo et al. 1996; Nilius and Wohlrab 1992; Pancrazio et
al. 1993; Pappas et al. 1994; Pappone and Ortiz-Miranda
1993; Puro et al. 1989; Wilson and Chiu 1993; Woodfork
et al. 1995). We recently reported an increase in the conductance of delayed rectifying K / channels in gliotic proliferative astrocytes in an in vitro model of spinal cord injury
(MacFarlane and Sontheimer 1997). A striking result from
our study is the hyperpolarized shift in the activation threshold for IDR to values close to 050 mV instead of 020 or
030 mV in other cell types, including rat hippocampal astrocytes, in situ (Bordey and Sontheimer 1997). It is possible
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that this change is due to some intracellular modulation affecting delayed rectifying K / channels (Chung and
Schlichter 1997; Cole et al. 1996; Koh et al. 1996; Perozo
and Bezanilla 1990). IDR were also present in comparison
human cells in our study and in normal rat hippocampal
astrocytes in situ.
Inwardly rectifying potassium channels
Our observation that astrocytoma cells lack IIR is in contrast with a previous report that studied glioma cell lines
(Brismar 1995). The reason for this discrepancy is unclear.
It is possible that cell lines (in vitro) display features that
differ from cells in situ or in vivo. Moreover, we studied
low-grade gliomas, whereas glioma cell lines used by others
were derived from high-grade gliomas (glioblastoma). Studies of rat C6 astrocytoma cells, believed to be more
astrocyte-like and highly GFAP positive, also lack inwardly
rectifying K / channels. The absence of IIR in pilocytic astrocytoma is consistent with other proliferating cell types that
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FIG . 4. Characterization of delayed rectifying potassium IDR in astrocytoma cells.
A: voltage command pulses from 070 to
80 mV from a holding potential of 070
mV evoked sustained outward currents. B:
mean current amplitudes obtained in 8 cells
from 2 pilocytic astrocytoma ( s ) and in
6 cells from comparison tissue ( ● ) were
plotted vs. corresponding membrane potential. I-V relationship suggested an activation threshold of Ç 050 mV in astrocytoma
cells and 030 mV in comparison cells. Currents activated at 050 mV (Fig. 3B). C:
bath application of 10 mM tetraethylammonium (TEA) reduced IDR by 32% (n Å 4).
D: tail current analysis was performed by
stepping the cell membrane from 0160 to
0 mV after a prepulse to 0 mV. Tail current
amplitudes were measured 500 ms after
stepping the membrane potential to 2nd
varying step potentials; then basal current
(‘‘leakage’’ current) was subtracted from
measured tail currents and resulting ‘‘tail’’
currents plotted against applied potential in
E. E: tail current/voltage curve yielded a
reversal potential of 086.6 mV (Nernst
EK Å 087.7 mV).
ELECTROPHYSIOLOGY OF HUMAN GLIOMA CELLS
TABLE
1.
Membrane properties and conductance densities in glioma cells and comparison astrocytes
Astrocytoma (6 cases)
4 Pilocytic astrocytoma
Vr , mV
R m , MV
035.5 { 18.5*** (53)
822.3 { 921.5 (40)
Cm , pF
INa /Cm , pA/pF
GNa /Cm , pS/pF
IDR /Cm , pA/pF
GDR /Cm , pS/pF
GIR /Cm , pS/pF
50.1 { 33.6 (53)
021.9 { 20.8* (37)
315.4 { 294.6 (37)
25.3 { 22.2 (38)
220.5 { 193.6 (38)
1 pilocytic: 2/11
743.1 { 195.6
1 pilocytic: 6/8
391.6 { 148.7
2.7 { 2.7*** (31)
2.3 { 2.5**
(0.04 to 9.0) (31)
032.6 { 20.3*** (35)
838.2 { 630.0*
(40 to 1886) (23)
36.4 { 29.7 (35)
027.8 { 19.9** (24)
394.4 { 276.0 (24)
32.3 { 22.9* (22)
281.5 { 199.5 (22)
GA /Cm , pS/pF
Desmoplastic
astrocytoma
049.2 { 18.9 (6)
960.6 { 742.0* (6)
19.0
034.0
525.3
37.5
326.8
{
{
{
{
{
14.8* (6)
18.9** (4)
267.6 (4)
20.9* (5)
182.1 (5)
Giant cell astrocytoma
041.9 { 16.1** (12)
421.3 { 465.3 (11)
84.0
00.8
11.5
7.0
61.2
{ 21.2*** (12)
{ 0.3 (9)
{ 4.7 (9)
{ 4.1* (11)
{ 36.0 (11)
Comparison
astrocytes
(3 cases)
063.9 { 12.5 (9)
288.0 { 188.3 (8)
44.7
04.4
81.1
14.4
111.5
408.7
{
{
{
{
{
{
18.4
5.9
89.4
7.3
65.9
277.4
(8)
(8)
(8)
(8)
(8)
(6)
248.5 { 197.0 (5)
1.2 {
1.3*** (18)
0.9 {
0.4** (4)
5.18 {
2.6** (9)
11.8 {
7.3 {
5.6 (8)
7.7 (8)
Values are means { SD; number of cells tested within parentheses. GA /Cm , transient outward potassium conductance; IIR /Cm , inwardly rectifying
potassium current divided by the membrane capacitance; GIR /Cm , inwardly rectifying potassium conductance divided by the membrane capacitance;
IDR /Cm and GDR /Cm , delayed rectifying potassium current and conductance divided by the membrane capacitance, respectively. * P õ 0.05, significant;
