Center for Neuropharmacology and Neuroscience and Division of Neurosurgery, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Zhou, Min and Harold K. Kimelberg. Freshly Isolated Astrocytes From Rat Hippocampus Show Two Distinct Current Patterns and Different [K+]o Uptake Capabilities. J. Neurophysiol. 84: 2746-2757, 2000. Whether astrocytes predominantly express ohmic K+ channels in vivo, and how expression of different K+ channels affects [K+]o homeostasis in the CNS have been long-standing questions for how astrocytes function. In the present study, we have addressed some of these questions in glial fibrillary acidic protein [GFAP(+)], freshly isolated astrocytes (FIAs) from CA1 and CA3 regions of P7-15 rat hippocampus. As isolated, these astrocytes were uncoupled allowing a higher resolution of electrophysiological study. FIAs showed two distinct ion current profiles, with neither showing a purely linear I-V relationship. One population of astrocytes had a combined expression of outward potassium currents (IKa, IKd) and inward sodium currents (INa). We term these outwardly rectifying astrocytes (ORA). Another population of astrocytes is characterized by a relatively symmetric potassium current pattern, comprising outward IKdr, IKa, and abundant inward potassium currents (IKin), and a larger membrane capacitance (Cm) and more negative resting membrane potential (RMP) than ORAs. We term these variably rectifying astrocytes (VRA). The IKin in 70% of the VRAs was essentially insensitive to Cs+, while IKin in the remaining 30% of VRAs was sensitive. The IKa of VRAs was most sensitive to 4-aminopyridine (4-AP), while IKdr of ORAs was more sensitive to tetraethylammonium (TEA). ORAs and VRAs occurred approximately equally in FIAs isolated from the CA1 region (52% ORAs versus 48% VRAs), but ORAs were enriched in FIAs isolated from the CA3 region (71% ORAs versus 29% VRAs), suggesting an anatomical segregation of these two types of astrocytes within the hippocampus. VRAs, but not ORAs, showed robust inward currents in response to an increase in extracellular K+ from 5 to 10 mM. As VRAs showed a similar current pattern and other passive membrane properties (e.g., RMP, Rin) to "passive astrocytes"in situ (i.e., these showing linear I-V curves), such passive astrocytes possibly represent VRAs influenced by extensive gap-junction coupling in situ. Thus, our data suggest that, at least in CA1 and CA3 regions from P7-15 rats, there are two classes of GFAP(+) astrocytes which possess different K+ currents. Only VRAs seem suited to uptake of extracellular K+ via IKin channels at physiological membrane potentials and increases of [K+]o. ORAs show abundant outward potassium currents with more depolarized RMP. Thus VRAs and ORAs may cooperate in vivo for uptake and release of K+, respectively.
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INTRODUCTION |
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With the application of
the patch-clamp technique for studying astrocyte ion channels, a large
repertoire of ion channels have been identified in astrocytes in
primary cell culture, acute brain tissue slices, and in acutely
dissociated cell preparations (Verkhratsky and Steinhäuser
2000). These findings have altered the early concept that
astrocytes only possess passive ohmic K+
conductances (Kuffler 1967
). However, astrocytes
displaying an ohmic I-V relationship, or "passive
astrocytes," have also been widely identified from different brain
regions in situ, e.g., corpus callosum (Berger et al.
1991
), hippocampus (D'Ambrosio et al. 1998
;
Steinhäuser et al. 1992
), spinal cord
(Chvatal et al. 1995
), and red nucleus (Akopian
et al. 1997
) using the patch-clamp technique. As a high
percentage of these passive astrocytes showed positive staining for
glial fibrillary acidic protein (GFAP), it has been suggested that
these represent mature astrocytes (Chvatal et al. 1995
;
Steinhäuser et al. 1994b
).
Recently, D'Ambrosio et al. (1998) found a high
percentage of passive astrocytes (72.5%) in the CA1 region as compared
with CA3 (12.5%), which correlated with a more intensive cell coupling in the CA1 region. In another study, when ATP was omitted from the
pipette solution, which should prevent the gating of gap junctions between coupled astrocytes in situ, no passive astrocytes were found in
the CA1 region (Bordey and Sontheimer 1997
). These two studies suggest that the existence of passive astrocytes in situ is
likely due to syncytial influences on the measured membrane current profiles.
In this study, we examined this question by studying the current
properties of freshly isolated astrocytes (FIAs) from the CA1 and CA3
regions of rat hippocampus. In our recent studies, GFAP(+) FIAs showed
different receptor expression profiles to GFAP(+) astrocytes in primary
culture and the former seemed to better represent the in vivo situation
(Cai and Kimelberg 1997; Kimelberg et al.
1997
; Schools and Kimelberg 1999
). By extension, the ion channel profiles analyzed in FIAs should better represent the
properties of astrocytes in vivo. Additionally, FIAs provide an
uncoupled astrocyte model, so that the voltage-clamp control should be
largely improved and the ion channels kinetics can be studied at a
higher resolution. Also, isolated cells allow a fast and complete
exchange of extracellular solution which makes the observation of fast
[K+]o uptake possible.
