Developmental Regulation of Calcium Channel-Mediated Currents in Retinal Glial (Müller) Cells

A. Bringmann, S. Schopf, and A. Reichenbach

Department of Neurophysiology, Paul Flechsig Institute of Brain Research, University of Leipzig, D-04109 Leipzig, Germany


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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Bringmann, A., S. Schopf, and A. Reichenbach. Developmental Regulation of Calcium Channel-Mediated Currents in Retinal Glial (Müller) Cells. J. Neurophysiol. 84: 2975-2983, 2000. Whole cell voltage-clamp recordings of freshly isolated cells were used to study changes in the currents through voltage-gated Ca2+ channels during the postnatal development of immature radial glial cells into Müller cells of the rabbit retina. Using Ba2+ or Ca2+ ions as charge carriers, currents through transient low-voltage-activated (LVA) Ca2+ channels were recorded in cells from early postnatal stages, with an activation threshold at -60 mV and a peak current at -25 mV. To increase the amplitude of currents through Ca2+ channels, Na+ ions were used as the main charge carriers, and currents were recorded in divalent cation-free bath solutions. Currents through transient LVA Ca2+ channels were found in all radial glial cells from retinae between postnatal days 2 and 37. The currents activated at potentials positive to -80 mV and displayed a maximum at -40 mV. The amplitude of LVA currents increased during the first postnatal week; after postnatal day 6, the amplitude remained virtually constant. The density of LVA currents was highest at early postnatal days (days 2-5: 13 pA/pF) and decreased to a stable, moderate level within the first three postnatal weeks (3 pA/pF). A significant expression of currents through sustained, high-voltage-activated Ca2+ channels was found after the third postnatal week in ~25% of the investigated cells. The early and sole expression of transient currents at high-density may suggest that LVA Ca2+ channels are involved in early developmental processes of rabbit Müller cells.


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INTRODUCTION
METHODS
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DISCUSSION
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Müller (radial glial) cells are the principal type of glial cells in the mammalian retina. One of the main functions of Müller cells is the spatial buffering of the extracellular K+ concentration (Newman and Reichenbach 1996; Newman et al. 1984). This function is mediated by the prominent K+ permeability of Müller cell membranes, in particular, by inwardly rectifying K+ channels (Brew et al. 1986; Newman 1993). In addition to the dominant expression of K+ channels, Müller cells may express other ion channels in their membranes; among them are voltage-gated Na+ (Chao et al. 1994; Francke et al. 1996) and Ca2+ channels (Newman 1985). In cultured human Müller cells, the presence of distinct types of voltage-gated Ca2+ channels has been described (Puro and Mano 1991; Puro et al. 1996). The functional roles of Na+ and Ca2+ channels in Müller cells are still unclear, although sparse previous data indicated their involvement in transdifferentiation and proliferative processes in cases of retinal pathobiology. Voltage-gated Na+ currents, for example, were found to be increased in their amplitude in human Müller cells obtained from patients with various eye diseases (Francke et al. 1996). L-type Ca2+ channels, on the other hand, have been implicated in the regulation of the proliferative activity of cultured Müller cells (Puro and Mano 1991; Uchihori and Puro 1991).

There is convincing evidence that, generally, glial cells may express voltage-gated Ca2+ channels of different distinct types (Sontheimer 1994; Steinhäuser 1993). Whereas the postnatal development of Ca2+ channel expression was extensively studied in neurons (for example, McCobb et al. 1989; Pirchio et al. 1990; Thompson and Wong 1989; Yaari et al. 1987), only a few data are available about developmental changes of voltage-gated Ca2+ channels in glial cells. Cultured glial precursor cells of the oligodendrocyte lineage express two types of Ca2+ currents that can be distinguished by the voltage dependence of activation: low- and high-voltage-activated Ca2+ currents (LVA and HVA currents) (Verkhratsky et al. 1990). Glioblasts and oligodendrocytes of the murine corpus callosum express Na+ and Ca2+ currents during early postnatal stages but not in the differentiated stage (Berger et al. 1992). In neurons, T-type LVA Ca2+ channels are predominantly expressed at early embryonic or neonatal stages when they generate spontaneous Ca2+ transients necessary for morphogenesis (Gu and Spitzer 1993); thereafter, their disappearance is accompanied by an increase in HVA Ca2+ currents (Carbone and Lux 1984; Kostyuk et al. 1993; McCobb et al. 1989; Tarasenko et al. 1998). In certain thalamic and hypothalamic neurons, however, T-type LVA Ca2+ channels play an important role in the organization of slow rhythmic activity in adult animals (Akaike et al. 1989; Huguenard and Prince 1992).

