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Neural and Vascular Biology, Ordway Research Institute, Albany, New York
Submitted 3 June 2005; accepted in final form 4 August 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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10% in the adult. They are GLAST(+) astrocytes from newborn to juvenile but NG2(+) glia in the adult. Electrophysiologically passive glia first appear at P4 and increase to 91% in adults, of which 85% are GLAST(+). These results indicate that glial electrophysiological diversity occurs predominantly in the developing brain. While most glia in the NG2 lineage preserve a certain amount of voltage-gated ion conductances, mature GLAST(+) astrocytes are electrophysiologically passive. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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) and astrocytes in slices (Chvatal et al. 1995; D'Ambrosio et al. 1998; Steinhauser et al. 1992). The concept of functional heterogeneity among astrocytes has been further supported by findings that show a segregated expression of ionotropic glutamate receptor AMPA subtypes and functional glutamate transporters by two or three electrophysiologically distinctive astrocyte phenotypes (Grass et al. 2004; Matthias et al. 2003; Zhou and Kimelberg 2001). Such differences contradict the homogenous view of astrocytes based on the pioneering electrophysiological studies of Kuffler et al. (1966) in amphibian optic nerve and later by others in the mammalian CNS (Picker et al. 1981; Somjen 1979), implying that astrocytes might be involved in brain function with greater complexity than had hitherto been envisioned. However, a critical issue is whether astrocyte heterogeneity occurs in the adult brain since all the studies to date have been limited to slices from juvenile rodents. If there is no heterogeneity at later ages, the electrophysiological heterogeneous properties of astrocytes may simply reflect a transition of astrocytes from their precursors to mature cells.
The question of what cells should be classified as astrocyte is made more complex by findings of heterogeneity. Proteins such as GFAP and the astrocyte-specific transporters GLAST and GLT-1 are the least equivocal markers for astrocytes (Walz 2000). Additionally, a chondroitin sulfate proteoglycan, NG2, has been found to selectively label another population of GFAP(–) glia that morphologically resemble astrocytes (Levine and Card 1987). However, recent studies show that NG2(+) glia differ significantly from classically defined astrocytes in terms of their lack of anatomical association with blood vessels, their electrophysiology, and the absence of glutamate transporters (Bergles et al. 2000; Butt et al. 2002; Levine and Card 1987; Peters 2004). Thus the association between electrophysiologically defined glial phenotypes and GFAP(+) and NG2(+) lineages as a function of age needs to be determined to better answer fundamental questions of astrocyte classification, functional heterogeneity, and the relationship between GFAP and NG2 lineage glia (Kimelberg 2004).
In this study, we examined the existence and the distribution of glial electrophysiological phenotypes in cells selected by their appearance under infrared differential interference contrast (IR-DIC) microscopy and their nonexcitability by in situ whole cell recording in the rat hippocampal CA1 region from newborn through adult [postnatal days 1–106 (P1–P106)]. To correlate electrophysiological glial phenotypes with the astrocytic and NG2 glial lineages, we loaded biocytin into the recorded cells and performed postrecording GLAST/NG2 double staining. We confined our studies to the striatum radiatum (SR) and striatum lacunosum moleculare (SLM) to exclude those glia in the dentate gyrus (DG) retaining neurogenetic potential in the adult hippocampus (Rietze et al. 2000).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Hippocampal slices were prepared from P1 to P106 rats. The procedure was performed in accordance with a protocol approved by the Wadsworth Center, New York State Department of Health Institutional Animal Care and Use Committee. Animals were anesthetized with 100% CO2 before decapitation, and their brains were removed from the skull and placed in an ice-cold, oxygenated (5% CO2-95% O2, pH = 7.4) slice preparation solution containing (in mM) 26 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 10 MgCl2, 10 glucose, 0.5 CaCl2, and 240 sucrose. Final osmolarity was 350 ± 2 mOsm. Coronal slices of 300 µm were cut with a Vibratome (Pelco 1500) and transferred to a nylon-basket slice holder in 20–22°C artificial cerebral spinal fluid (aCSF) containing (in mM) 125 NaCl, 25 NaHCO3, 10 glucose, 3.5 KCl, 1.25 NaH2PO4, 2.0 CaCl2, and 1 MgCl2 (osmolarity, 295 ± 5 mOsm). The slices were allowed to recover in ACSF with continuous oxygenation for 
60 min before recording.