We found two astrocyte types based on the membrane current profiles of
process-bearing FIAs. Neither of them showed purely linear
I-V curves. These two types of astrocytes also exhibited differences in cell size, resting membrane potential, ion current pharmacology, and regional distribution. The values of both
cell-membrane capacitance and resting membrane potential were bimodally
distributed and corresponded to the results obtained in situ
(D'Ambrosio et al. 1998), indicating that they are
distinct astrocyte types. Only the VRA type of astrocyte, characterized
by a resting membrane potential close to
EK and expressing abundant inward
potassium currents (IKin), was capable
of channel-mediated K+ uptake. By contrast, due
to a more positive resting membrane potential and lack of
IKin, the second type of astrocytes
[outwardly rectifying astrocytes (ORAs)] failed to show
K+ uptake under the same condition.
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METHODS |
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Cell isolation
Astrocytes were freshly isolated from rat hippocampus following
the procedure of Tse et al. (1992), with
modifications. Young rats (postnatal days 7 to 15) were anesthetized
with 50% O2-50% CO2 and
killed by decapitation. Their brains were dissected out and rinsed in
Ca2+-free bath solution (for composition, see
next section). The hemispheres were quickly cut into slices (around
500-µm thick) in coronal orientation using a razor blade. Slice
preparation was performed in Ca2+-free bath
solution at 6°C. The hippocampi were then dissected from the slices
and incubated in the same solution for 1 h at room temperature,
bubbled with 95% O2-5%
CO2. This "rest" step is critical for
obtaining astrocytes with the process-bearing morphologies shown in
Fig. 1, A-C. After that,
hippocampi were transferred for 30 min into normal aCSF solution
containing 24 U/ml papain at 22°C, supplemented with 0.24 mg/ml
cysteine as enzyme activator. After washing with
Ca2+-free bath solution, the hippocampi were
stored in the same oxygenated Ca2+-free bath
solution at room temperature for at least 1 h for recovery. Right
before the experiment, the CA1 or CA3 subregions were dissected out
from a slice and triturated onto the recording chamber in standard bath
solution using fire-polished Pasteur pipettes (tip diameter 200 µm)
under microscopic observation. To wash away unattached cells and tissue
debris, gravity perfusion (1-3 ml/min) of standard bath solution was
switched on 5 min after trituration and was continued throughout the
experiment. Recordings typically started 10 min after tissue
trituration.
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Solution and drugs
The standard bath solution contained 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM D-glucose. The pH of the bath solution was adjusted to 7.4 with NaOH. In Ca2+-free solution, CaCl2 was substituted by 1 mM sodium pyruvate. The pipette solution was 140 mM KCl, 0.5 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 3 mM Mg-ATP, and 0.3 mM Na-GTP. For mRNA analysis, all solutions were prepared in sterile milliQ water and were autoclaved. Phosphate buffered saline (PBS) consisted of 137 mM NaCl, 8 mM Na2HPO3, 1.5 mM KH2PO4, 2.7 mM KCl, 0.5 mM MgCl2, and 0.7 mM CaCl2. 4-aminopyridine (4-AP), tetraethylammonium (TEA), tetrodotoxin (TTX), and all other reagents were purchased from Sigma (St. Louis, MO).
Immunocytochemistry
The procedure for GFAP staining of the freshly isolated cells
has been described in detail in a previous report from our laboratory (Kimelberg et al. 1997). In brief, CA1 and CA3 regions
of hippocampus were triturated after enzymatic treatment in standard
bath solution onto coverslips precoated with 0.01%
poly-D-lysine. After the cells had adhered to the
coverslips (approximately 10 min), the attached cells were fixed by
immersion in 4% paraformaldehyde solution for 30 min. The fixed cells
were washed with cold PBS and then permeabilized with acetone at
20°C for 3 min. After washing in PBS, the cells were incubated in
3% normal goat serum (NGS) for 10 min. Following a rinse in PBS, the
cells were incubated with 1:400 primary antibody (Rabbit anti-GFAP,
DAKO, Carpinteria, CA) for 1 h. After further washing in PBS, the
cells were incubated for 1 h with a secondary,
fluorescein-conjugated goat antibody raised against rabbit IgG
(Biosource, Camarillo, CA) at a 1:75 dilution. After a final wash, the
coverslips were mounted onto slides with 50% glycerol in PBS and
viewed in a Nikon Epi-fluoresence microscope. To determine nonspecific
labeling the primary antibody was omitted.
