Regulation of Kv1 subunit expression in oligodendrocyte progenitor cells and their role in G₁/S phase progression of the cell cycle

Abstract

Proliferative oligodendrocyte progenitor cells (OPs) express large, delayed outward-rectifying K⁺ currents (I_K), whereas nondividing immature and mature oligodendrocytes display much smaller I_K. Here, we show that up-regulation of I_K occurs in G₁ phase of the cell cycle in purified cultured OPs and is the result of an RNA synthesis-dependent, selective increase of the K⁺ channel subunit proteins Kv1.3 and Kv1.5. In oligodendrocyte cells acutely isolated from developing rat brain, a decrease of cyclin D expression is observed as these cells mature along their lineage. This is accompanied by a decrease in Kv1.3 and Kv1.5 subunit expression, suggesting a role for these subunits in the proliferative potential of OPs in situ. I_K expressed in OPs in subventricular zone and developing white matter in acutely isolated slice preparations were selectively blocked by antagonists of Kv1.3, illustrating the functional presence of this subunit in situ. Interestingly, Kv1.3 block inhibited S-phase entry of both purified OPs in culture and in tissue slice cultures. Thus, we employ both in vitro and in situ experimental approaches to show that (i) RNA-dependent synthesis of Kv1.3 and Kv1.5 subunit proteins occurs in G₁ phase of the OP cell cycle and is responsible for the observed increase in I_K, and (ii) currents through Kv1.3-containing channels play a crucial role in G₁/S transition of proliferating OPs.

Oligodendrocytes, a major class of macroglia, are responsible for myelination in the central nervous system and primarily originate from highly proliferative oligodendrocyte precursor cells (OPs) in a spatially restricted central nervous system area termed the subventricular zone (SVZ; refs. 1 and 2). Under the influence of extrinsic trophic signals (3, 4), OPs migrate, proliferate, and the majority differentiate (by means of a number of intermediate cell stages) into myelinating oligodendrocytes.

A correlation exists between expression of the delayed, outward-rectifying voltage-gated K⁺ currents (I_K) and the proliferative potential of oligodendrocyte lineage cells (5–9). Proliferating OPs possess large I_K, whereas postmitotic oligodendrocytes do not express such currents (5–9). However, few studies have attempted to identify the cellular mechanisms responsible for these K⁺ channel changes in OPs. Furthermore, although the functional properties of these channels indicate that they are composed of subunits of the Kv1 subfamily (9, 10), the molecular identity of the K⁺ channel subunits involved in the developmental alterations of K⁺ channel expression in OPs has not been extensively analyzed.

We have previously used a cell culture model system that allows OPs to be synchronized in a nonproliferative state (G₀) and be induced to enter the cell cycle upon mitogen treatment (11, 12). By using this approach, we have previously shown that mitogen-induced entry of OPs into the cell cycle is accompanied by a marked increase in I_K.§ In the present study, we take further advantage of this culture system to: (i) analyze the time course of the mitogen-induced increase of I_K in OPs and determine the phase of the cell cycle at which these alterations occur; (ii) investigate the mechanism(s) responsible for the increase in I_K; and (iii) identify the subunits that underlie the observed increase in the functional expression of K⁺ channels. In addition to a purely in vitro approach, we also investigate the presence of Kv subunits and K⁺ channels in OPs in slice preparations to link the proliferative potential of these cells in situ to the expression of I_K and to confirm that previous observations in the culture system are relevant to a scenario more closely associated with an in vivo setting.

Materials and Methods

Materials.

Platelet-derived growth factor (PDGF; human, AB, heterodimer form), basic fibroblast growth factor (bFGF; human), and anti-cyclin-D antibody were obtained from Upstate Biotechnology (Lake Placid, NY). Anti Kv1.1–1.6 antibodies, r-agitoxin and r-margatoxin, were obtained from Alomone Labs (Jerusalem). Anti-NG2 polyclonal antibody was a gift of Bill Stallcup (Burnham Institute, La Jolla, CA). Anti-BrdUrd antibody was obtained from Dako. Rapamycin was obtained from Biomol (Plymouth Meeting, PA). All other reagents were obtained from Sigma.

Purified OP Cell Cultures and Organotypic Tissue Slice Cultures.

Rat OP cell cultures were prepared as described (12). Brain organotypic slice cultures were prepared from P3 coronal slices, containing the lateral ventricles of Sprague–Dawley rats, as described (13).