Electrophysiology
For in situ recording, individual hippocampal slices were placed in the recording chamber that was constantly perfused with oxygenated aCSF (2.5 ml/min). Glia located in the CA1 region were identified using IR-DIC video microscopy (Olympus BX51W1) with a x40 water immersion objective. The image was detected with an IR-sensitive CCD camera and displayed on a monitor. Whole cell membrane currents were amplified by a MultiClamp 700A amplifier, sampled by a DIGIDADA 1322A Interface, and data acquisition was controlled by PClamp 9.0 software (Axon Instruments, Foster City, CA) installed on a Dell personal computer. Low resistance patch pipettes (3–6 M
) were fabricated from borosilicate capillaries (OD: 1.5 mm; Warner Instrument Corporation, Hamden, CT) using a Flaming/Brown Micropipette Puller (Model P-87, Sutter Instrument Co., Novato, CA). Membrane potential was read in I = 0 mode, and the variation during the 20- to 30-min whole cell recording was always less than ±5 mV. Capacitance and series resistances (Rs) were measured using the "membrane test" protocol built into the PClamp 9.0. Patch pipettes were filled with a solution containing (in mM) 140 KCl, 0.5 Ca2Cl, MgCl2, 5 EGTA, 10 HEPES, 3 Mg-ATP, and 0.3 Na-GTP (pH = 7.3, 280 ± 5 mOsm). Biocytin (0.1–0.5%) was added into electrode solution to detect the recorded astrocyte and show its full morphology and coupling to other cells. All the experiments were conducted at room temperature (22–24°C).
Confocal immunocytochemical studies
After electrophysiological recording, the hippocampal slices containing the biocytin-filled cells were fixed for 45–60 min in 4% formaldehyde in phosphate-buffered saline (PBS; pH 7.4). After washing with PBS, the slices were permeabilized with 1.0% Triton X-100 in PBS (1 h). To visualize the biocytin-filled cells, the slices were treated with 1:1,200 Cy2-streptavidin in PBS for 4 h, followed by washing in PBS. Three percent normal goat serum in PBS (4 h) was used to block nonspecific binding of primary antibodies. Antibodies recognizing the GFAP (rabbit, Dako), GLAST (guinea pig, Chemicon) and NG2 (rabbit, Chemicon) antigens were diluted in 3% normal goat serum plus 0.1% Triton X-100 (NGS/TX). The slices were incubated for 18 h at room temperature with gentle shaking. Slices were washed successively in NGS/TX, 0.1% Triton X-100 in PBS, and PBS. Cy3- and Cy5-conjugated secondary antibodies were selectively used based on the minimal emission wavelength overlap. Three percent normal goat serum was used again to block nonspecific binding before incubation in the diluted secondary antibodies overnight. The specificities of all the secondary antibodies were tested with samples not exposed to primary antibodies, and little or no signal was seen. After the final washes, the slices were mounted on slides. Slides were viewed by confocal microscopy.
We used an inverted Zeiss LSM510 META confocal microscope with x10/0.3 NA, x25/0.8 NA, and x63/1.4 NA objectives. The recorded cell was initially found using a 50-W mercury arc lamp with a x10 objective and a filter set for Cy2. The intensity of the Cy2 signal and the location of the mark made by the recording electrode were used to identify the recorded cell from other cells coupled to this cell. In the laser scanning mode, the extent of the syncytium in the z-axis was determined, and detector gain for each fluorochrome in the sample was set to minimize saturated and black pixels. Each fluorochrome was excited, and its emission was acquired individually to minimize bleed through from channel to channel. A DIC image of each z-plane was also acquired.
Two-dimensional projections of the image stacks and three-dimensional reconstructions of the recorded and coupled cells were made using the LSM510 program. Colocalization of fluorescent labels within individual cells were displayed using the National Institutes of Health ImageJ program (Rasband 1997) with the Colocalization plugin (Bourdoncle 2003).
Statistics
All the data are given as means ± SD.