Electrophysiological recordings
Membrane currents were measured by the patch-clamp technique in
the whole-cell configuration. Current signals were amplified with an
Axopatch 200B (Axon Instruments, Foster City, CA) and sampled by a TL-1
DMA Interface (Axon Instruments). Data acquisition was carried out by
Pclamp 6.0.4 software (Axon Instruments) running on a Gateway 2000 Pentium II 233 computer. Low-resistance patch pipettes (3-5 M) and
cell harvesting pipettes were fabricated from borosilicate capillaries
(OD: 1.2 or 1.5 mm, Warner Instrument, Hamden, CT) using a
Flaming/Brown Micropipette Puller (model P-87, Sutter Instrument,
Novato, CA). Membrane capacitances (Cm)
and series resistances (Rs) were
determined by a depolarizing test pulse from
70 to
60 mV (10 ms,
filter at 10 kHz, sampling at 30 kHz). Input resistances
(Rin) were estimated in the
voltage-clamp mode from the currents induced by 10 mV hyperpolarizing
steps. For slow drug application, drugs were delivered to the
established whole-cell patches by gravity perfusion. For fast
application, test solutions were rapidly applied to the established
whole-cell patches through square tubes using the Perfusion Fast-Step
System (SF-77, Warner Instrument) controlled by programmed data
acquisition protocols. All experiments were performed at room
temperature (~20-24°C).
Single-cell RT-PCR for GFAP
The single-cell reverse transcription polymerase chain reaction
(RT-PCR) procedure for GFAP mRNA detection post recording has
been described in detail in our previous study (Zhou et al. 2000). The primers for the first round (GFAP-1) and the second nested primers (GFAP-2) for rat cDNA encoding GFAP were designed to
flank introns in genomic sequences according to the NCBI GenBank sequence data (accession number: L27219). The specificity of the
oligos/primers used in this study and the sensitivity of the single-cell RT-PCT procedure has been previously assessed in our laboratory (Schools and Kimelberg 1999
).
After membrane current recording, the same individual cell was
harvested by applying gentle suction with a second pipette (diameter
~10 µm), which was silanized to avoid RNA adherence, baked to
destroy Rnases, and filled with 1 µl RNase-free milliQ water. The
pipette contents were then expelled into a PCR tube filled with 10 µl
milliQ water, and the tube was immediately placed in a 80°C freezer.
RT-PCR for the cell was done on the same day after electrophysiological
recording using a SuperScript One-Step RT-PCR system (Gibco BRL, Life
Technologies) as previously described (Cai et al. 2000).
The reverse transcription (RT) step was run at 42°C for 30 min to
convert GFAP mRNA to cDNA. This was followed by the first-round
amplification (denaturation at 94°C for 30 s, annealing at
58°C for 30 s, extension at 72°C for 60 s; 40 cycles) with the GFAP-1 primer pair. Second-round amplification (denaturation at 94°C, annealing at 60°C, extension at 72°C; all 60 s; 35 cycles) was done with the nested primers, GFAP-2. First round product (1 µl) was used in each second-round reaction in a total volume of 50 µl. The PCR products were analyzed by 1.5% agarose gel
electrophoresis. The PCR products gave the expected products of 437 base-pair (bp) size after second-round amplification.
Data analyses
Steady-state activation of K+ currents was
obtained by dividing maximum currents by the corresponding driving
force, V EK, where
EK is the K+
equilibrium potential. Subsequently, data points were fitted by the
equation
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(1) |
Steady-state inactivation was fitted by a Boltzmann equation of the
form
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(2) |
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RESULTS |
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Morphology, GFAP staining, and GFAP mRNA of FIAs from CA1 and CA3
Figure 1, A-C, shows the morphology of freshly isolated astrocytes, as seen by phase contrast microscopy. Typically, the cell soma either had oval, elongated, or triangular shapes, with multiple, long, and bushy processes extending from the cell body. The morphology of astrocytes from CA1 and CA3 were not distinguishable.
The proportions of the different cell types in the fresh cell
suspension based on morphology are summarized in Table
1. The process-bearing astrocytes shown
in Fig. 1, A-C, amounted to ~19-25% of the total cells
regardless of CA1 and CA3 regions, whereas 22% of the cells present in
the suspension had a triangular appearance with several primary
dendritic branches. The electrophysiological properties of these cells
and absence of GFAP staining showed they were likely neurons
(Zhou et al. 2000). Forty-three percent of the
less-branched glia showed a small size and less-branched morphology. As
we have described in another report, this population of cells was
GFAP(
), but GFAP mRNA(+) (Zhou et al. 2000
). The 14%
remaining cells could not be classified into any of above morphologically defined cell populations due to atypical morphology, swelling, or general damage. We only studied process-bearing astrocytes for this report.
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For GFAP staining, CA1 and CA3 regions were dissected out from 30 hippocampi slices obtained from postnatal day 8 to 15 animals (n = 6) and dissociated. After staining, we found a total of 254 isolated cells which preserved the same morphology as shown in Fig. 1, A-C. All of them stained positively for GFAP antibody. An example of the GFAP immunoreactivity of these process-bearing cells is shown in Fig. 1E.