Immunocytochemistry.

For immunostaining of acutely isolated cells from brain slices, 400-μm cerebral coronal sections from P3 rats were dissociated, and total cell suspensions were prepared as described (13). For immunostaining of OP cultures, cells were fixed in 4% (wt/vol) paraformaldehyde for 15 min and permeabilized in 95% ethanol/5% acetic acid for 3 min. After incubation with anti-Kv1.3 and anti-Kv1.5 (1:50) overnight at 4°C, and with fluorescein-conjugated goat anti-rabbit IgG for 30 min at room temperature, cells were washed and mounted in Vectashield.

Western Blots.

Western blot analysis of cyclin-D and Kv channel subunit expression was performed as described (11, 14). Anti-Kv1.1–anti-Kv1.6 antibodies were used at a dilution of 1:1,000.

Cell Proliferation Assays in Purified OP Cultures and in Tissue Slice Cultures.

Cultured cells were incubated with BrdUrd (25 μM) for 24 h, fixed, permeabilized, and stained with anti-BrdUrd antibodies as described (13). In tissue slice cultures, coronal slices containing the lateral ventricles were incubated with BrdUrd (25 μM) for 24 h. After enzymatic dissociation (13), cells were stained with antibodies (NG2, O4, and anti-BrdUrd), and percentages of labeled cells were determined (13).

Electrophysiology in Cultured OPs and NG2⁺ Cells in Acutely Isolated Slices.

Whole-cell patch–clamp recordings were performed on OPs that were synchronized in G₀ or were treated with PDGF or bFGF (10 ng/ml). I_K and A-type transient K⁺ channel currents (I_A) in cultured OPs were isolated and measured as described (9). Measurements of I_K expressed by OPs in SVZ or in developing white matter of acutely isolated brain slices were performed as follows. Sprague-Dawley rats (P6–P10) were killed following National Institutes of Health Animal Welfare Guidelines, and 300 μM vibratome sagittal sections were obtained. Slices were perfused with extracellular solution of the following composition (in mM): 124 NaCl/3 KCl/2.5 CaCl₂/1.3 MgSO₄/26 NaHCO₃/1.25 NaH₂PO₄/15 glucose/0.001 tetrodotoxin, saturated with 95% O₂/5% CO₂. Whole-cell somatic recordings were made in SVZ and developing white matter dorsal to the lateral ventricles. Cells were voltage-clamped at −70 mV, and I_K or I_A was elicited as described for cultured OPs (9). Intracellular solution used was as described (9) but with the addition of 5 mg/ml biocytin. All membrane potentials were adjusted for a calculated junction potential of 13.7 mV. In both the culture and slice electrophysiological experiments, currents were recorded using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA), filtered at 5 kHz and digitized at 10 kHz. All offline analysis was performed by using the CLAMPFIT analysis program (Axon Instruments).

Biocytin and NG2 Double Immunostaining After Electrophysiological Recording from Acutely Isolated Slices.

Following electrophysiological recordings, slices were immediately fixed and processed as described (15). For NG2 staining, sections were incubated with a blocking solution [20% (vol/vol) goat serum in PBS], followed by anti-NG2 polyclonal antibody overnight at 4°C, and then mounted.

Statistical Analysis.

For all experiments described where statistical analysis was performed, an unpaired Student's t test was used (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Results

Time course of I_K up-regulation following mitogen treatment of quiescent OPs. Cultured OPs synchronized in G₀ phase (i.e., withdrawn from the cell cycle by culturing in serum-free DMEM-N1 for 24 h) displayed small I_K (Fig. 1A). On treatment with PDGF or bFGF (10 ng/ml), an up-regulation of I_K was observed (Fig. 1 B and C, respectively). Twenty-four hours after PDGF or bFGF treatment, I_K density increased approximately 2-fold (Fig. 1 D and E). The majority of the increase in I_K density occurred within approximately 15 h after mitogen treatment. Prolonged exposure to either PDGF or bFGF (48 h) did not lead to any further significant changes in I_K density (Fig. 1D).

Figure 1.