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RESULTS |
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The glia soma visualized under IR-DIC microscopy can be described as either round or irregular (Fig. 1, A and B) as reported before (Lin and Bergles 2004). We examined whether each shape correlates with GFAP and NG2 glial lineages. However, GFAP immunoreactivity could not be reliably detected from the recorded cell, although it readily detected in glia coupled to the recorded cell (see Supplementary Fig. 1)1 , we alternatively used the glutamate transporter isoform GLAST as a marker for GFAP lineage astrocytes because it stained reliably after whole cell recording. Possibly this is because it is expressed as an astrocyte membrane protein and thus should be less perturbed by the electrode solution during whole cell recording compared with GFAP. The fact that GLAST expression begins in the newborn and persists throughout life (Furuta et al. 1997), and GLAST expression is linked to a functional role of astrocytes in glutamate homeostasis, also makes it a good marker for astrocyte identification. In addition, GFAP/GLAST double-staining experiments in unrecorded cells showed an equal percentage of GFAP(+) glia among irregular and round soma shape cells (71%; Fig. 1E). Also, GLAST immunoreactivity was detected nearly identically from irregular (85%) and round soma (84%) glia (Fig. 1E). Except for one round soma glia, all the GFAP(+) glia of both soma shapes were GLAST(+) (n = 107); however, GFAP immunoreactivity was absent in 14% of GLAST(+) glia of both soma shapes (n = 107; Fig. 1E). These results indicate that, although the number of GLAST(+) glia are slightly higher than GFAP(+) glia, GLAST(+) overlaps excellently with GFAP(+) glia and therefore is an equivalent marker for GFAP lineage glia.
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20% of all the cells studied (Fig. 1, E and F). Importantly, GLAST and NG2 immunoreactivity were never colocalized in the same cell, regardless of soma shapes. The quantitations in Fig. 1, E and F, were calculated from P10 rats, and the same percentages were observed for P35 rats (data not shown). The identification of soma shapes by DIC and the correlation of GLAST and GFAP immunoreactivities with these DIC soma shapes images were performed by different individuals in a blinded fashion. Therefore we conclude that 1) the variation in glial soma shapes observed with two-dimensional IR-DIC cannot be applied, at least in the CA1, to discriminate GFAP/GLAST from NG2 lineage glia; and 2) GLAST and NG2 are specific markers for GFAP and NG2 lineage glia identification. Based on these findings, glial cells having both round and irregular soma shapes were included in the following experiments, as specified.
Diverse electrophysiological astroglial phenotypes identified by whole cell recording
To understand the developmental changes in glial electrophysiological phenotypes, 561 whole cell recordings were taken from glial cells in the hippocampal CA1, SR, and SLM regions from P1 to P106. Three current profiles in voltage-clamp mode were distinguished (Fig. 2, A–C). Two of them resemble what has been described from freshly isolated hippocampal astrocytes (Zhou and Kimelberg 2000) and from hippocampal slices (D'Ambrosio et al. 1998; Steinhauser et al. 1992). Figure 2A corresponds to what we termed (Zhou and Kimelberg 2000) an outwardly rectifying astrocyte (ORA), characterized by a depolarization-induced voltage-gated inward Na+ (INa), outward transient K+ (IKa), and delayed rectifier K+ (IKdr) channel currents. Figure 2B corresponds to what we termed a variably rectifying astrocyte (VRA) with expression of both inward and outward K+ currents, no inward Na+ currents, and with multiple rectification points in its voltage-current relationship. However, because postrecording staining showed that these cells actually represented both GLAST(+) and NG2(+) glia, we will now term the electrophysiological phenotypes without postrecording identification outwardly rectifying glia (ORG) and variably rectifying glia (VRG). The third electrophysiological glial phenotype that we found (Fig. 2C) is distinguished by a linear current-voltage relationship in whole cell recording, which has been previously described in in situ hippocampal glial studies from P10 to P30 rat hippocampus (D'Ambrosio et al. 1998; Steinhauser et al. 1992).
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(n = 216) in PGs is significantly lower than either ORGs (870.3 ± 705.7 M, n = 111), VRGs (114.6 ± 112.4 M, n = 134), or neurons (147.2 ± 170.0, n = 10). Thus the PG cells would have been largely omitted in the systematic development study of Bordey and Sontheimer (1997), who selected for cells with RM 50 M, and were, in fact, not represented. The mean membrane potential of their selected population was –59 mV, RM was 222 M, and CM was 28pF; this corresponds to the characteristics of ORGs, and to a lesser extent VRGs, in our study (Table 1).