The first aim of the present study was to detect voltage-gated Ca2+ currents in freshly isolated Müller cells (and/or their precursors) of the rabbit. Using Ba2+ or Ca2+ ions as charge carriers, transient (T-type) LVA currents were found in cells from young postnatal animals. The second aim was to determine whether the expression level of Ca2+ channels in Müller cells is developmentally regulated. If the Ca2+ channels are implicated in the proliferative activity of Müller cells (Puro and Mano 1991; Uchihori and Puro 1991), one may assume that the activity of Ca2+ channels decreases during the postnatal differentiation of mitotically active precursor cells, via immature radial glial cells, into mature Müller cells. Since the amplitudes of the Ba2+ and Ca2+ currents were very small, it was advantageous to maximize the currents through voltage-gated Ca2+ channels. For this purpose, their developmental regulation was investigated by recording currents of monovalent cations. As previously described (Almers and McCleskey 1984; Hess and Tsien 1984; Kostyuk et al. 1983; Lux et al. 1989, 1990), voltage-gated Ca2+ channels mediate currents of monovalent ions when divalent cations are largely absent in the extracellular solution.


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Cell isolation and identification

Rabbits were kept in the animal center of the Leipzig University Medical School. Animals were deeply anesthetized by urethan (2.0 g/kg) before decapitation and enucleation of the eye balls. Isolated retinae were incubated at 37°C for 30 min in Ca2+- and Mg2+-free phosphate-buffered saline (PBS), pH 7.4, which contained papain (15 U/ml; Boehringer, Mannheim, Germany) and desoxyribonuclease I (200 U/ml; Sigma, Deisenhofen, Germany). After washing in PBS, the tissue was titrated by a wide-pore pipette. The resulting suspensions of dissociated singular cells were stored at 4-8°C in serum free modified Eagle's medium (Sigma) until use within 3 h after isolation.

Müller cells were identified by their unique bipolar morphology and size (>100 µm length). While this discriminated them unequivocally from bipolar neurons and photoreceptor cells (which may roughly resemble immature Müller cells in shape but are much shorter), it was impossible to distinguish immature Müller cells from late progenitor cells that are still present during the first few postnatal days. The latter, producing both glial (Müller) and neuronal (bipolar and photoreceptor cells) by their last division, share the same set of antigenic molecules with the Müller cells (Reichenbach 1993 and references therein) and cannot be distinctly labeled by any known antibody or marker. Thus both (a majority of) immature Müller cells and (less and less) late progenitor cells may have been grouped together between postnatal days (P) 2 and 6. However, as there seems to be a gradual rather than a clear-cut transition between the two cell types (cf. Bringmann et al. 1999), this was not considered as a major obstacle of our study.

Electrophysiological recordings

Whole cell voltage-clamp currents (Hamill et al. 1981) were measured using a List EPC-7 amplifier (List Electronics, Darmstadt, Germany) and the TIDA 5.72 computer program (HEKA elektronik, Lambrecht, Germany). High frequencies >4 kHz were cutoff. The series resistance (13-16 MOmega ) was compensated by 30-50%. Records were made at room temperature (22-25°C). Patch electrodes of 4-7 MOmega resistance were pulled from borosilicate glass (GB150F8P, Biologic, Science Products, Frankfurt/M., Germany). The Ca2+ channel-mediated currents were evoked by a standard step protocols (Vh, -80 mV; depolarizing voltage steps from a 500-ms prepulse). For LVA current activation, depolarizing voltage steps were applied to voltages between -100 and +20 mV with an increment of 10 mV, after a prepulse to -120 mV. For HVA current activation, voltage steps were applied to voltages between -100 and +20 mV, after a prepulse to -60 mV. To assess LVA current inactivation, voltage steps to -50 mV were applied after prepulses to different potentials (between -120 and 0 mV; increment, 10 mV). For HVA current inactivation, voltage steps to -20 mV were applied after prepulses to potentials between -80 and +40 mV (increment, 10 mV). The traces were not leak subtracted. Leak currents were subtracted when I-V curves were calculated. Data were not corrected for liquid junction potentials since they did not exceed 3 mV. The membrane capacitance of the cells was measured by integrating the uncompensated capacitive artifact evoked by a hyperpolarizing voltage step from -80 to -90 mV. For recording the capacitive artifact, the sampling rate was 30 kHz, and the frequencies 10 kHz were cutoff. Measurements of time-dependent changes of the Ca2+ channel-mediated currents indicated that, after disruption of the membrane, dialysis of the cell interior was completed within 3 min, and, thereafter, current amplitudes remained stable for ~10 min. Thus the "run-down" of the Ca2+ channel-mediated currents was considered to be negligible within this period of time.

