Differential Expression of High- and Two Types of Low-Voltage-Activated Calcium Currents in Rod and Cone Bipolar Cells of the Rat Retina

Zhuo-Hua Pan

Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan 48201


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pan, Zhuo-Hua. Differential Expression of High- and Two Types of Low-Voltage-Activated Calcium Currents in Rod and Cone Bipolar Cells of the Rat Retina. J. Neurophysiol. 83: 513-527, 2000. Whole cell voltage-clamp recordings were performed to investigate voltage-activated Ca2+ currents in acutely isolated retinal bipolar cells of rats. Two groups of morphologically different bipolar cells were observed. Bipolar cells of the first group, which represent the majority of isolated bipolar cells, were immunoreactive to protein kinase C (PKC) and, therefore likely to be rod bipolar cells. Bipolar cells of the second group, which represent only a small population of isolated bipolar cells, did not show PKC immunoreactivity and were likely to be cone bipolar cells. The validity of morphological identification of bipolar cells was further confirmed by the presence of GABAC responses in these cells. Bipolar cells of both groups displayed low-voltage-activated (LVA) Ca2+ currents with similar voltage dependence of activation and steady-state inactivation. However, the activation, inactivation, and deactivation kinetics of the LVA Ca2+ currents between rod and cone bipolar cells differed. Particularly, the LVA Ca2+ currents of rod bipolar cells displayed both transient and sustained components. In contrast, the LVA Ca2+ currents of cone bipolar cells were mainly transient. In addition, the LVA Ca2+ channels of rod bipolar cells were more permeable to Ba2+ than to Ca2+, whereas those of cone bipolar cells were equally or less permeable to Ba2+ than to Ca2+. The LVA Ca2+ currents of both rod and cone bipolar cells were antagonized by high concentrations of nimodipine with IC50 of 17 and 23 µM, respectively, but largely resistant to Cd2+ and Ni2+. Bipolar cells of both groups also displayed high-voltage-activated (HVA) Ca2+ currents. The HVA Ca2+ currents were, at least in part, to be L-type that were potentiated by BayK-8644 (1 µM) and largely antagonized by low concentrations of nimodipine (5 µM). The L-type Ca2+ channels were almost exclusively located at the axon terminals of rod bipolar cells but expressed at least in the cell soma of cone bipolar cells. Results of this study indicate that rod and cone bipolar cells of the mammalian retina differentially express at least two types of LVA Ca2+ channels. Rod and cone bipolar cells also show different spatial distribution of L-type Ca2+ channels.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bipolar cells are the second-order neurons that bridge the signal from photoreceptors to amacrine and ganglion cells in the retina. Bipolar cells do not simply relay the signal from photoreceptors to third-order neurons but play an important role in retinal information processing. The conversion of sustained responses from the outer retina to transient responses in the inner retina is believed to be due, in part, to mechanisms taking place at the bipolar cell level (Maguire et al. 1989; Tachibana and Kaneko 1988). Two types of bipolar cells, ON and OFF, with distinct glutamate receptors generate the segregation of ON and OFF signaling in the visual system (Kaneko 1970; Slaughter and Miller 1981; Werblin and Dowling 1969). On the basis of their synaptic inputs, bipolar cells are divided into rod and cone bipolar cells. In mammals, only a single type of rod bipolar cell, ON type, was reported (Boycott and Dowling 1969; Boycott and Kolb 1973; Dacheux and Raviola 1986; Greferath et al. 1990). On the other hand, multiple subtypes of cone bipolar cell have been reported (Euler and Wässle 1995; Famiglietti 1981; Kolb et al. 1981; Pourcho and Goebel 1987). In the rat, nine subtypes of cone bipolar cell have been described on the basis of their terminal stratification in the inner plexiform layer (Euler and Wässle 1995; Hartveit 1997).