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; Steinhauser et al. 1992) (data not shown). No cells that might correspond to microglia (Boucsein et al. 2003) were observed. Voltage-clamp measurements were always followed by measurements in the current-clamp mode to check for excitability and possible postsynaptic electric events (Fig. 3, A–C). Only ORGs from newborn rats (P1–P3) showed some immature spiking when positive current was injected. These were characterized by a slow rise but no overshoot (Fig. 3B). At later postnatal and adult stages, we never saw immature spikes in current clamp for any of the three electrophysiological glial phenotypes. Although, in current-clamp mode, some ORGs in the early developmental stage show fluctuations in their basal membrane potential, no clear postsynaptic events were measurable (Fig. 3C). Although interneurons are distributed throughout the same brain region and some of them are similar in soma shape and size to glia, they always showed miniature excitatory postsynaptic potential (mEPSP) and spontaneous electric spikes (Fig. 3F). Thus differences in electrical excitability are one essential criterion discriminating glia from interneurons.
Developmental changes in ion current profiles
We systematically examined the existence and distribution of the electrophysiological phenotypes recorded in voltage-clamp mode, in this case from irregular soma shaped glia, in slices from newborn to adult rat (Fig. 4A). In light of the dramatic changes we found occurring in the first postnatal week, we studied cells on each successive day. For each day, 25 glial cells were recorded to determine the relative ratio among the different electrophysiological glial phenotypes. In an additional study (Fig. 4B), recordings from round soma glia were taken from P2, P7, and P38. The electrophysiological developmental profile of round soma clearly follows the pattern for irregular soma glia, further supporting the view that irregular and round soma represent the same glial population.
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25 recordings were taken.
During the juvenile period, ORGs continually decreased in number and completely disappeared at the end of the juvenile period. Concomitantly, the proportion of VRGs increased and peaked at P5 and declined gradually to a stationary level at the end of the juvenile period (10% of total cells examined). After the first appearance of PG at P4, the number of PG increased and rapidly became the dominant glial type at the end the juvenile period.
For the round shape soma glia (Fig. 4B), the electrophysiological glial phenotypes followed the same pattern that we found for irregular soma glia, which is consistent with the conclusion from the staining results that both round or irregular soma equally represent GLAST and NG2 lineage glia, and therefore the same cell population (Fig. 1).
Interestingly, 70% of VRGs (n = 19) after the third postnatal week expressed INa (Fig. 5), which was never seen in VRA-type isolated astrocytes, and has been used by us as one of the diagnostic features discriminating ORA from VRA (Zhou and Kimelberg 2000). The cellular identity of VRGs after the third postnatal week was further explored with postrecording GLAST/NG2 double-staining studies, as described in the next section.
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Together, the analysis of glial electrophysiological phenotypes as a function of animal age led to the conclusions that 1) electrophysiological glial heterogeneity occurs mainly within the first 3 postnatal wk in the hippocampal CA1; 2) PGs originate postnatally, and in the adult brain, starting from the fourth postnatal week, PG becomes the dominant electrophysiological glial phenotype that amounts to 92% of the total cells examined from P22 to P106; and 3) the apparent VRG-like glia in the adult hippocampal CA1 region exhibits features similar to ORGs and therefore appears as a variant of the ORGs seen in the earlier developmental stages (see Fig. 2A), but additionally with inward K+ channel currents. We never saw any ORGs after the third postnatal week, and the VRG-like glia were always <10% of the total cells in every week after the third postnatal week.
Immunocytochemical identification of different electrophysiological glial phenotypes
To determine the association between the different electrophysiological glial phenotypes and the GLAST and NG2 glia lineages as a function of age, postrecording double immunofluorescent staining was done.
To identify recorded cells after postrecording immunocytochemistry, 0.1–0.5% biocytin was included in the electrode solution so that recorded cells could be located based on Cy2-streptavidin labeling of biocytin. The hippocampal slice was fixed immediately in 4% formaldehyde and subjected to immunocytochemistry, and the resulting staining was viewed by laser scanning confocal microscopy (see METHODS). Representative GLAST/NG2 double-staining results are shown in Fig. 6, A–D, and the quantitative data for all the results are shown in Fig. 6, E–G.
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All the VRGs (Fig. 6F) from the newborn and juvenile period were GLAST(+) glia. In the adult, however, the VRGs were exclusively NG2(+) glia. Given that VRGs in the adult also express INa, a characteristic feature of ORGs, it suggests that the VRG phenotype in the adult brain derives from ORG NG2(+) glia (see Fig. 7).