Solutions

The pipette solution consisted of (in mM) 10 NaCl, 130 CsCl, 1 CaCl2, 1 MgCl2, 10 ethyleneglycolbis(aminoethyl)-(ether)tetra-acetate (EGTA), and 10 N-2-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid (HEPES). The pH of 7.2 was adjusted with Tris-base. The Ba2+ currents were recorded with a bath solution containing (in mM) 20 BaCl2, 130 CsCl, 5 HEPES, and 10 glucose (pH 7.4 adjusted with Tris-base). The Ca2+ currents were recorded using a bath solution that consisted of (in mM) 105 NaCl, 10 CaCl2, 1 MgCl2, 10 HEPES, and 11 glucose (pH 7.4). To increase the amplitude of the currents through Ca2+ channels, currents of monovalent cations in divalent cation-free bath solutions were recorded. The bath solution consisted of (in mM) 113 NaCl, 10 HEPES, 11 glucose, and 1 EGTA (pH 7.4). To obtain Na+-free solution, Na+ was equimolarly replaced by choline+. To investigate the Ca2+ dependence of the Na+ currents, the control data were obtained in Ca2+- and Mg2+-free solution containing 1 mM EGTA. The solutions with 0.1, 1, 5, and 10 µM free Ca2+ were made by adding 0.625, 0.944, 0.993, and 1.004 mM CaCl2, respectively, to the control solution. The effect of other divalent cations was tested in solutions without EGTA.

Substances

Nimodipine and flunarizine were from Calbiochem (Bad Soden, Germany). Tetrodotoxin was obtained from Alomone Labs (Jerusalem, Israel). All other substances were from Sigma. Lipophilic drugs were dissolved in dimethylsulphoxide. Vehicle controls were prepared as above without addition of the drug. Drugs were applied by changing the perfusate within the recording chamber.

Data presentation

Single-channel currents depicted as negative (downward deflections) represent cation fluxes from the extra- into the intracellular compartment. Amplitude histograms of single-channel currents were established by means of the TIDA 5.72 computer program. Single-channel currents were evaluated from the current steps between peaks of the amplitude histograms. Statistical analysis (regression analysis, Bonferroni corrected P values using ANOVA) and curve fits were made using the Prism program (Graphpad Software, San Diego, CA). Data are expressed as means ± SD.


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Ba2+ and Ca2+ currents

To isolate Ba2+ currents through Ca2+ channels in Müller cells of young rabbits [postnatal days (P) 10-11], voltage step protocols were applied using 20 mM extracellular Ba2+ as the charge carrier, in K+-free bathing and pipette solutions. Under these conditions, a depolarization-activated inward current was found to be present in the whole cell records (Fig. 1A). The mean peak amplitude of this current was small, with 10.6 ± 4.5 pA (n = 14). The current activated at potentials positive to -60 mV; the peak was at -22.9 ± 7.5 mV (n = 32; Fig. 1B). The inward current was completely blocked by exposure to extracellular Cd2+ (1 mM; n = 9) and is, therefore assumed to be mediated by Ca2+ channels. The Ba2+ currents showed transient time-dependent activation kinetics (Fig. 1A) similar to the T-type currents previously described in cultured human Müller cells (Puro and Mano 1991). Moreover the relatively low activation threshold is indicative of currents through LVA Ca2+ channels. Transient inwardly directed currents were also observed when Ca2+ ions (10 mM) were used as the charge carrier (Fig. 1C). The Ca2+ currents activated at potentials positive to -60 mV, peaked at -28.1 ± 8.8 mV (n = 5) and were blocked by external Cd2+ ions (1 mM). Again, the peak amplitude of the Ca2+ currents was small, with 8.2 ± 3.2 pA (n = 5).



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Fig. 1. Freshly isolated rabbit Müller cells display inwardly directed currents through voltage-gated Ca2+ channels of small amplitude when Ba2+ ions (20 mM) or Ca2+ ions (10 mM) are used as the charge carrier. A: Ba2+ currents evoked in a cell from a P10 rabbit. B: current-voltage curve of the peak Ba2+ currents from the example shown in A. C: mean current-voltage curve of the peak Ca2+ currents evoked in 5 cells from a P11 rabbit.

Since the amplitudes of the Ca2+ channel-mediated currents were very small when Ba2+ or Ca2+ ions were used in the extracellular solution, we used Na+ ions as the main charge carrier in all further experiments to increase the amplitude of the currents flowing through Ca2+ channels. Therefore bath solutions without divalent cations were used.

Na+ currents through Ca2+ channels

Using Ca2+-, Mg2+-, and K+-free bath solutions, large inwardly directed currents were evoked by depolarizing voltage steps. Figure 2A shows records in a cell from a P24 rabbit. The voltage-gated, inwardly directed currents were strongly diminished in their amplitudes when Ca2+ and Mg2+ ions were present in the bath solution at physiological concentrations (2 and 1 mM, respectively; middle). The amplitude of the inwardly directed currents recovered after a washout of the divalent cations (right). The inwardly directed currents recorded in divalent cation-free bath solution were mediated by external Na+ ions because they were absent when the bath solution was Na+ free (Fig. 2B).