** P õ 0.01, very significant; *** P õ 0.001, extremely significant.
also lack IIR , for example, leukemia cells (Missiaen et al.
1997), neuroblastoma cells (Arcangeli et al. 1995), early
mouse embryos (Day et al. 1993), and HeLa cells (Takahashi et al. 1994). In addition, injury induced proliferation
of spinal cord astrocytes in situ correlates with a downregulation of IIR (MacFarlane and Sontheimer 1997). In hippocampal slices IIR channel expression is developmentally
regulated and its expression appears to characterize primarily
FIG . 5. Transient current recordings in
pilocytic astrocytoma cells. Transient currents were isolated by sequentially activating currents from a prepulse potential of
0110 mV (A) and subsequent subtraction
of currents activated from a prepulse potential of 070 mV (B), yielding a transient
current component that activates at potentials ú 060 mV (C). Mean values of peak
currents were plotted as a function of applied potential (D) for 5 cells. Steady-state
inactivation of the transient current was analyzed by varying the prepulse potential
from 0180 to 020 mV (E). Mean values
of the subtracted transient currents from 5
cells were plotted as a function of applied
prepulse potential in Fig. 5D ( s ).
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GK:gNa
GDR :GNa
2789
2790
A. BORDEY AND H. SONTHEIMER
FIG . 6. Action potential in astrocytoma
cell. A: tumor cell in a pilocytic astrocytoma was recorded in current-clamp mode.
Action potential was elicited by applying
100-pA current injection pulse. B: currentclamp traces in another cell from same tumor after shorter depolarizing current
pulse. Spike amplitude was 52 mV, threshold was Ç 033 mV, rise-time (beginning
of pulse to peak value) was 15.1 ms, duration of spike from threshold was 11.3 ms,
and corresponding ratio for the same recorded cell was 1.1.
Transient outward potassium channels
In glioma cells, transient A-type K / channels are largely
absent. Like the loss of IIR , the absence of transient outward
potassium currents appears to be an early feature that accompanies the transformation of a normal astrocyte to become
a tumor cell. The role of these channels in glial cells is
not known. In neurons they modulate frequency of action
potential discharge (Rudy 1988). Interestingly, A-currents
were recently implicated in the extension of neurites (Wu
and Barish 1996). Glioma cells displayed fewer processes
than normal glial cells and show numerous alterations in
extracellular matrix (McKeever et al. 1997) and membrane
architecture (Kharbanda et al. 1993; Nagano et al. 1993).
In analogy to the role of A-currents in neurite extension,
a loss of A-currents in glioma cells might be a result of
perturbations in extracellular matrix.
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Na / channels
Our data suggest a two- to fivefold up-regulation of Na /
channel density in astrocytoma cells without any significant
biophysical or pharmacological alterations. This could have
been due to a functional modulation of sodium channels or
an increase in channel expression. Considering the second
possibility and assuming a single-channel conductance of 20
pS and an open probability of 0.25 (Barres et al. 1989),
mean sodium channel densities in pilocytic astrocytoma and
desmoplastic astrocytoma had mean values of 0.8 { 0.5
channels/ mm2 (ranging from 0.1 to 1.75) and 1.0 { 0.5
channels/ mm2 , respectively. These values are significantly
higher than values of 0.16 { 0.18 channels/ mm2 (0.02 to
0.4) in comparison human glial cells. This increase in INa in
conjunction with the suppression of IIR accounted for the
unusual spiking behavior of astrocytoma cells after current
injection.