After patch-clamp recording, 25 similar process-bearing astrocytes were harvested for GFAP mRNA measurement by RT-PCR. Twenty-one out of 25 cells (84%) were GFAP mRNA positive (n = 25) (Fig. 1F). These experiments together indicated that the process-bearing cells under study were both GFAP(+) and GFAP mRNA(+) astrocytes.
Freshly isolated GFAP(+) astrocytes show two membrane current profiles
The FIAs were found to exhibit two classes of membrane current profiles. One was characterized by a dominant expression of outward transient and sustained K+ currents, plus inward sodium currents (Fig. 2A). Because currents were mainly inducible in response to depolarizing voltage steps, and the I-V relationship showed a strong outward rectification (Fig. 2C), we term these cells outwardly rectifying astrocytes (ORA). These comprised 60% of the cells examined (n = 80) from CA1 and CA3. The other type of astrocyte was characterized by a relatively symmetric inducible current pattern (Fig. 2B) and a variably rectifying I-V curve (Fig. 2D). We therefore term them variably rectifying astrocytes (VRA). These comprised the remaining 40% of cells recorded.
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The ionic selectivity of both outward transient and sustained and
inward sustained currents were determined from the reversal potentials
of tail currents (Erev). For outward
currents, the membrane potential was stepped to +60 mV (both transient
and sustained currents were found to be almost maximally activated at
this voltage) for varied periods (10, 50, 100 ms), and then to
different test potentials (120 to +20 mV in 10 mV increments). A
representative recording from a VRA is shown in Fig.
3A. As summarized in Table 2, the reversal potentials
(Erevs) of outward currents were
weakly dependent on prepulse duration. As the prepulse was prolonged to
100 ms, the tail currents still reversed at
74 mV. This
Erev is close to the theoretical
potassium equilibrium potential calculated from pipette and bath
solutions
[K+]i/[K+]o
=140/5 mM (EK =
88 mV),
indicating that even though IKa were almost completely inactivated at this time, the outward currents were
still dominated by potassium channels (presumably
IKdr). The involvement of nonspecific
currents to the outward currents appears negligible as they will have
an Erev around 0 mV. The Erev for inward sustained currents was
also tested (n = 5). As shown in Fig. 3B,
the cell was first stepped to
180 mV for 50 ms and then to the same
test potentials as for the outward currents. The mean
Erev of
84 ± 5 mV for inward
sustained currents was very close to the
EK of
88 mV. These data
demonstrate that both inward sustained and outward transient and
sustained currents are predominantly carried by potassium. In this
analysis, we also noticed that, in some VRAs, depolarization
prepulse-evoked tail currents showed a time-dependent activation (Fig.
3A), implying the presence of endothelial
Iha like currents (Guatteo et
al. 1996
). A further clarification of these
Iha currents will need further study.
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The fast inward transients evoked from ORAs by depolarization steps
typically activated when potentials were more positive than 40 mV and
peaked at
10 mV. The time to peak at
10 mV averaged 450 ± 50 µs (n = 20). These currents were completely blocked
by 100 nM TTX (n = 6, Fig. 3, D-F).
Application of 0.1 µM TTX also produced a 12 ± 6 mV
hyperpolarization in resting-membrane potential in four ORA cells. Both
the kinetics and TTX sensitivity of these sodium currents were similar
to the astrocytes studied in situ by Bordey and Sontheimer
(1997)
.
Different passive membrane properties between ORAs and VRAs
Resting membrane potentials (RMP) were measured in the
current-clamp mode within 30 s after establishment of a stable
whole-cell configuration, with a 140 mM KCl pipette solution and 5 mM
K+ bath solution. For all the cells, these RMPs
spanned a wide range (25 to
85 mV). They were bimodally distributed
and well fitted by a double Gaussian function, yielding two peak values
of
66 and
43 mV (coefficient of correlation r = 0.87, Fig. 4A). These values
closely match the two mean RMP figures found in VRAs (
65 ± 10 mV) and ORA (
40 ± 10 mV) astrocytes, respectively (Table 3). A wide variation in astrocyte RMPs is
consistent with the in situ patch-clamp study by McKhann et
al. (1997)
. Also, the two average peak RMP values from FIAs
were similar to the bimodal peak values of
69 and
51 mV found by
D'Ambrosio et al. (1998)
recorded in astrocytes of the
hippocampal CA1 + CA3 regions in situ. A slightly lower value of the
second peak of
43 mV for the FIAs perhaps reflects a developmental
influence as 3-5 wk rats were used in the slice study. RMPs did not
differ between the CA1 and CA3 regions for the two cell types. Input
resistance (Rin) for ORAs (5.0 ± 3.5 G
) was significantly higher than for VRAs (0.6 ± 0.3 G
)
in cells from both CA1 and CA3 (Table 3). However, although the
Rin values roughly followed a bimodal
distribution, the data failed to be fitted by a double Gaussian
function due to some outlying values (Fig. 4B). In both CA1
and CA3 regions, the Cm values of VRAs
were 3.5 times greater than ORAs (Table 3).