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Fig. 2. Na+ currents through transient Ca2+ channels in Müller cells from young rabbits. A: example of whole cell records in a cell from a P24 rabbit. The currents were evoked from different prepotentials to a test potential of -50 mV in K+-free solutions. In Ca2+, Mg2+-free bath solution, inwardly directed currents could be observed (left). Addition of Ca2+ (2 mM) and Mg2+ ions (1 mM) to the bath solution strongly decreased the amplitude of the inwardly directed currents (middle). The amplitude increased again when the divalent cations were omitted from the bath solution (right). B: records in a cell from a P19 rabbit. The currents were evoked in Ca2+-, Mg2+-, and K+-free bath solutions from a prepulse to -120 mV. While in Na+-containing bath solution, depolarizing voltage steps evoked transient currents (left), these inwardly directed currents were fully absent in Na+-free bath solution (middle). Right: the difference between both records, representing the currents depending on extracellular Na+. C: the transient Na+ current was sensitive to flunarizine. Example of records in a cell from a P16 rabbit. The currents were evoked by a voltage step from -120 to -50 mV before and during extracellular exposure to the T-type Ca2+ channel blocker flunarizine (5 µM). D: dose-dependent relations for the block of the peak transient Na+ currents by flunarizine and nimodipine. The points represent the average of 3-6 experiments. |, SD. The curves were fitted with f = (b*x)/(x + a) with a = 0.6 µM (flunarizine) or 3.6 µM (nimodipine), and b = 100%. E: the transient Na+ currents were not diminished during exposure to tetrodotoxin (10 µM). Mean peak current-voltage curves of 3 cells from P17 to P20 rabbits are shown.

Because the activation kinetics of the inwardly directed currents were transient (Fig. 2), we assumed that these currents represent Na+ fluxes through transient (T-type) Ca2+ channels. Therefore we tested whether blockers of voltage-gated Ca2+ channels may affect the currents. As shown in Fig. 2C, the T-type channel blocker flunarizine (at a concentration of 5 µM) almost completely blocked the inwardly directed currents. The dose-dependence of the peak current inhibition revealed that 0.6 µM flunarizine inhibited 50% of the transient currents (Fig. 2D). Nimodipine also blocked this current, although at significant higher concentrations (IC50 = 3.6 µM). A blocking effect of nimodipine (10 µM) was also observed on the Ba2+ currents through LVA Ca2+ channels (not shown). Tetrodotoxin, a blocker of fast Na+ channels, did not inhibit the transient Na+ currents (n = 9; Fig. 2E). As indicated by both the sensitivity to Ca2+ channel blockers and the insensitivity to tetrodotoxin, the transient Na+ currents are mediated by T-type Ca2+ channels.

The Na+ currents through Ca2+ channels were blocked by extracellular divalent cations (Fig. 2A). Ca2+ ions decreased the amplitude of the transient currents in the low micromolar range, in a dose-dependent manner (Fig. 3A). The current inhibition by external Ca2+ ions was prominent over the entire voltage range with slightly greater inhibiting effects at more depolarized voltages (Fig. 3B). The dose dependences of the LVA-current block by various divalent cations are illustrated in Fig. 3C. The peak current was reduced to 50% by 0.6 µM Cd2+, by 0.7 µM Ca2+, by 1.7 µM Ni2+, by 3.0 µM Cu2+, by 31.4 µM Ba2+, and by 35.9 µM Mg2+.



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Fig. 3. The Na+ currents through low-voltage-activated (LVA) Ca2+ channels are inhibited by external divalent cations. A: example of the inhibition of the LVA current by increasing external Ca2+ recorded in a Müller cell from a P10 rabbit. The currents were evoked by voltage steps to -50 mV after prepulses to -120 mV. The control record was made in K+-, Ca2+-, and Mg2+-free bath solution containing 1 mM EGTA. B: mean current-voltage relations of the peak current densities obtained in 3 cells from P10 rabbits. C: concentration-dependent divalent cation-mediated inhibition of the peak LVA current. The points represent the average of 3-6 experiments. |, SD. The curves were fitted with f = (b*x)/(x + a) with a = 0.7 µM (Ca2+), 1.7 µM (Ni2+), and 0.6 µM (Cd2+), respectively, and b = 100%.