Bipolar cells in various species have been reported to express voltage-activated Ca2+ currents, outward K+ currents, inward rectifier currents or H currents, and Ca2+-activated K+ and Cl- currents (Connaughton and Maguire 1998; Heidelberger and Matthews 1992; Kaneko et al. 1989; Kaneko and Tachibana 1985; Karschin and Wässle 1990; Lasater 1988; Maguire et al. 1989; Okada et al. 1995; Tessier-Lavigne et al. 1988). The properties of voltage-activated Ca2+ channels could be particularly important in bipolar cell synaptic transmission because the influx of Ca2+ at presynaptic terminals is believed to be directly correlated to transmitter release (Llinas et al. 1995). In one group of goldfish retinal bipolar cells, transmitter release has been demonstrated to be triggered by Ca2+ influx through dihydropyridine-sensitive L-type Ca2+ channels (Heidelberger et al. 1994; Tachibana et al. 1993). On the other hand, the role of T-type Ca2+ channels in bipolar cell function remains unknown.

Both L-type high-voltage-activated (HVA) and low-voltage-activated (LVA), or T-type, Ca2+ channels have been reported in mammalian bipolar cells. T-type Ca2+ currents were earlier characterized in isolated mouse bipolar cells with patch-clamp recordings (Kaneko et al. 1989). L-type Ca2+ responses were later revealed by Ca2+ imaging studies at the axon terminals of isolated rat bipolar cells (Pan and Lipton 1995). The presence of L-type and T-type Ca2+ currents in bipolar cells was also reported by patch-clamp recordings in mouse retinal slice preparations (de la Villa et al. 1998; Satoh et al. 1998). Bipolar cells studied in the isolated preparations from rodent were reported to be mostly rod bipolar cells (Greferath et al. 1990). However, a more recent study reported that rod bipolar cells in rat retinal slice preparation displayed only L-type Ca2+ currents, but no transient T-type Ca2+ currents (Protti and Llano 1998). On the other hand, the same study reported that some cone bipolar cells appeared to display transient Ca2+ currents (Protti and Llano 1998). Thus there is still controversy regarding the expression of T-type Ca2+ channels in mammalian rod bipolar cells. Furthermore, the detailed properties of voltage-activated Ca2+ currents in the cone bipolar cells of mammals are less clear.

The purpose of this study was to characterize and compare voltage-activated Ca2+ currents among rod and cone bipolar cells in the mammalian retina. Whole cell voltage-clamp recordings were mostly performed on freshly isolated rat bipolar cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dissociation of bipolar cells

Bipolar cells were dissociated from Long Evans rats >= 4 wk of age by dissociation methods previously described (Pan and Lipton 1995). Some bipolar cells were also dissociated from adult mice (C57BL/6). In brief, animals were deeply anesthetized with CO2 and killed by decapitation. Retinas were removed and placed in a Hanks' solution (in mM): 138 NaCl, 1 NaHCO3, 0.3 Na2HPO4, 5 KCl, 0.3 KH2PO4, 1.25 CaCl2, 0.5 MgSO4, 0.5 MgCl2, 5 HEPES-NaOH, and 22.2 glucose, with phenol red, 0.001% vol/vol; adjusted to pH 7.2 with 0.3 N NaOH. The retinas were incubated for ~50 min at 34-37°C in an enzyme solution that consisted of the normal Hanks' described above, supplemented with 0.2 mg/ml DL-cysteine, 0.2 mg/ml bovine serum albumin, and 1.6 U/ml papain, adjusted to pH 7.2 with 0.3 N NaOH. Following several rinses in Hanks' solution, the retinas were mechanically dissociated by gentle trituration with a glass pipette. The resulting cell suspension was plated onto culture dishes. Cells were kept at room temperature and used for recordings within 5 h after dissociation.

Bipolar cell identification and protein kinase C (PKC) immunocytochemistry

The bipolar cells were identified based on their characteristic morphology: oval shape soma with dendrites emerging from one end and an axon at the other end (Karschin and Wässle 1990; Pan and Lipton 1995; Yeh et al. 1990). Rod and cone bipolar cells were recognized by their distinct morphological properties (see details in RESULTS).