Cms also followed a bimodal distribution
which was well fitted by a double Gaussian function (r = 0.98, Fig. 4C). The two peak values of 10 and 30 pF were
close to the mean values for ORA- and VRA-type astrocytes (see Table
3). Since the mean values of RMPs and Cms
for ORA and VRA closely matched the peak values of the double Gaussian
function and also the values seen for astrocytes in slices, the results
seem best interpreted as that the ORAs and VRAs belong to two GFAP(+)
astrocyte subpopulations with distinct current profiles and passive
properties and were not produced by the isolation procedure.
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Abundant IKohm conductance exists in VRAs
The ion channel identities of different membrane current
components can be assessed by their distinct pharmacology. We used prepulse activation/inactivation plus off-line digital subtraction strategies to isolated different current components. To isolate outward
transient IKa and sustained
IKdr, prepulses at 110 and
40 mV
(300 ms for each) prior to the test potentials were used, respectively
(see shadowed inset for Fig. 5). With
this strategy, we found that the current density of
IKa was lower in VRAs than in ORAs.
The current density in ORAs was 63 ± 12 pA/pF (n = 25, peak response to a +60 mV step) versus 6 ± 5 pA/pF in VRAs
(n = 7). We next used millimolar concentrations of 4-AP
(Bordey and Sontheimer 1997
, 1999
; Tse et al.
1992
) to test the sensitivity of
IKa seen in both cell types. As shown
in Fig. 5, B1-B3, 4 mM 4-AP almost completely blocked
IKa in VRAs. At a +60-mV step, 4-AP
blocked the peak IKa current amplitude
by 91 ± 15% compared with controls (n = 4).
However, the same concentration of 4-AP showed only 63 ± 13%
inhibition of IKa in ORAs (Fig. 5,
A1-A3, n = 10). From studies of recombinant
K+ channels, it is now clear that currents
resembling IKa can be mediated by a
number of cloned K+ channel subunits (see review
by Coetzee et al. 1999
). Heterogeneity of 4-AP-sensitive
current has been recently reported in other cell types
(Honjo et al. 1999
) and also in astrocytes in situ (Bordey and Sontheimer 1999
). The differential
inhibitory effect of 4-AP on IKa
implies that a different subunit composition may exist between ORAs and
VRAs.
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To examine the contribution of IKdr in
outward sustained currents, we tested the sensitivity of outward
sustained currents to the selective Kdr channel
blocker TEA (Bordey and Sontheimer 1997; Tse
et al. 1992
). Sustained outward potassium currents were isolated by a set of depolarizing steps with a 300-ms prepulse at
40
mV to avoid activating IKa (see
shadowed insets in Fig. 5). TEA (15 mM) inhibited the outward sustained
potassium currents in a voltage-dependent manner. At the +60 mV step,
TEA blocked outward sustained potassium currents by 71 ± 11% in
ORAs (n = 6; Fig. 5, C1-C3). The same
concentration of TEA blocked only 25 ± 9% (n = 5) of the sustained outward currents in VRAs (Fig. 5,
D1-D3). These data suggest that in VRAs other current
components besides IKdr also
contribute to the total outward sustained currents. The likely
candidates are the unidentified leak potassium channels (IKohm), e.g., TREK, which was
recently shown to be abundant in hippocampus (see review by
Coetzee et al. 1999
).
To further clarify the contribution of
IKohm to the sustained inward
potassium currents in VRAs, we tested the sensitivity of inward
potassium currents to extracellular 1 mM CsCl. At this concentration,
IKir currents observed in cultured
astrocytes were almost completely blocked (Ransom and Sontheimer
1995). Sustained inward potassium currents were induced by a
set of hyperpolaring steps with a prepulse at 0 mV for 500 ms to remove
the activation of IKa (see shadowed
inset in Fig. 6). We found that in 17 out of 32 VRAs, these currents did not show a typical
Kir-like voltage- and time-dependent
inactivation. Pharmacologically, only in 3 out 10 VRAs were these
currents sensitive to 1 mM Cs+ (Fig. 6,
A1-A3), whereas another 7 VRAs showed only a weak
sensitivity to Cs (inhibited by <20%, n = 7, Fig. 6,
B1-B3). The noninactivating kinetics and the resistance of
these inward potassium currents to Cs+ suggests
an abundant coexistence of IKohm in
VRAs. Besides IKohm, other channels,
e.g., ERG (Emmi et al. 2000
) and
Iha (Guatteo et al. 1996
),
are also likely involved. Therefore, inward currents of VRAs are likely
composed of several K+ components. For this
reason, we term the current IKin to
cover this complexity. In summary, the pharmacological properties of IKa,
IKdr are different between ORAs and
VRAs. Notably, VRAs can possess abundant
IKohm or other
Cs+-insensitive IKin,
which is similar to the properties of "passive cells" in situ
(D'Ambrosio et al. 1998
).