Although the vast majority of cells expressed only currents through transient LVA channels (623 out of 653 investigated cells), cells from rabbits older than two weeks also showed currents through sustained, long-lasting HVA channels. In three of five cells from adult animals, both LVA and HVA currents were found. However, the amplitudes of the HVA currents were regularly very small, as compared with the amplitudes of the LVA currents. An example of records in a cell expressing both types of currents is shown in Fig. 4A. The two components of currents were separated by a variation of the prepulses from -120 to -60 mV. Depolarizing voltage steps after prepulses to -120 mV evoked both current components, while the transient component was inactivated after prepulses to -60 mV. The difference between both records represents the transient component. Figure 4B shows the mean peak current-voltage curves for the sum (prepulse to -120 mV), for the HVA (prepulse to -60 mV), and for the LVA currents (difference) in four cells. The transient component had an activation threshold of -82.0 ± 5.4 mV and peaked at -43.5 ± 3.9 mV, while the noninactivating, sustained current component activated at -52.7 ± 6.4 mV and showed a maximum at -12.0 ± 5.1 mV. According to their voltage dependence, these two types of currents could be attributed to LVA and to HVA Ca2+ currents, respectively. The LVA currents gated ~20 mV more negative in divalent cation-free solutions when compared with Ba2+- or to Ca2+-containing solutions (Fig. 1, B and C) due to alterations of the membrane surface charges. The currents of monovalent cations through Ca2+ channels reversed between +25 and +30 mV (Fig. 4B). The true reversal potential of these currents was not determined since omitting the divalent cations from the bath solution increased the "leak" conductance of the records, possibly reflecting the opening of unspecific cation channels that were normally blocked by divalent cations (not shown). The flow of cations through these channels would shift the reversal potential of the Ca2+ channel-mediated currents toward more negative values. When the Cs+ ions within the pipette solution were equimolarly replaced by N-methyl-D-glucamine, no shift of the reversal potential was observed, indicating that the reversal potential was not influenced by a possible flow of Cs+ ions through K+ channels.



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Fig. 4. Rabbit Müller cells may express different types of Ca2+ channel-mediated currents. A: example of whole cell records in 1 cell from a P29 rabbit that expressed both types of currents (left). Depolarizing voltage steps after prepulses to -60 mV evoked only the sustained current component (middle). The difference between both traces shows the transient current component (right). B: average peak current-voltage curves of the sum currents (prepulses to -120 mV), of the sustained high-voltage-activated (HVA) currents (prepulses to -60 mV), and of the transient LVA currents calculated as difference. Means of 4 cells. C: voltage dependence of the steady-state inactivation and activation of the transient LVA currents and of the sustained HVA currents.

The voltage dependences of the steady-state inactivation and activation of both components of monovalent cation currents are illustrated in Fig. 4C. For establishing activation curves, the current amplitudes at different potentials were transformed into conductances (g) using a reversal potential of the currents of +25 mV based on the data shown in Fig. 4B. Conductances were then normalized to the maximum conductance and fitted by a Boltzmann relation. The voltages at which half-maximal activation occurred were -63.3 ± 6.3 mV for the transient LVA current and -29.7 ± 5.2 mV for the sustained HVA current. The slopes characterizing the voltage sensitivity of the channels were comparable for the activation of the LVA current (4.4 ± 1.8 mV/e-fold change) and of the HVA current (5.6 ± 1.6 mV/e-fold change). To record the steady-state inactivation, the peak currents at different prepotentials (V) were normalized to the peak currents recorded from V = -120 mV (LVA) and from V = -80 mV (HVA), respectively (Fig. 4C, open circle ). The fractional currents, normalized to the maximum currents, were fitted with a Boltzmann equation. The voltages at which half-maximal inactivation occurred were -88.4 ± 6.1 mV (LVA) and -39.2 ± 3.5 mV (HVA). The slopes of the inactivation curves were -5.4 ± 1.5 mV for the LVA current and -8.3 ± 3.2 mV for the HVA current. The "window currents," representing the overlap of the activation and inactivation curves, were significantly different and ranged from about -90 to -60 mV in the case of the transient LVA currents and from about -60 to -20 mV in the case of the sustained HVA currents. Despite the difficulty to determine the reversal potential of the currents of monovalent cations through voltage-gated Ca2+ channels (see preceding text), the voltage range of LVA current activation and inactivation was well within the range previously reported for these currents in other cell types (Lux et al. 1989, 1990).

Cells from early postnatal stages had a rather small cell membrane area and the amplitude of the Ca2+ channel-mediated currents was small (see following text), indicating that they may express only few Ca2+ channels in their membranes assuming that no developmental alterations of the channel conductance occurred. In some of these cells, it was possible to record single-channel activity in the whole cell mode. Examples of such records in cells from P3 and P4 rabbits are shown in Fig. 5A, at different depolarizing steps from a prepulse to -120 mV. Channel openings as well as time-dependent inactivation increased at stronger depolarizations. Figure 5B shows the mean current-voltage relation of single-channel currents in six cells. The mean slope conductance was 16.2 pS; the extrapolated reversal potential was at about +30 mV.