PKC immunocytochemistry was performed according to the previously published procedures of Karschin and Wässle (1990) and the instruction manual of Vector Labs (Burlingame, CA). In brief, cells were fixed with 4% paraformaldehyde in 0.1 mM sodium phosphate buffer (PBS) for 15 min, rinsed with PBS, and blocked for 1 h in PBS-T (1 × PBS plus 0.5% Triton X-100) containing 10% horse serum. The cells were incubated for 2 h in PBS-T containing 3% horse serum and a monoclonal anti-PKC antibody at a dilution of 1:100 (RPN 536; Amersham, Arlington Heights, IL). After several rinses, cells were incubated for 1 h with biotinylated horse anti-mouse antibody (Vector Labs) at a dilution of 1:100 in PBS-T, and, after more rinses, then incubated for a half hour with avidin and biotinylated horseradish peroxidase reagents using a Vectastain Elite ABC kit (Vector Labs). Peroxidase activity was visualized by an AEC substrate kit (Vector Labs).

Electrophysiological recordings

Recordings with patch electrodes in the whole-cell configuration were made by standard procedures (Hamill et al. 1981) at room temperature (20-25°C) with an EPC-9 amplifier and PULSE software (Heka Electronik, Lambrecht/Pfalz, Germany). Electrodes were fabricated from borosilicate microcapillary tubes (VWR Scientific, West Chester, PA), coated with silicone elastomer (Sylgard; Dow Corning, Midland, MI), and fire-polished. The resistance of the electrode was 7-14 MOmega . Series resistance ranged from 12 to 40 MOmega and was not routinely compensated because the Ca2+ currents were small (<= 100 pA). Cell capacitance was automatically canceled and recorded by EPC-9 amplifier and PULSE software. Leak and capacitance currents were usually subtracted with an on-line P/4 protocol provided by PULSE software. Test pulses were delivered once every 5-10 s for all recordings. To facilitate Ca2+ current recordings, voltage-activated K+ currents were suppressed by the inclusion of Cs and tetraethyl-ammonium (TEA) in the recording electrode and extracellular solutions. Unless otherwise indicated, the recordings were made in high-Ca2+ (10 mM) extracellular solution containing (in mM) 95 NaCl, 5 KCl, 10 Cs-Acetate, 20 TEA-Cl, 1 MgCl2, 10 CaCl2, 5 HEPES, and 22.2 glucose, with phenol red, 0.001% vol/vol; pH 7.2. In some recordings, 0.5-1 µM tetrodotoxin (TTX) was included in the extracellular solutions to block potential voltage-activated Na+ currents. The electrode solution contained (in mM) 40 CsCl, 80 Cs-acetate, 20 TEA-Cl, 1 MgCl2, 0.5 CaCl2, 5 EGTA, 10 HEPES, 0.5 Na-GTP, and 2 Na-ATP, pH adjusted with CsOH to 7.4. Under these recording conditions, the voltage-activated K+ currents in bipolar cells were mostly blocked because no significant outward current was observed when Ca2+ (10 mM) in the extracellular solution was replaced by 4 mM Co2+ and 6 mM Mg2+. The chloride reversal potential was around -20 mV under above intracellular and extracellular recording solutions. Liquid junction potentials were measured (7.3 mV) according to the procedure described by Neher (1992) and corrected.

Chemical agents were applied either by local perfusion or through bath application. In the local perfusion, chemicals were applied to the cells by five-barreled gravity-driven superfusion pipettes (modified from Carbone and Lux 1987) placed ~200-300 µm away from the cell being recorded. In bath application, the extracellular recording solution was completely replaced by the solution containing the indicated concentration of chemicals before recordings were made. Data were analyzed off-line using PULSE-FIT (Heka Electronik) and ORIGIN programs (Microcal Software, Northampton, MA). Each result reported in this study was based on the observations obtained from at least five bipolar cells unless otherwise indicated.