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One of the differences between NG2(+) glia and GFAP(+) astrocytes is that GFAP(+) glia form syncytia through gap junction channels, whereas NG2(+) glia are never coupled (Bergles et al. 2000; Lin and Bergles 2004), which we also observed in slices from mature rats. The association between the electrophysiological phenotypes, GLAST and NG2 lineage, and the degree of coupling as a function of age will be the subject of a separate paper (unpublished data).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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As noted in the introduction, the relationships between the three different electrophysiological glial phenotypes and the GFAP and NG2 lineages are currently unclear. For example, the ORG phenotype has been shown to be exhibited by GFAP(+) astrocytes (Bordey and Sontheimer 1997; Zhou and Kimelberg 2000), GFAP mRNA(+) "complex glia" (Zhou et al. 2000), and NG2(+) glia (Schools et al. 2003) in the rat hippocampus. Our postrecording GLAST/NG2 double-staining results clarify this issue. For the first time, our studies show that both GLAST (+) and NG2 (+) glia are present at P1, and some of them share the same electrophysiological phenotype, ORG, during the first 3 postnatal weeks but not at a latter time (Fig. 7). We show that the progression of GLAST lineage glia follow the electrophysiological phenotype order of ORG, VRG, and PG during development (Fig. 7). This leads to the inference that the electrophysiologically passive glial phenotype is the mature form of classically defined GFAP(+) astrocytes, similar to the early studies on amphibian optic nerve (Kuffler et al. 1966) and later in mammalian glia (Picker et al. 1981; Somjen 1979).
Because GFAP and NG2 lineage glia show no cross-lineage relationship in the postnatal brain (Bergles et al. 2000; Butt et al. 2002; Peters 2004), the postrecording immunostaining results also indicate that the development of NG2 lineage glia follows the electrophysiological phenotype order of ORG in the newborn and juvenile brain and VRG-like in the adult (Fig. 7). However, although 100% of VRG cells are NG2(+) (n = 5) in the adult, 15% of PGs (n = 13) were also NG2(+) in the adult brain, showing that PG also represents NG2 glia but to a much lesser extent. Why NG2 glia persistently express voltage-gated K+ and Na+ currents in the adult brain, or alternatively why some of them are electrophysiologically passive, remains to be determined. NG2 glia give rise to interneurons and other glia postnatally (Aguirre and Gallo 2004), and the persistence of voltage-gated ion channels may be involved in this proliferative capacity.
Other groups studying astrocyte heterogeneity have used a transgenic mouse line that expresses enhanced green fluorescent protein (GFP) under the control of the human GFAP promoter. This facilitates selection of astrocytes for in situ patch-clamp recordings. However, the GFP signal is not limited to astrocytes but also labels NG2(+) cells (Grass et al. 2004; Matthias et al. 2003) and a varying number of GFAP(–) cells throughout the brain (Nolte et al. 2001). In this study, we chose to identify cell lineages, after initial morphological identification, by performing postrecording immunohistochemistry. With the mutually exclusive nature of GLAST and NG2 labeling in the rat and mouse, there is little chance for ambiguous results. Thus our postrecording GLAST/NG2 double staining allows us to correlate glia lineage and electrophysiological phenotypes with good confidence.
Relative ratio of GLAST(+) versus NG2(+) glia in CA1 region
Although NG2(+) glia are considered to be numerous in the brain, no quantitative data are currently available. We show that from P22 on, 100% of VRGs and 15.4% of PGs are NG2(+) glia in the CA1 region (Fig. 6). From P22 on, 212 PGs and 19 VRGs were identified (Fig. 4A). Based on this, the total number of NG2(+) glia should be 52, which amounts to 22% of the total glia in the CA1 region. This is reasonably close to a preliminary analysis on the relative numbers of GFAP(+) and NG2(+) cells in slices from P31 rat. From five images of the CA1 region from two slices, we counted a total of 63 GFAP(+) and 21 NG2(+) cells, or a ratio of 75/25% GFAP(+)/NG2(+) (data not shown).
Developmental regulation of K+ and Na+ channel expression
Our systematic analysis of the astroglial electrophysiological phenotypes clearly shows a developmental switch of current profiles in both GLAST and NG2 lineage glia. Specifically, in the newborn and regardless of GLAST or NG2 lineage, glia expresses the voltage-gated K+ channel currents, IKa and IKdr. Additionally, ORGs also express voltage-gated INa. Our study precisely shows that the first appearance of cells expressing predominant leak type K+ currents starts at P4. We refer to these as electrophysiologically passive glia or PG, and the number of PGs increases rapidly to become the predominant glia at the end of the third postnatal week. Given that astroglia in CA1, SR, and SLM are postmitotic at birth (Rietze et al. 2000), the switch in ion channel currents could be reasonably inferred as an intrinsic transition of ion channel expression among the same population of glia instead of different glia proliferating from a common precursors.