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A more limited voltage-clamp control occurs in VRAs compare to ORAs
As noted in the INTRODUCTION, there has been controversy regarding whether there are any mammalian astrocytes in situ which show purely linear I-V curves. We found that no FIAs showed a linear I-V relationship. We therefore explored the possible reasons accounting for this difference between FIAs and some of the findings for astrocytes in situ.
In situ astrocytes are extensively connected by gap junctions. This
leads to space-clamp problems and restricted voltage control. As
discussed by Steinhäuser et al. (1994a), poor
space clamp makes the precise analysis of
INa kinetics impossible because INa has a rapid activation and
inactivation time course. Therefore, they studied astrocytic
INa exclusively by using freshly
isolated astrocytes.
As VRAs showed a significantly larger size than ORAs (as reflected by
Cm), this suggested that the smaller sized
ORAs could be better clamped than the larger sized VRAs. To study this,
we measured IKa, as it has a fast
kinetics and is present in both astrocyte types. We analyzed the
activation kinetics of IKa for both
VRAs and ORAs (Fig. 7, A-C)
and fitted the IKa activation kinetics by the Boltzmann equation (see METHODS, Eq. 1). We found the half-maximum activation of
IKa,
Vn,0.5 was 20 mV positive in VRAs as
compared with ORAs without a change in slope (Kn) (Fig. 7C). These different
Vn,0.5 values corresponded well with
the thresholds for IKa seen in ORAs
and VRAs. When 100 nM TTX was added to block
INa, the
IKa threshold in ORAs was 60 mV
(n = 5, data not shown). This is very close to the
value of
70 mV seen in cultured spinal cord astrocytes (Bordey
and Sontheimer 1999
). However, it was also 20 mV more negative
than for VRAs (n = 3, data not shown). We also analyzed
the steady-state inactivation of IKa
by the Boltzmann equation (see METHODS, Eq. 2).
As shown in Fig. 7, D-F, this analysis did not
reveal any difference in both half-maximum inactivation,
Vg,0.5, and slope,
Kg, between ORAs and VRAs, showing
that differences in voltage-clamp control mainly affect the activation
kinetics of IKa. In the same analysis,
we found that the IKa amplitude
reached a peak within 1.4 ± 0.3 ms (n = 12) in
response to a +60 mV step in ORAs. In contrast, for VRAs the
corresponding time was 6 ± 2 ms (n = 6), or four
times longer than for ORAs. These analyses indicate that the different
Ka activation kinetics between ORA and VRA could well be due to altered space-clamp control in these different sized
astrocytic subpopulations.
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Only VRAs showed a rapid response to a fast increase in [K+]o
We next addressed the question of whether these
IKin channels seen in VRAs are capable
of fast K+ uptake when
[K+]o is rapidly increased. We
directly tested the K+ uptake capabilities of
both VRAs and ORAs by a fast change of [K+]o from 5 to 10 mM, a
concentration likely to be achieved physiologically during intense
neuronal activity. As shown in Fig. 8,
only the VRA-type astrocytes (n = 3 for VRAs,
n = 6 for ORAs) showed strong inward currents under
these conditions. As the membrane potential was clamped at 70 mV in
this experiment, this indicates that the
IKin channels have a high conductance
for K+ under these conditions. Further, the fast
onset and offset kinetics in response to high K+
application suggests that VRAs are capable of rapidly removing K+ released from neurons, in agreement with a
role of VRAs in [K+]o
homeostasis.
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Ion current profiles and occurrence of ORAs and VRAs in CA1 and CA3 regions
The distribution of the four ion current components, namely,
INa,
IKa,
IKdr,
IKin, in the CA1 and CA3 regions of
ORAs and VRAs is shown in Fig.
9A. There were no clear
differences between CA1 and CA3 when the currents of the two types of
FIAs were averaged. However, as shown in Fig. 9B, there were
differences between ORAs and VRAs. One hundred percent of the ORA type
astrocytes possessed IKdr
(n = 48), 92% (44/48)
IKa, and 90% (43/48)
INa. Inward potassium currents were
observed in only 6 out of 48 ORAs, and all of these current amplitudes
were <40 pA at 160 mV. As it is difficult to distinguish these small
currents from nonspecific leak currents, so they were not registered as
channel-mediated inward potassium currents. In contrast, all the VRAs
possessed IKin, 53% (17/32) had
IKa, and only one cell showed a
detectable but very small INa (1/32)
(Fig. 9B).
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As shown in Fig. 9C, the occurrence of VRAs and ORAs showed regional differences between CA1 and CA3. In the CA1 region, ORA and VRA current patterns were recorded in essentially similar proportions of cells (52 versus 48%). However, ORAs were more frequently recorded than VRAs in FIAs prepared from the CA3 region (71 versus 29%).