Chemicals

Nimodipine, nifedipine, S-(-)Bay K 8644, omega -conotoxin-MVIIC, and tetrodotoxin (TTX) were purchased from Research Biochemicals (Natick, MA). Isradipine was provided by Research Biochemicals (Natick, MA) as part of the National Institute of Mental Health Chemical Synthesis Program. Other chemicals were purchased from Sigma (St. Louis, MO).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Over 1,000 rat retinal bipolar cells were patch-clamp recorded in this study. Two distinct LVA Ca2+ currents were observed in two groups of morphologically different bipolar cells. Similar results were also observed in a limited number of mouse bipolar cells (n = 65). All the detailed studies were carried out in rat bipolar cells.

Morphological properties of two groups of bipolar cells

Bipolar cells of the first group represented the vast majority of identified bipolar cells after dissociation. Several representative bipolar cells of this group are shown in Fig. 1A. Bipolar cells of this group had a similar morphological appearance in several respects. These bipolar cells all had long and thick axons and usually retained axon terminals with several clearly visible synaptic boutons. Their dendritic trees were thick and bushlike. In fact, their dendrites were so characteristic that, in many cases, these cells could be easily recognized even without axons or axon terminals, which had been lost during dissociations. On the other hand, the dendrite branching patterns were found to vary. The six bipolar cells shown in Fig. 1A illustrate the typical dendrite branching patterns for the bipolar cells in this group.



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Fig. 1. Morphology and protein kinase C (PKC) immunoreactivity of 2 groups of isolated rat bipolar cells, which display distinct voltage-gated Ca2+ channel properties. A: representative PKC-positive bipolar cells, which represent the majority of isolated bipolar cells. B: representative PKC-negative bipolar cells, which represent only a small population of isolated bipolar cells. C: in the same field, a PKC-positive bipolar cell (right) and a PKC-negative bipolar cell (left) are shown. The PKC immunoreactivity is displayed by reddish color. Scale bars are 10 µm.

Bipolar cells of the second group represented a very small population of the total identified bipolar cells. The overall morphological appearance of bipolar cells in this group was different from those of bipolar cells in the first group (Fig. 1B). Bipolar cells of this group had sparser dendritic trees. Their dendrites and axons were thinner. Most bipolar cells of this group appeared to show slightly smaller soma than those in the first group. Synaptic boutons were small or absent. These features made it quite easy to distinguish this group of bipolar cells from the first group under the microscope. Occasionally, bipolar cells without axons in this group could still be recognized based on their dendritic feature and soma shape. On the other hand, the length of the axons of these bipolar cells was variable. Some bipolar cells had short axons.

It has been reported that bipolar cells immunoreactive to PKC are rod bipolar cells (Greferath et al. 1990; Negishi et al. 1988; Wood et al. 1988). To determine the types of isolated bipolar cells under the dissociation conditions of this study, PKC immunocytochemistry was performed. Bipolar cells in the first group were found to show PKC immunoreactivity (Fig. 1A). Consistent with a previous report (Greferath et al. 1990), the vast majority of isolated bipolar cells displayed PKC immunoreactivity. On the other hand, bipolar cells of the second group were not shown PKC immunoreactivity (Fig. 1B). In Fig. 1C, a PKC-negative bipolar cell (left) is shown together with a PKC-positive bipolar cell (right) in the same field. These results suggest that bipolar cells in the first group were likely to be rod bipolar cells, whereas those in the second group were likely to be cone bipolar cells.