Our studies are consistent with an assumption that VRG is an intermediate phenotype between ORG and PG in GLAST(+) astrocyte lineage. The evidence is 1) the decrease in GLAST(+) ORGs in the newborn and juvenile is accompanied by an increase in GLAST(+) VRGs; 2) the decrease in GLAST(+) VRGs also parallels the progressive increase in GLAST(+) PGs; and 3) the GLAST(+) VRGs almost completely disappear after the third postnatal week. The 
10% of VRG-like glia in the adult brain are actually NG2(+) glia.
Mature astrocytes show electrophysiologically passive phenotypes
We believe that one of the most significant findings derived from our studies is that the electrophysiologically passive glia are the mature form of GLAST(+) astrocytes and are the predominant glia in the adult rat hippocampal CA1 region. Although whole cell current profile of PGs shows a linear I-V relationship, this does not rule out some contributions of other voltage-gated ion/anion conductance to the apparent overall linear conductance (Barres et al. 1990; Pannicke et al. 2005). The underlying ion/anion species that contribute to the passive conductances are clearly a subject for future work. It is noticeable that the average membrane potential of PGs measured under whole cell conditions (–73 ± 8, n = 275) deviates significantly from the theoretical prediction based on [Ko]/[Ki] = 3.5/140 (EK = –96 mV), showing that PGs may have contributions from other conductances. Because the Cl– concentration was symmetrical in bath and electrode solutions, Cl– conductance may contribute to the deviation from EK. Consistent with this idea, our unpublished observations show that ClC-2–like chloride currents are present in freshly isolated astrocytes (unpublished observations), and a similar finding has been reported from an in situ study on astrocytes (Makara et al. 2003).
Comparison with isolated astrocytes
A major difference between these results in slices and our previous data in freshly isolated cells is that passive cells were never seen in the freshly isolated astrocytes, even when the cells were isolated from P21 to P35 (Zhou and Kimelberg 2000) when significant passive cells from the same region are seen in situ. This could be because of removal of passive K+ channel–rich regions, the destruction of the astrocytic syncytium, or failure of passive cells to survive the isolation procedure. However, a recent study showed that neither the passive current profile nor the membrane resistance of hippocampal PGs was affected by gap junction blockers (Wallraff et al. 2004). This implies that the passive phenotype in situ reflects the intrinsic K+ channel properties of PGs instead of syncytial coupling. These different possibilities need further study.
In conclusion, the various reports of astrocyte electrophysiological heterogeneity can now be understood on the basis that the previous studies, even if they were in slices, primarily focused on astroglia from the critical developmental period of 1–3 wk. Our studies show that starting from the fourth postnatal week, all the GLAST(+) astrocytes are passive glia. This amounts to 78% of the total glial population. Coincidently, thrombospondins, which are synthesized by astrocytes to promote neuronal synaptogenesis, are expressed at high levels during the first 3 postnatal weeks and decline in the adult brain (Christopherson et al. 2005), implying only immature astrocytes are involved in synaptogenesis. Other conclusions implied by our studies are 1) that two glial lineages recognized by GLAST or NG2 marker expression, are fate-determined at birth; 2) GLAST lineage astrocytes are always the predominant glial type, whereas NG2-lineage glia constitute a smaller population of 22% of the total glial population in the rat hippocampal CA1 region; 3) during brain maturation, the GLAST lineage astrocytes undergo a progressive loss of voltage-gated ion channels that have been predominantly substituted by leak-type K+ conductances; and 4) around 50% of the NG2-lineage glia persistently express voltage-gated K+ and Na+ channels in the mature brain, implying the potential of NG2-lineage glia for cell proliferation and differentiation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The Supplementary Material for this article (a Figure) is available online at http://jn.physiology.org/cgi/content/full/00570-2005/DC1. 
Address for reprint requests and other correspondence: M. Zhou, Neural and Vascular Biology, Ordway Research Inst., 150 New Scotland Ave., Albany, NY 12208 (E-mail: mzhou{at}ordwayresearch.org)
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