I_K is up-regulated following 24-h (B) PDGF and (C) bFGF treatment (10 ng/ml), when compared with I_K in OPs in G₀ (DME-N1 medium) (A). (D and E) Time course of up-regulation of I_K in OPs following PDGF and bFGF (total cells = 131 and 147, respectively) treatment. (F and G) The PDGF- and bFGF-induced increase is not prevented by the G₁/S transition blockers, rapamycin (30 nM) and aphidicolin (3 μM), after 12 or 24 h following mitogen treatment (data represent means of 8–11 OP cell recordings per condition from at least three different individual cultures ± SEM). In F and G, I_k density in OP cells cultured in N1 medium without PDGF or bFGF was the same as in D and E.

The Mitogen-Induced Up-Regulation of I_K Occurs in G₁ Phase of the Cell Cycle.

Rapamycin and aphidicolin cause cell arrest in the late G₁ phase of OP cell cycle (11). In the presence of either agent, an up-regulation of I_K density similar to that observed in cells treated with PDGF or bFGF alone was observed, showing that late G₁ arrest did not prevent the mitogen-induced up-regulation (Fig. 1 F and G). These observations, coupled with the time course of the increase in I_K and estimates of cell cycle duration in cultured OPs (24 h) (16, 17), indicate that I_K is up-regulated during G₁ phase of the cell cycle.

Mitogen-Induced Increase in I_K Is Mediated by a Selective Up-Regulation of Kv1.3 and Kv1.5 Subunit Protein.

Western blot analysis of Kv subunit proteins was performed in cultured OPs, either in G₀ or 24 h after incubation with PDGF or bFGF. Of the Kv subunits tested (Kv1.1 through Kv1.6), no detectable Kv1.1 protein was observed, and only low levels of Kv1.2 were present. Furthermore, PDGF or bFGF (data not shown) did not affect expression of Kv1.2. In contrast, OPs expressed significant levels of Kv1.3, Kv1.4, Kv1.5, and Kv1.6 proteins (Fig. 2). Following PDGF or bFGF treatment (24 h), a selective increase was only apparent in Kv1.3 (Fig. 2A) and Kv1.5 (Fig. 2C) expression. No significant change in Kv1.4 (Fig. 2B) or Kv1.6 (Fig. 2D) was observed.

Figure 2.

Western blot analysis of Kv1.3–Kv1.6 expression in cultured OPs in DME-N1 medium and 24 h after PDGF (P) or bFGF (bF) treatment (10 ng/ml). A selective and significant increase of normalized protein expression (**, P < 0.01) is apparent following either PDGF or bFGF treatment of Kv1.3 (A) and Kv1.5 (C) but not of Kv1.4 (B) and Kv1.6 (D) (see Insets for single examples of band staining). Data were normalized with respect to cells cultured in DME-N1 and represent means from three to seven individual cultures ± SEM. Equal protein loading was verified by Ponceau S solution reversible staining of the blots.

Up-Regulation of Kv1.3/Kv1.5 Subunit Protein Expression and I_K Are RNA Synthesis-Dependent.

The RNA polymerase inhibitor α-amanitin (20 μg/ml) (18) prevented the mitogen-stimulated increase in Kv1.3 and Kv1.5 expression (Fig. 3 A and B). Similarly, no up-regulation of I_K was observed in the presence of α-amanitin (Fig. 3 C and D). RNA synthesis-inhibition in mitogen-treated OPs reduced Kv1.3/Kv1.5 protein expression and I_K density to levels comparable with those of OPs in G₀ (DME-N1 medium) (Fig. 3 A–D). α-Amanitin caused no change in I_K in G₀ cells (data not shown), suggesting that under quiescent conditions the tonic levels of K⁺ channel expression were not dependent on RNA synthesis. The effects of α-amanitin on Kv1.3/Kv1.5 and I_K up-regulation were not because of a failure of OPs to enter G₁ phase, as demonstrated by the increase in the levels of the G₁ phase marker, cyclin D (11, 19) (Fig. 3E). In OPs cultured with PDGF alone, cyclin D levels decreased between 18 and 24 h, whereas in OPs treated with α-amanitin cyclin D levels remained constant between 18 and 24 h (Fig. 3E), indicating that preventing Kv1.3 and Kv1.5 up-regulation caused cell cycle arrest in G₁ phase (11, 12).

Figure 3.

