Freshly Isolated Astrocytes From Rat Hippocampus Show Two Distinct Current Patterns and Different [K+]o Uptake Capabilities

Min Zhou and Harold K. Kimelberg

Center for Neuropharmacology and Neuroscience and Division of Neurosurgery, Albany Medical College, Albany, New York 12208


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Morphology, glial fibrillary acidic protein (GFAP) staining, and GFAP mRNA of freshly isolated astrocytes (FIAs). A-C: phase micrographs showing the characteristic morphologies of freshly isolated astrocytes from the hippocampal CA1 regions of P12 rats. These cells are characterized by an oval (A), elongated (B), or triangular (C) cell body, with multiple processes extending from the cell body (the scale bar below B also applies to A and C). D: phase contrast and E: corresponding fluorescent micrographs, respectively, of a freshly isolated GFAP(+) astrocyte. F: gels of single-cell GFAP mRNA reverse transcription polymerase chain reaction (RT-PCR) products obtained after electrophysiological experiments on the same type of cells from P8 rat. Standard DNA markers with 100 base-pair (bp) difference are shown on the left side of the gel. The second-round PCR products using the nested GFAP primer-2s were expected to give a cDNA band at the position indicated by the left-hand arrow. Lanes 4, 6 were RT-PCR results obtained from the recordings shown in Fig. 2, B and A, respectively. Lane 1 was a negative media control. Lane 3 is a negative control from a recorded neuron (electrophysiological recording not shown). Lanes 2 and 5 were from another 2 recorded astrocytes (recordings not shown).

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 MOmega ) 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
<IT>g</IT><IT>/</IT><IT>g</IT><SUB><IT>max</IT></SUB><IT>=</IT>{<IT>1+exp</IT>[(<IT>V</IT><SUB><IT>n</IT><IT>,0.5</IT></SUB><IT>−</IT><IT>V</IT>)<IT>/</IT><IT>k<SUB>n</SUB></IT>]}<SUP><IT>−1</IT></SUP> (1)
with V being the test potential, Vn,0.5 the voltage of half-maximum activation, and kn a slope factor.

Steady-state inactivation was fitted by a Boltzmann equation of the form
<IT>I</IT><IT>/</IT><IT>I</IT><SUB><IT>max</IT></SUB><IT>=</IT>{<IT>1+exp</IT>[(<IT>V</IT><IT>−</IT><IT>V</IT><SUB><IT>g</IT><IT>,0.5</IT></SUB>)<IT>/</IT><IT>k<SUB>g</SUB></IT>]}<SUP><IT>−1</IT></SUP> (2)
where I is the voltage-dependent current amplitude, Imax maximum current, V the prepulse potential, Vg,0.5 the voltage of half-maximum inactivation, and kg the slope factor. Significant differences between data were evaluated by the use of Student's t test. All the data are given as means ± SD. The level of significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1. Proportion of different morphological cell types in CA1 and CA3 suspensions

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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Fig. 2. Ion channel profiles of FIAs measured by whole-cell recordings. The cells were preconditioned at -110 mV for 300 ms to remove the inactivation of IKa. Subsequently, the cells were stepped for 50 ms to test potentials ranging from -180 to +60 mV in 10 mV increments (for voltage commands, see shadowed inset). Two distinct astrocyte types, as defined by their ion channel profiles, were seen. A: representative recording of one type of astrocytes characterized by a dominant expression of outward transient (IKa) and sustained delayed rectifier potassium currents (IKdr). This cell showed also initial inward transient sodium currents (INa). These INa currents are shown in a expanded scale in the inset below A. As the corresponding I-V curve showed a dominant outward rectification (C), these type of cells were termed outward rectifying astrocytes (ORAs). B: the other astrocyte type was distinguished by a more symmetrical inducible potassium currents in response to de- and hyperpolarizing steps. The I-V curve of this type of astrocytes showed several regions of rectification (D); therefore, we term them variably rectifying astrocytes (VRA). These two cells were all obtained from the CA1 region of a P8 rat. The membrane potentials measured <= 30 s after establishment of a whole-cell seal was -50 mV for recording A and -74 mV for recording B. The post recording RT-PCRs for GFAP mRNA are shown in Fig. 1F, lanes 4 and 6, for A and B, respectively.

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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Fig. 3. Ion selectivity of membrane conductance of FIAs. A and B: recordings of tail current analysis from a CA1 VRA-type astrocyte from a P14 rat, for both outward (A) and inward (B) currents. TTX (100 nM) was added to block voltage-gated INa. As shown in the shadowed voltage command below A and B, the cell was first clamped to 60 mV (A) or -180 mV (B) for 50 ms. Subsequently, the membrane was stepped to a series of test potentials between -120 to +20 mV (in 10 mV increments). C: corresponding I-V curves for A () and B () shown in an expanded scale for the Erev region. These I-V curves were constructed by taking current amplitudes 5 ms after the onset of the test pulses (shown as the vertical dotted line in A and B). The Erevs determined for this cell were -67 and -85 mV for outward (Erev out) and inward (Erev in) currents, respectively. Average values for several cells are given in Table 2. D and E: sensitivity of transient inward currents of a CA3 ORA type astrocyte to 100 nM TTX. D was the recording in control bath solution, and E with addition of 100 nM TTX. F: the TTX-sensitive sodium current obtained by subtracting E from D. This TTX-sensitive current was activated at -60 mV and peaked at -10 mV.