To provide nonmorphological evidence that the cells described above were all bipolar cells, the pharmacology of GABA-evoked responses were examined. The reason to examine the GABA response pharmacology is that only bipolar cells but not third-order neurons in rats have been reported to express significant GABAC receptors (Euler and Wässle 1998; Feigenspan et al. 1993). More than 40 morphologically identified rod bipolar cells and 26 cone bipolar cells were tested. All of them displayed GABA-evoked currents in the presence of bicuculline, a GABAA receptor antagonist. A typical example for a cone bipolar cell is shown in Fig. 2A. When GABA (100 µM) and bicuculline (200 µM) were co-applied, a sustained inward current was observed when the cell was held at -70 mV (top trace). The current was reversed around -20 mV (data not shown) at the predicted Cl- reversal potential under the recording conditions of this study (see METHODS). Application of GABA itself evoked a larger but rather transient current (bottom trace in Fig. 2A). For comparison, the GABA response was also tested in third-order neurons (n = 9) under the same recording conditions. The third-order neurons were identified by their multipolar morphology and the presence of large voltage-activated Na+ currents (data not shown). As a typical example shown in Fig. 2B, co-application of GABA and bicuculline did not evoke any significant currents in third-order neurons (top trace), although application of GABA itself always evoked large currents (bottom trace). These results further confirm the validity of morphological identification of bipolar cells described in this study.



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Fig. 2. Pharmacology of GABA-evoked currents of morphologically identified bipolar cells and 3rd-order neurons. A: co-application of 100 µM GABA and 200 µM bicuculline evoked sustained inward currents in a morphologically identified cone bipolar cell (top trace). Application of GABA itself evoked larger but transient inward currents (bottom trace). B: no significant currents were observed in a 3rd-order neuron when 100 µM GABA and 200 µM bicuculline were co-applied (top trace). GABA itself evoked large inward currents (bottom). Recordings were made in normal Hanks' solution. Cells were held at -70 mV.

Properties of voltage-activated Ca2+ currents of rod bipolar cells

To record voltage-activated Ca2+ currents, bipolar cells were usually held at -80 mV and depolarized by a series of 200- to 400-ms test pulses with potentials ranging from -60 to +40 mV or by a voltage ramp from -80 to +40 mV at a speed of 100 mV/s.

A typical example of the Ca2+ currents of rod bipolar cells is shown in Fig. 3. When rod bipolar cells were depolarized from the holding potential of -80 mV, inward Ca2+ currents were evoked at around -60 mV and reached their peak around -40 to -30 mV (Fig. 3, A and B). The average peak Ca2+ current of rod bipolar cells is 34.8 ± 10.3 pA (mean ± SD; n = 37), and the average whole cell capacitance of rod bipolar cells is 4.01 ± 0.64 pF (n = 37). Thus the average Ca2+ current density of rod bipolar cells is 8.75 ± 2.68 pA/pF (n = 37). At test potentials of -40 mV or more negative, activation of the inward Ca2+ current was slow (see top trace in Fig. 3B). The Ca2+ currents did not show significant inactivation during the 400-ms testing pulse. At more positive potentials, for example at -30 and -10 mV, the Ca2+ currents displayed an initial transient phase followed by a plateau (see bottom 2 traces in Fig. 3B). In addition, at the termination of the test pulses, large tail currents were observed. The current-voltage (I-V) relationships of the inward Ca2+ currents measured at the peak and at the end of a 400-ms stimulation pulse are shown in Fig. 3C. Inward Ca2+ currents evoked by a voltage ramp displayed a single peak around -40 mV. When rod bipolar cells were depolarized after being held at -45 mV for several seconds, no significant inward Ca2+ currents were observed by pulse or ramp stimulation (Fig. 3, E and F), suggesting that the observed Ca2+ currents in rod bipolar cells were mainly LVA Ca2+ currents.



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Fig. 3. Typical properties of the voltage-activated Ca2+ currents of rod bipolar cells. A: Ca2+ currents evoked by test pulses ranging from -60 to +40 mV for 400 ms from the holding potential of -80 mV. B: 3 individual traces are shown from A evoked by test potentials of -50, -30, and -10 mV, respectively. C: current-voltage (I-V) relationships of the Ca2+ currents in A. The currents measured at the peak () and at the end of 400-ms pulses (black-triangle) were plotted vs. test potentials. D: Ca2+ currents evoked by a voltage-ramp from -80 to +40 mV from the holding potential of -80 mV at a speed of 100 mV/s. E: Ca2+ currents evoked by test pulses ranging from -40 to +40 mV from the holding potential of -45 mV. F: Ca2+ currents evoked by a voltage ramp from -45 to +40 mV from the holding potential of -45 mV at a speed of 100 mV/s. All the recordings were made from the same cell.