(A and B) Densitometric measurement of Western blot band (see Insets) intensity illustrates that Kv1.3 and Kv1.5 protein expression is significantly prevented (*P < 0.05) by α-amanitin (+A; 20 μg/ml) in the presence of PDGF (P) and bFGF (bF) to levels that are comparable with those seen in OPs in G₀ (i.e., cultured in DME-N1). Data were normalized with respect to cells cultured in DME-N1 and represent means from three to five individual cultures ± SEM. (C and D) α-Amanitin also significantly prevents (*, P < 0.05) the mitogen-induced up-regulation (12- or 24-h treatment) of I_K density in OPs. Data represent the means of the calculated I_K density elicited by a test pulse to 50 mV and are from 15–21 OPs from at least three individual cultures. (E) Inhibition of RNA synthesis does not prevent the PDGF-induced entry of cultured OPs into the cell cycle, as assessed by cyclin D expression. Samples were processed for Western blot analysis with anti-cyclin D antibody at varying time points following PDGF treatment. ctr, purified cyclin D protein. Equal protein loading was verified by Ponceau S solution reversible staining of the blots.

Kv1.3 and Kv1.5 Are Developmentally Regulated with Progression Through the Oligodendrocyte Lineage.

Immunocytochemical staining of oligodendrocyte lineage cells acutely isolated from tissue slices demonstrated that expression of Kv1.3 and Kv1.5 subunits was higher in NG2⁺ and O4⁺ progenitors than in mature O1⁺ oligodendrocytes (Fig. 4A). Furthermore, expression of the same subunits within the NG2⁺, O4⁺, and O1⁺ populations paralleled that of cyclin D (Fig. 4B). Immunoreactivity for Kv1.4 and Kv1.6 was low both in NG2⁺ and O4⁺ cells (data not shown). These data demonstrate that both Kv1.3 and Kv1.5 expression is down-regulated as OPs progress through the lineage to mature oligodendrocytes and is correlated with the proliferative potential of these cells.

Figure 4.

(A) Acutely isolated cells were immunostained with oligodendrocyte lineage antibodies and anti-Kv1.3 or anti-Kv1.5 antibodies. A significant decrease in Kv1.3 and Kv1.5 expression was observed in O4⁺ and O1⁺ cells, as compared with NG2⁺ cells (**, P < 0.01; ***, P < 0.001). Data shown are means ± SEM obtained from three independent experiments. The number of total cells counted for each antibody ranged between 226 and 700. (B) Acutely isolated cells were immunostained with NG2, O4, O1, and anti-cyclin D antibodies. A significant decrease in cyclin D expression was observed in O4⁺ and O1⁺ cells, as compared with NG2⁺ cells (***, P < 0.001). The data shown are means ± SEM obtained from of three independent experiments. The total number of cells counted ranged between 456 and 1,042.

OPs in Acutely Isolated Slices Display I_K That Are Partially Sensitive to Kv1.3 Block.

Whole-cell voltage–clamp recordings were performed from OPs located in the SVZ and in developing white matter of acutely isolated brain slices in rats aged P6–P11. To unequivocally identify these cells as OPs, both immunocytochemical and electrophysiological characterizations were performed. I_K was detected in NG2⁺ OPs (Fig. 5 A–C). These cells had a mean resting membrane potential of −42 ± 3 mV (cf. −50 ± 3 mV in cultured OPs) (9). In addition, 64% of the cells tested expressed I_A (Fig. 5C Bottom). This proportion was comparable to that seen previously in cultured OPs (6, 9, 20). The voltage dependence of I_K in OPs of the SVZ was composed of an overlap of two separate current components (Fig. 5D). The two current components displayed V_1/2 activation values of −23 ± 1.4 mV and 19 ± 2.4 mV, respectively (Fig. 5D), similar to those found in cultured OPs (9). The relative contribution of the two current components to the total K⁺ current was 40/60% in NG2⁺ cells in brain slices and 37/63% in cultured OPs (9). These data illustrate the striking similarity between the functional properties of I_K in NG2⁺ cells in brain slices and in cultured OPs.

Figure 5.