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Fig. 5. In some radial glial cells from early postnatal stages, it was possible to record single-channel activity in the whole cell mode. A: examples of records in cells from a P3 and from a P4 rabbit. From a prepulse to -120 mV, the cells were depolarized to various test potentials. left-arrow , the closed-state level of the channels. B: mean current-voltage relation of single channel currents recorded in 6 cells.

Developmental changes of Ca2+ channel-mediated currents

In the developing rabbit retina, proliferative activity is present up to about P7 (Germer et al. 1997; Reichenbach et al. 1991). Thus within the first postnatal week, postmitotic radial glial cells develop from mitotically active progenitors. This was reflected by an increasing number of cells in the cell suspensions that displayed a unique bipolar morphology, involving a so-called endfoot at the end of one stem process, and a length of >100 µM (Bringmann et al. 1999). Between P6 and P20, radial glial cells differentiate into Müller cells as indicated by the enhanced expression of inwardly rectifying K+ currents, the main membrane current expressed by adult cells (Bringmann et al. 1999). Figure 6A (left) illustrates whole-cell records of two typical cells that were found in the cell suspension derived from a retina of a P2 rabbit. The records were made in Ca2+-, Mg2+-, and K+-free bath solution; only depolarization-induced inwardly directed Na+ currents are shown. While the ganglion cell displayed three different Na+ currents (through fast Na+ channels and through both transient LVA and sustained HVA channels), the radial glial cell displayed only currents through transient LVA Ca2+ channels. The expression of fast Na+ currents and different types of Ca2+ currents in early postnatal ganglion cells was previously described for the rat retina (Schmid and Guenther 1996). In the further course of development, the amplitude of the transient LVA currents in radial glial cells increased, as shown by the example of the record in a cell from a P29 rabbit (Fig. 6A, right).



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Fig. 6. Developmental regulation of LVA Ca2+ channel-mediated Na+ currents in radial glial/Müller cells from rabbits between postnatal days 2 and 37. A: records of inwardly directed Na+ currents in a ganglion cell and in a radial glial cell from the retina of a P2 rabbit (left). Right: currents in a Müller cell from a P29 rabbit. Note the different current scalings. B: mean cell membrane capacitance as a function of the postnatal age. C: amplitude of the peak LVA currents in dependence on the postnatal age. Each point represents the mean of 3-53 cells. D: density of the peak LVA currents in dependence on the postnatal age. E: mean peak density-voltage curves of the sum currents recorded in cells from rabbits of various postnatal stages.

During the maturation of Müller cells, the cell membrane capacitance increased (Fig. 6B) (Bringmann et al. 1999). The mean membrane capacitance of the radial glial cells was small between days 2 and 4 (5.1 ± 2.3 pF, n = 38) and increased to 47.4 ± 12.4 pF after day 30 (n = 36; P < 0.001, ANOVA).

All investigated radial glial/Müller cells from rabbit retinae between P2 and P37 (n = 647) expressed currents through transient LVA Ca2+ channels. The amplitude of the LVA currents was found to increase during the postnatal development. Figure 6C illustrates the mean amplitudes of the peak LVA currents in dependence on the postnatal age. Between P2 and P7, the peak amplitude of the LVA currents increased from 45.3 ± 22.7 pA at day 2 to 145.9 ± 99.6 pA at day 7 in correlation to both the postnatal age (r = 0.44, n = 111 cells, P < 0.001) and to the cell membrane capacitance (r = 0.64, P < 0.001). After day 6, the peak amplitude of the LVA current remained largely constant (130.5 ± 86.7 pA, n = 552 cells from P7 to P37), indicating that the number of LVA Ca2+ channels per radial glial cell did not significantly change after the first postnatal week when the channel conductance remained unaltered during development. On the other hand, the peak density of the LVA currents was found to be maximal at early postnatal stages (Fig. 6D). Between days 2 and 5, the mean density was 12.9 ± 7.6 pA/pF (n = 61); thereafter, the density decreased to a mean of 2.6 ± 1.7 pA/pF after day 18 (n = 163; P < 0.001). Between days 3 and 19, the peak current density decreased in correlation to both the postnatal age (r = -0.55, n = 492, P < 0.001) and the cell membrane capacitance (r = -0.46, P < 0.001). After day 18, the density remained constant; no correlations were found to the postnatal age or to the membrane capacitance. In cells from adult animals, the LVA currents displayed a peak density of 2.0 ± 0.7 pA/pF (n = 6). The postnatal decrease of the LVA current density was not accompanied by significant changes of the steady-state current inactivation and activation (not shown) nor by changes of the current density-voltage curves (Fig. 6E).