The sustained Ca2+ currents in rod bipolar cells could last for seconds without becoming totally inactivated (Fig. 4A). The sustained Ca2+ currents were still present in rod bipolar cells that had lost their axon or axon terminals during dissociation (n = 35), suggesting that the sustained Ca2+ currents were not located exclusively at axon terminals. An example is the recordings of a rod bipolar cell without axon shown in Fig. 4B. The sustained Ca2+ currents were also observed in recording solutions containing 4-10 µM nimodipine, as shown in Fig. 4C (n = 26). Similar results were observed in recording solutions containing 10 µM nifedipine (n = 11) or 10-15 µM isradipine (n = 8; not illustrated). These results indicate that the sustained component of Ca2+ currents was not L-type. The sustained Ca2+ currents were also present in the recording solution containing 10 µM omega -conotoxin-MVIIC (n = 3; not illustrated). omega -Conotoxin-MVIIC at this concentration was reported to block N-, P/Q-type HVA Ca2+ currents (McDonough et al. 1996), suggesting that the significant presence of these other types of Ca2+ currents in rod bipolar cells is unlikely. Furthermore, the sustained Ca2+ currents were unlikely to be significantly contaminated by other currents for the following reasons. First, the observed inward currents had to be Ca2+-related because no inward current was observed when Ca2+ in the extracellular solution was replaced by 4 mM Co2+, as shown in Fig. 4D. Second, the Cl- reversal potential was around -20 mV (see METHODS), whereas the sustained currents were prominent at test potentials around the Cl- reversal potential (see bottom 2 traces in Fig. 3B). Third, similar patterns of Ca2+ currents were observed in extracellular solution where Ca2+ was replaced by Ba2+, as shown in Fig. 4E. Ba2+ was reported to less effectively activate Ca2+-activated Cl- currents (Okada et al. 1995). In addition, as shown in Fig. 4F, the Ca2+ currents were not altered by quinine (30 µM), which was reported to block the Ca2+-activated K+ currents (Kaneko and Tachibana 1985). Finally, replacing extracellular Na+ with Li+ or choline did not alter the Ca2+ currents (Fig. 4G), suggesting that the presence of significant Na+ or Na+-related currents was unlikely. Taken together, these results suggest that the observed Ca2+ currents in rod bipolar cells are mainly LVA Ca2+ currents, which contain both transient and sustained components.



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Fig. 4. Demonstration of the presence of the sustained component of low-voltage-activated (LVA) Ca2+ currents in rod bipolar cells. Each trace was recorded from a different rod bipolar cell. Currents were evoked by a test pulse at -30 mV from the holding potential of -80 mV for 2 s in A and 400 ms in B-G. All the recordings were made in the high-Ca2+ solution with the exception in D and E, where Ca2+ was replaced by Co2+ (D) or Ba2+ (E). In G, Na+ was replaced by Li+. The current showed in B was recorded from a rod bipolar cell without an axon. Nimodipine (C) or quinine (F) was applied by bath application.