(A) A representative example of a biocytin-filled cell from which electrophysiological recordings were made. (B) Illustration that this cell (see arrow) was immunoreactive for NG2 (scale bars = 20 μM). (C) All cells expressed I_K (Middle) and, in some cases, I_A (64% of cells; n = 25, Bottom). Insets in top and middle panels illustrate voltage protocols used. (D) Activation curves for I_K illustrate the presence of two current components; data yielded a significantly better fit when described by the sum of two Boltzmann distributions (single Boltzmann vs. double Boltzmann fit; χ² values = 0.0013 ± 0.0001 vs. 0.0006 ± 0.00007; P < 0.001; n = 16). Data for the activation plots are expressed as means ± SEM from 25 cells. I_K was partially inhibited by either AgTx (50 nM) (E) or MgTx (50 nM) (F). Subsequent addition of TEA (10 mM) caused a further inhibition. Data are means ± SEM obtained from five to six cells. The traces in E and F are individual examples of I_K taken at the times indicated by the numbers.

To examine whether I_K expressed by NG2⁺ OPs in the SVZ/developing white matter contain or comprise Kv1.3 subunits, we studied the effects of the Kv1.3 channel blockers, r-agitoxin (AgTx) and r-margatoxin (MgTx), at concentrations shown to be selective for Kv1.3-containing channels (21–24). Both of these toxins only partially inhibited I_K in these cells (Fig. 5 E and F). Subsequent addition of tetraethylammonium (TEA) caused a further inhibition of I_K (Fig. 5 E and F), suggesting that not all I_K is attributable to channels containing or comprising Kv1.3.

Kv1.3 Block Inhibits Proliferation of NG2⁺ Cells in Tissue Slice Cultures and Prevent G₁/S Transition in Cultured OPs.

In brain tissue slice cultures, AgTx and MgTx significantly reduced proliferation of NG2⁺ OPs and O4⁺ preoligodendrocytes, as assessed by BrdUrd staining (Fig. 6A). Because it is not possible to easily determine the phase of the cell cycle at which Kv1.3 block affects cell proliferation in tissue slice cultures, we examined the effects of AgTx and MgTx on PDGF-induced proliferation of cultured OPs (Fig. 6B). These toxins also inhibited proliferation of cultured OPs (Fig. 6B) but did not affect PDGF-induced G₀/G₁ transition, as measured by cyclin D expression (Fig. 6C). These data suggest that blockade of currents through Kv1.3-containing channels inhibited cell cycle progression in the G₁ phase.

Figure 6.

(A) Cortical coronal slices were cultured for 48 h in the absence or presence of TEA (10 mM), r-agitoxin (AgTx; 50 nM), or r-margatoxin (MgTx; 50 nM) and incubated with BrdUrd for the last 24 h. Acutely isolated cell suspensions from these slices were immunostained with NG2 or O4 antibodies, together with anti-BrdUrd antibodies. All K⁺ channel blockers significantly inhibited cell proliferation in the NG2⁺ and O4⁺ cell populations (**, P < 0.01; ***, P < 0.001). Data shown are means ± SEM obtained from three to four experiments. Total number of NG2⁺ and O4⁺ cells counted for the different treatments ranged between 654 and 958. (B) Treatment of cultured OPs with AgTx or MgTx inhibits cell proliferation to the same extent as TEA. Data are means ± SEM of four to five independent experiments run in duplicate. The total number of cells counted for each treatment ranged between 2,843 and 3,616. (C) Time course of cyclin D expression following PDGF treatment of OPs in the absence or presence of r-agitoxin (AgTx; 5 and 50 nM) or r-margatoxin (MgTx; 5 and 50 nM).

Discussion

Here, we show that Kv1.3–Kv1.6 subunit protein expression is detectable in quiescent and proliferating OPs, consistent with previous observations demonstrating that OPs and mature oligodendrocytes express Kv1.3–Kv1.6 mRNA transcripts (20, 25). We demonstrate that transition of quiescent OPs into G₁ phase of the cell cycle is accompanied by a selective increase in Kv1.3 and Kv1.5 protein expression. This process is RNA synthesis-dependent and is accompanied by an up-regulation of I_K. Therefore, the insertion of newly transcribed and translated subunit proteins into the cell membrane produces the increase in I_K.

Changes in the expression levels of various channel proteins following alterations in the rate of mRNA translation are mainly governed by the protein turnover rate. The half-lives of other voltage-gated channels (Na⁺ and Ca²⁺) have been estimated to be 20–48 h (26, 27). Such long half-lives would not allow for the rapid changes in the mRNA synthesis-dependent up-regulation of K⁺ channel expression noted in this study. However, unlike voltage-gated Na⁺ and Ca⁺ channels, previous studies have indicated mechanisms that produce a dynamic regulation of Kv transcript levels (28). These temporal differences have been attributed to the relatively short half-lives of Kv mRNA and protein (35 min and 4 h, respectively) (29, 30). Importantly, these changes translated to a corresponding rapid increase in the functional expression of the voltage-gated K⁺ current (29, 30). Thus, it is clear that under certain conditions, K⁺ channel activity can be rapidly regulated under the control of mRNA transcription, and the time course of up-regulation noted in the present study is in agreement with previous observations.