Observations of astrocytes isolated from older P20-30 rats showed that ORA and VRA remained the two astrocyte profiles (data not shown). However, some of the ORAs additionally expressed a more pronounced IKin but retained INa (n = 31, unpublished observations). A detailed analysis of acutely isolated astrocytes from older animals (P20-30) will be the subject of a future report.
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DISCUSSION |
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Freshly isolated astrocytes
Acutely, or freshly isolated astrocytes (FIAs) as we term
them, have been used on a limited basis to study astrocyte properties (reviewed in Barres et al. 1990; Kimelberg et al.
2000
; Sontheimer 1994
; Verkhratsky and
Steinhäuser 2000
), especially in relation to the new
findings on current and other properties of astrocytes determined in
situ in slices. The FIAs offer the advantage of being able to clearly
determine the properties of astrocytes without having to control for
indirect effects, as is critical for work in slices. The widely used
primary astrocyte cultures prepared from neonatal rodents have the same
advantages but suffer from unpredictable changes occurring due to
long-term culture (Kimelberg et al. 2000
). The major
potential disadvantages of the FIAs are alterations and damage during
isolation and to what degree they are a selected sample of astrocytes
in situ. The present studies, in showing a good correspondence in
current profiles between FIAs and astrocytes recorded from the same
region in slices, further support the use of FIAs as representative models.
Heterogeneity and regional differences of GFAP(+) astrocytes subtype in hippocampus
In this study, two types of GFAP(+) astrocytes were identified
based on distinct ion channel profiles, passive membrane properties, and the good fit of their RMPs and Cms to
a double Gaussian function. At this developmental stage (P7-P15), the
major differences in the current profiles are the presence of
INa (90%) but absence of
IKin in ORAs and presence of
IKin but absence of
INa in VRAs. In hippocampus, numerous
GFAP() "complex" astrocytes also showed a similar current profile
to ORAs (Seifert et al. 1997
; Steinhäuser et al. 1994a
; Zhou et al. 2000
). However, both
morphology and lack of GFAP staining distinguish them from ORAs.
Furthermore, INa in such complex glia
almost disappears after P20 (Kressin et al. 1995
), but
it is continuously present in ORAs at least until P30 (unpublished observations).
We found essentially an equal frequency of occurrence of VRAs and ORAs
in CA1. However, the ORAs were 2.4-fold more frequent than VRAs in the
CA3 region (Fig. 9C). This agrees with the finding by
D'Ambrosio et al. (1998) that astrocytes with different
current profiles are anatomically segregated between the CA1 and CA3
regions with passive cells, which may correspond to our VRAs (see
predominantly in CA1 region). However such heterogeneity differs from
the conclusions of Bordey and Sontheimer (2000)
. As
astrocytes in CA1 and CA3 regions have a different structural
relationship with neurons and blood vessels (Coyle 1978
;
McBain et al. 1990
), these regional differences may
reflect different physiological functions.
VRAs share several similarities with passive astrocytes recorded in situ
Passive astrocytes in situ are characterized by a linear
I-V relationship, low Rin,
and an RMP close to EK. However,
whether these cells carry voltage-gated conductances is not clear
(Chvatal et al. 1995). It is noteworthy that the VRAs
are quite similar in many respects to the passive astrocytes recorded
in situ. The VRAs show a relatively symmetrical potassium current
pattern, the mean RMP of
65 ± 10 mV is close to the
EK and the average Rin value of 0.6 G
is comparable to
the low Rin recorded for passive
astrocytes in situ. Although VRAs showed large
IKin currents, 17 out of 32 VRAs
(53%) IKin failed to show
time-dependent inactivation at potentials more negative than
130 mV
(Fig. 6), which are the typical IKir
kinetics seen in cultured astrocytes (Ransom and Sontheimer
1995
). This suggests an expression of other inward potassium
current components besides IKir.
Finally, 70% (7/10 cells) of IKin in
VRAs were less or insensitive to Cs+, and TEA
blocked only a fraction (30%) of sustained outward potassium currents
in VRAs. These data together indicate a coexistence of abundant
IKohm and/or other
K+ channel-mediated currents with voltage-gated
conductances in VRAs. Therefore, under the conditions in situ,
especially the effects of extensive gap junction couplings (see next
section), at least some of VRAs could represent the passive astrocytes
recorded in situ.
Syncytia likely obscure the apparent activation of voltage-gated ion channels in situ
VRAs have an apparently large cell-surface area, as their mean
Cm value of 33.5 pF is not only 3.5 times
larger than ORA-type astrocytes, but also 1.7 times larger than freshly
isolated hippocampal pyramidal neurons (19.2 ± 6.6 pF,
Seifert et al. 2000). This larger size is
accompanied by a more positive IKa
activation threshold, half-maximal activation parameter
(Vn,0.5), and a longer time to peak
of maximal current activation as compared with the smaller ORAs. It is
possible that the K+ channel subunits that
mediate IKa are different between ORAs and VRAs as they showed different sensitivities to 4-AP. However, this
difference is unlikely to account for the different
IKa activation kinetics between VRAs
and ORAs. A number of cloned K+ channels,
including Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3, show the current
kinetics resembling IKa
(Baldwin et al. 1991
; Schroter et al.