Currents through HVA channels were scarcely observed in cells from the first three postnatal weeks. With the exception of two cells from a P12 and a P14 rabbit, all other investigated cells from rabbits up to P23 (n = 524) expressed only LVA currents. After the first three postnatal weeks, a subpopulation of the cells displayed both LVA and HVA currents. In cells that displayed both current types, the density of the HVA currents was always smaller than that of the LVA currents (Fig. 4B). In 118 investigated cells from P24 to P37 rabbits, 25 cells (21%) expressed both current types. In these cells, the peak HVA current displayed a mean density of 0.6 ± 0.5 pA/pF while the peak LVA current had a mean density of 3.3 ± 3.1 pA/pF. An age-dependent increase of the incidence of cells that expressed both current types was not observed between P24 (28%) and P37 (20%).


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In cultured human Müller cells, using Ba2+ ions as the charge carrier, the presence of transient LVA and of sustained HVA currents was previously described (Puro and Mano 1991; Puro et al. 1996). Here, we demonstrate that freshly isolated Müller cells of the rabbit retina may express both types of Ca2+ channel-mediated currents with significantly different expression patterns during the postnatal development. The inwardly directed Ba2+ and Ca2+ currents in cells from young postnatal age activated at potentials positive to -60 mV, peaked at about -25 mV and displayed a transient time-dependent activation kinetics (Fig. 1). These features are consistent with the expression of T-type Ca2+ channels. However, since the peak amplitudes of the Ba2+ and Ca2+ currents were small, the postnatal development of Ca2+ channel expression was investigated using Na+ ions as the main charge carrier in divalent cation-free external solution. The increased amplitude of Na+ currents through T-type Ca2+ channels may be partly explained by the greater single-channel conductance (16 pS) as compared with Ba2+ or to Ca2+ currents through the same channels (5-9 pS) (for a review, see Ertel et al. 1997). The omission of the divalent cations from the bath solution caused a negative shift of the activation kinetics of the T-type channels by ~20 mV when compared with Ba2+ currents via alteration of the membrane surface charges. The transient Na+ currents were blocked in the low micromolar range by various divalent cations (Fig. 3). The values of half-maximal inactivation of the Na+ currents through Ca2+ channels by Ca2+ (0.7 µM) and Mg2+ ions (35.9 µM) are in the range previously described for neuronal Ca2+ channels (0.7 and 39 µM, respectively) (Carbone et al. 1997).

The transient Na+ currents described in this paper were not mediated by fast transient Na+ channels, for the following reasons: although Müller cells from different mammalian species, including man, express fast transient Na+ channels (Chao et al. 1994; Francke et al. 1996), rabbit Müller cells were never shown to express this channel type; currents mediated by fast transient Na+ channels in human cells displayed a significantly faster activation and inactivation kinetics as compared with the transient Na+ currents described in the present paper; and the Na+ channel-mediated currents in human cells were largely reduced in their amplitudes by tetrodotoxin (10 µM) (Francke et al. 1996) while the transient Na+ currents in rabbit Müller cells were insensitive to tetrodotoxin. The inwardly directed transient Na+ currents in young postnatal rabbit Müller cells were blocked by low micromolar concentrations of the T-type channel blocker flunarizine while the L-type channel blocker nimodipine depressed the current amplitude with an IC50 value significantly larger than flunarizine (Fig. 2D). Dihydropyridine-sensitive LVA Ca2+ channels were previously described to be present in various neuronal and glial cell preparations (Akaike et al. 1989; Akopian et al. 1996; Koike et al. 1993; Takahashi and Akaike 1991).

From the above-mentioned data, it can be concluded that rabbit retinal Müller (glial) cells may express both LVA and HVA type Ca2+ channels. These two types of Ca2+ channel-mediated currents displayed a significantly different developmental regulation during the postnatal differentiation of immature precursor cells into mature Müller cells, i.e., during the first three postnatal weeks (Bringmann et al. 1999). While the transient currents were early expressed at high densities in developing cells, their density fell to a stable moderate level in the course of further differentiation into mature Müller cells (Fig. 6D). When the channel conductance did not change during development, the data indicate that the number of LVA channels per cell increases within the first postnatal week but remains constant thereafter (Fig. 6C). As a result, the density of the LVA currents decreases along with the increasing cell membrane area after the first postnatal week. While all investigated rabbit cells from all developmental stages displayed transient LVA currents, HVA currents were expressed only at later developmental stages. The different expression patterns probably indicate that the two currents have different functional roles in Müller cells.