A correlation between the cyclin E/cdk2-associated kinase activity and OP cell proliferation has been observed (14). Thus, cdk2-associated kinase activity is a reliable marker of the proliferative state of OPs. Here, we found that mitogen-induced alterations of I_K in OPs and cdk2 activity (14) follow a similar time course, strengthening the link between K⁺ channel expression and the proliferative state of OPs. Because K⁺ channel blockers inhibit cyclin E/cdk2 activity (14), one possible function of outward K⁺ channels is to sustain cdk2 activity in OPs, thus regulating G₁/S progression through a mechanism yet to be determined.

Nonselective block of K⁺ channels with TEA causes a failure in G₁/S transition (11). Here, we show that selective block of Kv1.3-containing channels alone with AgTx and MgTx is sufficient to also elicit G₁ arrest. Furthermore, the increase in both Kv1.3 subunit protein and I_K occurs during G₁ phase. These data illustrate that Kv1.3 up-regulation during G₁ is a prerequisite for G₁/S transition. In fact, Kv1.3 has been implicated in the proliferation of other cell types (24, 31, 32).

To extend these observations to a scenario that is closer to an in vivo situation, we demonstrate that I_K expressed by OPs in acutely isolated slices is partially sensitive to Kv1.3 block. These data show that OPs in an intact system express Kv1.3, and this subunit functionally contributes to I_K. Kv1.3 block with AgTx and MgTx also prevents OP cell proliferation in tissue slice cultures to a similar extent as TEA. This is observed despite a TEA-sensitive component of I_K in the presence of the Kv1.3-selective toxins in OPs from acutely isolated slices. Furthermore, in acutely isolated cells, a decrease in Kv1.3 expression occurs as OPs progress through the lineage and become less proliferative. These latter experiments strengthen the initial observations in cultured OPs and illustrate that the observations in this study are relevant to OPs in situ.

Unfortunately, we could not assess the role of Kv1.5 in G₁/S transition, as no selective pharmacological agents are available. In a previous study, treatment of OPs with Kv1.5 antisense oligonucleotides caused a reduction in Kv1.5 protein expression and I_K density (20), but no effect on proliferation was observed (20). Here, we show that Kv1.3 block prevents OP proliferation to the same extent as TEA, suggesting that this subunit exerts a functionally dominant role in G₁/S cell cycle progression. Furthermore, the concentration of TEA used in the cell proliferation assays was close to the IC₅₀ for Kv1.3-containing channels (10) and blocked 50% of outward K⁺ currents in the electrophysiological experiments.

In contrast, channels formed by Kv1.5 are insensitive to block by TEA (10). Therefore, the concentration of TEA used in the OP proliferation assays (10 mM) would be expected to exert no effects on Kv1.5-containing channels. Thus, although Kv1.5 subunit is up-regulated during G₁ phase of the cell cycle, it is conceivable that this subunit plays a relatively minor role when compared with Kv1.3 in OP G₁/S cell cycle progression (20). Conversely, treatment of astrocytes with Kv1.5 antisense oligonucleotides does lead to an inhibition of their proliferation (33). These data indicate that differing Kv subunits are involved in the proliferation of distinct glial cell types.

In conclusion, functional K⁺ channels comprising Kv1.3 are expressed in OPs in culture and in vivo. These channels are crucial for G₁/S transition and proliferation of OPs and thus play an important role in the processes leading to the generation of mature oligodendrocytes. Future studies are required to determine the intracellular targets that K⁺ channel expression impinges upon to modulate cell cycle progression.

Abbreviations

AgTx

r-agitoxin

bFGF

basic fibroblast growth factor

I_A

A-type transient outward K⁺ channel current

I_K

delayed outward-rectifying K⁺ channel current

MgTx

r-margatoxin

PDGF

platelet-derived growth factor

SVZ

subventricular zone

TEA

tetraethylammonium

OPs

oligodendrocyte precursor cells