1991
; Serodio et al. 1994
, 1996
; Stuhmer
et al. 1989
). The biophysical properties of
IKa seen in ORAs were very similar to
Kv1.4, Kv4.1, Kv4.2, and Kv4.3
(Vn,0.5 =
10 to
22 mV and
Vg,0.5 =
45 to
69 mV). VRA showed
a similar Vn,0.5 value of 8 mV to
Kv3.4 (Vn,0.5 =13 to 19 mV), but a
more negative Vg,0.5 of
65 mV as
compared with Kv3.4 (Vg,0.5 =
20 to
32 mV). If Kv3.4 was a major participant in VRAs, VRAs should also
have a distinct Vg,0.5 around
23 to
32 mV. In our analysis, however,
Vg,0.5 showed no difference between
VRAs and ORAs.
Because imperfect voltage clamp is an unavoidable limitation for whole-cell study, all the analyses generated from this study cannot be taken as absolute values. However, relative effects are reliable and our analyses indicate that the IKa activation kinetics analyzed from VRAs were shifted to the positive compared to ORAs, likely caused by the difference in their cell sizes. It is reasonable to infer that the voltage-clamp control will be further reduced and activation of voltage-gated conductances will be further obscured by the coexistence of a large leak potassium conductance experienced in situ due to syncytial coupling. Thus, we speculate that the "passive" properties of astrocytes in situ likely result from their widespread electronic coupling.
Functional implications
The oldest and most frequently discussed role for the dominant
K+ conductances seen in astrocytes in the CNS is
spatial buffering of K+ released from active
neurons (Orkand et al. 1966). In Müller cells and
astrocytes, Kir was suggested to be the most
suitable channel for this role as it has a high open probability at the resting potential and the conductance increases with increasing [K+]o (Newman and
Frambach 1984
; Ransom and Sontheimer 1995
).
Similarly, VRAs carried abundant IKin
and showed robust [K+]o
uptake (Fig. 8B). It is noteworthy that the
IKin in VRAs are at least partially
mediated by Kohm, suggesting an important role of
these leak potassium channels in K+ uptake. In
astrocytes, these are likely important functional channels, but their
molecular identities and function in astrocytes have not yet been
defined (Coetzee et al. 1999
). Additionally, the
channels participating in IKin seems
more complicated as another two currents, namely,
Iha (Guatteo et al.
1996
) and ERG (Emmi et al. 2000
), have been
identified in astrocytes, suggesting a further complexity in astrocyte
K+ channel expression. ORAs lacked
IKir and also failed to show any
[K+]o uptake capability
(Fig. 8A). However, the finding that
INa is present in 90% of ORAs, and
TTX induced a moderate hyperpolarization (12 mV) of membrane potential
in ORAs (unpublished data) supports the idea that astrocyte
Na+ channels are open at the RMP, as described by
Sontheimer et al. (1994
, 1996
) for cultured spinal cord
astrocytes. If ORAs are coupling with VRAs in vivo, their depolarized
RMP likely gate their abundant expressed outward voltage-gated
potassium channels, particularly, Ka, which has
an activation threshold around
60 to
70 mV. Thus, open
Ka channels could contribute to spatial buffering
to release [K+]i to an
area where neuronal activity is less intense.
In conclusion, numerous studies have demonstrated that the study of astrocytes in situ is a powerful approach to address the questions of astrocyte properties and function in the CNS. FIAs provide a useful additional single-cell model, allowing a study of astrocytes properties that are close to those exhibited in situ, but without having to account for the effects of the cell-cell coupling and influence of the surrounding neurons and other cells. Taking advantage of studying FIAs, we demonstrated the existence of two distinct astrocyte types, but neither of them behaved precisely like the passive astrocytes described in situ. We demonstrated that one type, VRAs, are capable of fast [K+]o uptake, and the second type, ORAs, showed a more depolarized RMP and abundant expression of outward potassium currents and does not take up [K+]o with a moderate physiological increase of [K+]o from 5 to 10 mM. For future, these and other data from FIAs can be integrated with in situ studies to further advance our understanding of the roles that astrocytes play in the CNS.
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ACKNOWLEDGMENTS |
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The authors thank Y. Goto for contributing to some of the experiments, Dr. M. W. Fleck, Dr. A. A. Mongin, and G. P. Schools for stimulating discussions and comments on the manuscript, and C. J. Charniga for excellent technical assistance.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-19492 to H. K. Kimelberg.
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FOOTNOTES |
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Address for reprint requests: H. K. Kimelberg, Center for Neuropharmacology and Neuroscience, MC-60, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: Kimelbh{at}mail.amc.edu).
Received 12 May 2000; accepted in final form 28 August 2000.
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REFERENCES |
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