There are two possible functions of the early postnatal expression of LVA currents at high-density. First, the activity of LVA channels may be necessary for the precursor proliferation that occurs in the rabbit retina up to postnatal day 7 (Germer et al. 1997). According to this idea, the postnatal decrease of the density would reflect the cessation of retinal proliferation. This assumption is supported by the fact that proliferation of cultured Müller cells is dependent on the activity of voltage-gated Ca2+ channels. In cultured Müller cells of the guinea pig, the proliferation induced by epidermal growth factor was found to be blocked by flunarizine and by nimodipine, with flunarizine being the more effective substance (own unpublished results). Second, LVA channels may have functional roles in early differentiation processes of retinal radial glial cells. In neuronal development, T-type Ca2+ channels were implicated in the regulation of the morphogenesis and of the circuit specification via mediating spontaneous Ca2+ transients (Gu and Spitzer 1993) and autorhythmic oscillatory activity, respectively (Bertolino and Llinas 1992). Waves of synchronous bursting activity were described to occur in the immature retina (Meister et al. 1991; Wong et al. 1992; Zhou 1998), and it may be possible that immature radial glial cells are involved in this oscillatory electrical activity, which may enhance the activity of voltage-gated Ca2+ channels in these cells. LVA channels may mediate the Ca2+ entry into developing radial glial cells, an event that, in turn, may trigger intracellular events necessary for cell differentiation such as for the outgrowth of glial side branches. Further investigations, however, are necessary to determine whether the activity of LVA channels contribute to the morphogenesis of Müller cells. On the other hand, the expression of both LVA and HVA currents in cells from adult animals indicates that voltage-gated Ca2+ channels may also have, yet unknown, functions in mature Müller cells.

The Ca2+ channel-mediated currents develop in relation to various types of K+ currents in Müller cells. Figure 7 summarizes the densities and activities, respectively, of distinct membrane conductances during the postnatal development from late retinal progenitor cells into radial glial, and into immature and mature Müller cells, respectively (for staging of the postnatal radial glia development, see Bringmann et al. 1999). Developmental alterations of distinct radial glia membrane conductances occurred partly in relation to different markers of the retinal activity; for example, the amplitude of the LVA currents increased up to about day 6, then, the density of the inwardly rectifying K+ currents begins to increase, presumably along with the light-induced ganglion cell activity. The main indicator of differentiation of immature radial glial cells into mature Müller cells is the strong up-regulation of the density of inwardly rectifying K+ currents that occurs between postnatal days 6 and 20 that may mainly underlie the hyperpolarization of the Müller cell membrane observed during postnatal maturation (Bringmann et al. 1999). The hyperpolarization of the cell membrane may cause a decrease of the opening probability of depolarization-activated channels. Indeed a developmental decrease of the activity of Ca2+-activated K+ channels of big conductance (BK) was previously described (Bringmann et al. 1999). It was suggested that both BK and voltage-gated Ca2+ channels may work together to enhance the Ca2+ entry from the extracellular space after receptor activation, for example, when both channel types would be co-localized in Müller cell membranes (Bringmann et al. 2000). The stabilization of the membrane potential at hyperpolarized values should also decrease the activity of LVA Ca2+ channels; a decreased LVA channel-mediated Ca2+ influx may contribute to the observed decrease in the activity of BK channels.



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Fig. 7. Developmental regulation of LVA channel-mediated currents in relation to changes of K+ conductances in retinal radial glial/Müller cells and to distinct markers of retinal development, respectively. The postnatal development of the K+ conductances was described previously (Bringmann et al. 1999). Following markers of retinal development were adapted from the literature: cell proliferation within the rabbit retina (Germer et al. 1997; Reichenbach et al. 1991), light-induced ganglion cell activity (Masland 1977), the density of ribbon synapses in the inner plexiform layer (McArdle et al. 1977), and the electroretinogram (ERG) b-wave amplitude (Noell 1958). All data are given as mean relative values. Big conductance (BK), Ca2+-activated K+ channels of big conductance; KIR, inwardly rectifying K+ currents; KA, transient (A-type) K+ currents.


    ACKNOWLEDGMENTS

This study was supported by grants from the Bundesministerium für Bildung, Forschung und Technologie (BMBF), Interdisciplinary Center for Clinical Research at the University of Leipzig (01KS9504, Project C5) and from the Deutsche Forschungsgemeinschaft (Bonn, Germany; Re 849/8-1).


    FOOTNOTES

Address for reprint requests: A. Bringmann, University of Leipzig, Paul Flechsig Institute of Brain Research, Dept. of Neurophysiology, Jahnallee 59, D-04109 Leipzig, Germany (E-mail: bria{at}server3.medizin.uni-leipzig.de).

Received 27 April 2000; accepted in final form 6 September 2000.


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ABSTRACT
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DISCUSSION
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