On the other hand, rod bipolar cells did express L-type HVA Ca2+ channels, but these currents were small and, in most cases, were masked by LVA Ca2+ currents under the normal recording conditions of this study. The presence of HVA Ca2+ currents in rod bipolar cells was noticed by a weak inward current when rod bipolar cells were depolarized from the holding potential of -45 mV (see Fig. 3F). Furthermore, L-type HVA Ca2+ currents could be clearly revealed by BayK-8644. Application of BayK-8644 (1 µM) enhanced a prominent second component of inward current with a peak at around -10 mV as demonstrated by ramp stimulation (Fig. 5A; middle panel). Application of nimodipine at a concentration of 5 µM did not alter the Ca2+ currents significantly (Fig. 5A; bottom panel). The HVA Ca2+ currents enhanced by BayK-8644 were sustained as demonstrated by a depolarizing pulse from the holding potential of -45 mV (Fig. 5B), consistent with the property of L-type Ca2+ currents. BayK-8644 was found to enhance large L-type HVA Ca2+ currents in all 23 rod bipolar cells that retained their axon terminals. When tested in 17 bipolar cells that retained axons but not terminals, BayK-8644 did not induce any detectable HVA Ca2+ currents in 15 of these cells (Fig. 5C) and induced only a barely detectable HVA component in the remaining two cells. Thus L-type Ca2+ channels appear to be almost exclusively located at the axon terminals of rod bipolar cells.



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Fig. 5. Demonstration of the presence of L-type high-voltage-activated (HVA) Ca2+ currents at the terminals of rod bipolar cells. A: current evoked from a rod bipolar cells retaining axon terminals by a voltage ramp from -80 to +40 mV from the holding potential of -80 mV in control (top), in 1 µM BayK-8644 (middle), and in 5 µM nimodipine (bottom). B: current evoked from a rod bipolar cells by a voltage pulse at 0 mV from the holding potential of -45 mV in control (top trace) and in 1 µM BayK-8644 (bottom trace). C: current evoked from a rod bipolar cell without an axon terminal by a voltage ramp in control (top) and in 1 µM BayK-8644 (bottom). Nimodipine and BayK-8644 were applied by local superfusion.

Properties of voltage-activated Ca2+ currents of cone bipolar cells

Under the same recording conditions, voltage-activated Ca2+ currents were observed in cone bipolar cells. Two typical Ca2+ currents of cone bipolar cells are shown in Fig. 6, A and B, respectively. The Ca2+ currents were activated around -60 to -50 mV and reached their peak around -20 mV (a-c in Fig. 6, A and B), suggesting the presence of LVA Ca2+ currents in cone bipolar cells. The properties of the Ca2+ currents of cone bipolar cells were different from those of rod bipolar cells in several aspects. First, the magnitudes of Ca2+ currents in cone bipolar cells were larger than that of rod bipolar cells. The average peak Ca2+ current of cone bipolar cells is 65.7 ± 18.4 pA (n = 67), which is significantly larger than that of rod bipolar cells (P < 0.05, by t-test). On the other hand, the average whole cell capacitance of cone bipolar cells is 2.61 ± 0.48 pF (n = 67), indicating the cell membrane of cone bipolar cells is significantly smaller than that of rod bipolar cells (P < 0.05, by t-test). The average Ca2+ current density of cone bipolar cells was 25.6 ± 7.92 pA/pF (n = 67). Thus the Ca2+ current density of cone bipolar cells is about three times that of rod bipolar cells. More markedly, the Ca2+ currents of cone bipolar cells evoked by all test potentials were transient (Fig. 6, Ab and Bb). In the majority of cone bipolar cells, Ca2+ currents were completely inactivated within 200- to 400-ms pulses (see top 2 traces in Fig. 6Ab). The inactivation kinetics, however, were found to vary among cone bipolar cells. In some cone bipolar cells, as the cell shown in Fig. 6B, complete inactivation of the Ca2+ currents was not reached at the end of the 400-ms test pulse (see traces in Fig. 6Bb). Furthermore, at more depolarized voltages (greater than -30 mV), prominent sustained Ca2+ currents could be observed in many cone bipolar cells, as in the example shown in Fig. 6A (see the bottom trace in Fig. 6Ab), suggesting the presence of HVA currents in these cone bipolar cells. The presence of both LVA and HVA Ca2+ currents in these cells was further evident in the voltage-ramp stimulation where two peaks of inward currents were observed (Fig. 6Ad). In addition, when the cell was depolarized from the holding potentials of -45 mV, only the sustained HVA Ca2+ current was evoked (Fig. 6, Ae and Af), further confirming that the transient Ca2+ currents were LVA Ca2+ currents.