                              
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Table 2. Erev of tail currents at different prepulse duration and potentials of in- and outward currents in VRAs and ORAs

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 GOmega ) was significantly higher than for VRAs (0.6 ± 0.3 GOmega ) 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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Fig. 4. Fitting of RMP, Rin, and Cm to double Gaussian models. A-C: RMPs, Rins, and Cms from 80 cells were plotted as a function of observed frequencies. The distributions of RMP and Cm were clearly bimodal as they were well fitted by a double Gaussian function (smooth lines, with the coefficient of correlation r = 0.87 for A, and r = 0.98 for C), yielding a pair of RMP values of -66 and -43 mV, and two peak Cm values of 10 and 30 pF, respectively. However, Rins could not be well fitted by this model (B).


                              
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Table 3. Passive membrane properties of FIAs

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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Fig. 5. Sensitivities of IKa and IKdr in ORAs and VRAs to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). A1-2 and B1-2 are IKa currents isolated with the protocols shown in the shadowed inset between A and B and described in the text. A1-2 and B1-2 show the 4-AP (4 mM) sensitivity of IKa from a representative ORA and VRA cell, respectively. TTX was not present in these experiments. A3 and B3 are the corresponding I-V curves showing the voltage-dependent inhibition. As shown in A2 and B2 at +60 mV voltage step, 4-AP inhibited the ORA IKa by 52%, but essentially completely blocked VRA IKa. C1-2 and D1-2 are the outward sustained potassium currents induced by voltage commands with prepulse at -40 mV to remove the activation of IKa (see shadowed inset in the middle of C and D). C1 and D1 were the recordings in control bath solution; C2 and D2 were the recordings with 15 mM TEA, and C3 and D3 were the corresponding I-V curves for ORA and VRA cells, respectively. The same concentration of TEA blocked 70% outward sustained currents in an ORA cell (C3), whereas it inhibited only 30% of sustained outward potassium currents in a VRA cell at +60 mV (D3).

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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Fig. 6. VRAs show both Cs+-sensitive and Cs+-resistant IKin. The IKin were evoked by the voltage commands shown in the shadowed inset. A1-3 show Cs+ sensitive IKin of a VRA cell. B1-3 show a VRA cell with Cs+ insensitive IKin. The kinetics of both IKins failed to show a time-dependent inactivation. For the I-V curves, the current amplitude values were taken close to the end of each 50-ms pulse as indicated by the vertical lines identified by open and filled circles and filled squares corresponding to the symbols used for the I-V curves, respectively. In cell A, Cs+ blocked IKin by 85%, and in cell B IKin was only inhibited by 17% (all at -180 mV voltage step). The cells in recordings A and B were obtained from the CA1 region of P12 and P10 rats, respectively.

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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Fig. 7. Kinetic properties of IKa in VRA and ORAs. To determine steady-state activation of IKa, this current component was isolated by the procedure shown in the shadowed inset below A and B. Recording A was obtained from an ORA, and B from a VRA cell. The membrane conductances were calculated by dividing peak currents elicited at different membrane potentials (-70 to 60 mV) by the corresponding driving force, V - EK. Subsequently, conductances were normalized to values at 60 mV from the same cell (g/gmax) and averaged (n = 10 for ORA and n = 3 for VRA). The open and filled circles in C and F stand for average values for VRAs and ORAs, respectively. The best fittings (smooth lines in C) by the Boltzmann equation (see METHODS, Eq. 1) yielded Vn,0.5 values of -12 and 8 mV for ORA and VAR, respectively (C), with no difference in slope factor Kn (C). This analysis indicates that the Vn,0.5 value of VRA is 20 mV positive compared with ORA. D and E were the recordings for IKa inactivation analysis. Inactivation of IKa was recorded by changing the prepulse potential (300 ms) between -120 and -20 mV and evoking currents at a test potential of 60 mV (50 ms). Subsequently, the current response after the -20 mV prepulse was subtracted from all traces to compensate for the sustained component. For the detailed procedure, see shadowed inset below D and E. Individual peak currents were normalized to maximal current induced at prepulse of -120 mV from the same cell and averaged (n = 8 for ORA and n = 3 for VRA). The best fittings (smooth lines in F) by the Boltzmann equation (see METHODS, Eq. 2) yielded the same Vg,0.5 of -65 associated with a same slope factor (Kg) of 11 mV for both ORA and VRA.

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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Fig. 8. ORA and VRA show different responses to a rapid, moderate increase in [K+]o. To determine the acute [K+]o uptake capability of ORAs and VRAs, 10 mM K+ (normal bath K+ 5 mM) was fast applied to an identified ORA (A) and a VRA astrocyte (B), respectively. At the holding potential of -70 mV, ORA did not show any response to the moderate increased [K+]o. In contrast, the same increase in [K+]o induced a rapid rise and steady-state inward potassium currents in the VRA cell, suggesting a robust K+ uptake capability. A was an ORA astrocyte obtained from CA1 region of a P10 rat, and B a VRA astrocyte from the CA1 region of a P11 rat.

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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Fig. 9. Comparison of ion current profiles and heterogeneity of occurrence of ORAs and VRAs in CA1 and CA3 regions. A: occurrences of the four ion current components in the CA1 and CA3 regions. When expressed this way, the average of each current component from both ORAs and VRAs did not show a regional difference between CA1 and CA3. B: the ion current components show a marked difference between ORAs and VRAs. While 90% of ORAs expressed INa (n = 48) and 92% expressed IKa, none of them expressed IKin. By contrast, all the VRAs expressed large IKin (n = 32), and only one VRA expressed a tiny INa. C: in the CA1 region, ORAs and VRAs were recorded in essentially equal proportions (52 vs. 48%). However, ORAs were 2.4-fold more abundant (71%) than VRAs in the CA3 region. All the data are from P7-15 rats.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 GOmega 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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