Biophysical ‘‘spikes’’ are a consequence of altered
channel complement
Our data suggest that the lack of IIR allows inducement
of spikes by current injection. Our experiments with spinal
cord astrocytes in vitro corroborate this. These cells have
the adequate ion channel complement, both Na / channels
and inwardly rectifying K / channels. Blocking IIR by an
application of low concentrations of barium turned ‘‘passive’’ glial cells into spiking glial cells, suggesting that the
activity of inwardly rectifying K / channels prevented such
spikes in Na / channel-bearing glial cells.
Functional consequences and implications
The role of these slow glial spikes is an enigma. We
believe that they probably never occur in vivo. However,
others (Labrakakis et al. 1997) suggested that the genesis of
tumor (glioblastoma multiform)-associated epileptic seizure
may be due to spiking glioma cells. However, at the resting
potential of glioma cells, Na / channels are essentially fully
inactivated, curtailing activation. The cases studied here did
not present with seizure symptoms.
Much has been speculated about the potential role of glial
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differentiated nonproliferating astrocytes (Bordey and
Sontheimer 1997).
The lack of IIR currents in astrocytoma cells is due either
to a modulation of the channel protein or to the down-regulation of its expression. Our detection methods do not allow
to discriminate between these possibilities and, for purposes
of our study, this difference is not germane. However, the
loss of functional IIR currents may have important physiological consequences. This channel class is known to participate
in the potassium homeostasis in the brain (Amedee et al.
1997; Newman and Reichenbach 1996) and one would predict that in the absence of this channel, potassium buffering
would be impaired in tumors. In addition, these potassium
channels, in conjunction with chloride channels, are involved
in cell-volume regulation and, consequently, volume regulation may also be compromised in tumor cells. Interestingly,
glioma cells had a more rounded-up morphology and appeared swollen (phase image). Inwardly rectifying K / channels are thought to establish the hyperpolarized resting potential typical of most astrocytes. Its absence in glioma thus
explains the depolarized resting potential of tumor cells.
Depolarized membrane potentials are considered to be a
characteristic of tumor cells (Cone 1970).
ELECTROPHYSIOLOGY OF HUMAN GLIOMA CELLS
2791
Na / channels (Ritchie 1992). These speculations include
roles in fueling the Na / /K / -ATPase (Sontheimer et al.
1994), ephaptic coupling to neurons (Chao et al. 1994), or
channel biosynthesis and transfer to neurons (Bevan et al.
1987). We do not want to expand on these suggestions.
Instead, we are compelled to speculate that the most intriguing finding is the loss of IIR amplifying the relative contribution of Na / currents to the whole cell conductance. The
latter, we believe, is an important aspect in cell differentia-
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tion. We speculate that inwardly rectifying K / channels are
expressed in differentiated, nondividing astrocytes, whereas
they are down-regulated in dividing cells for unknown reasons. Indeed, this was observed in numerous cell types, including astrocytes, and our recent study showed the rapid
loss of IIR in conjunction with gliotic proliferation (MacFarlane and Sontheimer 1997).
Thus the observed changes are consistent with enhanced
proliferation of transformed glial cells. Glial spikes can be
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FIG . 7. Glial spikes can be induced in normal astrocytes after pharmacological blockade of IIR . A, top panel: under
voltage-clamp configuration currents recorded in control (left) and in the presence of bath application of 10 mM Ba 2/ (right).
Holding potential was 080 mV. Currents were activated after depolarizing cell to 0 mV for 100 ms, before application of
hyperpolarizing voltage step from 0180 to 010 mV for 40 ms. Bottom panel: under current-clamp configuration, membrane
potentials recorded from the same cell as in top panel in control (left) and in presence of 10 mM Ba 2/ (right). Ba 2/ blocked
the IIR and allowed recorded cell to spike in response to a current injection (100-pA step). B: under current clamp, a 200pA current was injected every 5 s from a membrane potential of 070 mV (resting potential of the recorded cell). Ba 2/ was
applied at trace number 6, depolarized the cell, and turned its square passive response into a spiking response ( left). Right
panel: resting potential of the cell ( ● ) and peak amplitude of the current-injected responses ( s; measured between 10 and
20 ms) were measured every 5 s and plotted against time of recording. This graph illustrates the depolarizing and spikinginduced effects of Ba 2/ onto the cell.
2792
A. BORDEY AND H. SONTHEIMER
experimentally induced as a consequence of these changes
in channel complement. However, they are a biophysical
exercise with probably little physiological significance.
This work was supported by National Institutes of Health Grants RO1NS-31234, NS-36692, and P50-HD-32901.
Address for reprint requests: H. Sontheimer, Dept. of Neurobiology,
The University of Alabama at Birmingham, 1719 6th Ave. South, Cintan
International Research Center, Room 545, Birmingham, AL 35294.
Received 20 October 1997; accepted in final form 12 January 1998.
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