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Fig. 6. Properties of voltage-activated Ca2+ currents of cone bipolar cells. Ca2+ currents recorded from two representative cone bipolar cells were shown in A and B, respectively. Aa and Ba: Ca2+ currents evoked by test pulses ranging from -60 to +40 mV for 400 ms from the holding potential of -80 mV. Ab and Bb: 3 individual traces from A evoked by test potentials of -50, -30, and -10 mV for A and -40, -30, and -10 mV for B. Ac and Bc: I-V relationships of the Ca2+ currents in Aa and Ba, respectively. The currents measured at the peak () and at the end of 400-ms pulses (black-triangle) were plotted vs. test potentials. Ad and Bd: Ca2+ currents evoked by a voltage ramp from -80 to +40 mV from the holding potential of -80 mV. Ae and Be: Ca2+ currents evoked by test pulses ranging from -40 to +40 mV from the holding potential of -45 mV. Af and Bf: Ca2+ currents evoked by a voltage ramp from -45 to +40 mV from the holding potential of -45 mV. All recordings were made in high-Ca2+ extracellular solution containing 500 nM TTX.

It should be mentioned that the transient nature of the LVA Ca2+ currents in cone bipolar cells was unlikely to be due to contamination of voltage-activated K+ currents because voltage-activated K+ currents in cone bipolar cells were also mostly blocked under the recording conditions of this study. In addition, the Ca2+ currents in cone bipolar cells were unlikely to be significantly contaminated by Ca2+-activated K+ because the same properties of Ca2+ currents were observed in recording solutions containing 30 µM quinine. Furthermore, similar properties of Ca2+ currents in cone bipolar cells were observed when either Ca2+ was replaced by Ba2+ or Na+ was replaced by Li+, suggesting that significant contamination of Ca2+-activated Cl- currents or Na+-related currents is unlikely.

The HVA Ca2+ currents in cone bipolar cells were largely blocked by 5 µM nimodipine and enhanced by 1 µM BayK-8644 (Fig. 7A). These results indicate that the HVA Ca2+ currents are mainly L-type. Nimodipine and BayK-8644 at these concentrations did not affect the LVA Ca2+ currents significantly (Fig. 7A). It also should be mentioned here that the amplitude of L-type Ca2+ currents was found to vary among cone bipolar cells. For many cone bipolar cells, prominent HVA Ca2+ currents could be clearly observed in the voltage-ramp stimulation under the normal recording conditions (e.g., Figs. 6A and 7A). But in some cone bipolar cells the L-type Ca2+ current was small, as shown in Figs. 6B and 7B, and only became noticeable after the application of BayK-8644 (e.g., see Fig. 7B). The variation in the amplitude of L-type Ca2+ currents was not found to be closely correlated to the presence of axon terminals. These results suggest that the expression level of the L-type Ca2+ channels among cone bipolar cells is likely to be heterogeneous.



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Fig. 7. Demonstration of the presence of L-type HVA Ca2+ currents in cone bipolar cells. A: a typical cone bipolar cell displayed large L-type HVA Ca2+ currents. Ca2+ currents evoked by a voltage ramp in control (top), after the application of 5 µM nimodipine (middle), and 1 µM BayK-8644 (bottom). B: BayK-8644 evoked prominent L-type HVA Ca2+ currents in an axonless cone bipolar cell. Ca2+ currents recorded evoked by a voltage-ramp in control (top) and after the application of 1 µM BayK-8644 (bottom).

Furthermore, BayK-8644 was found to enhance the HVA Ca2+ currents for all cone bipolar cells (n = 19); eight of these cells did not retain axon terminals, whereas two did not retain axons. The recordings shown in Fig. 7B were from one of these cells without an axon. These results indicate that the L-type Ca2+ channels are expressed in the soma of cone bipolar cells.

Activation and steady-state inactivation

The voltage dependence of activation for the LVA Ca2+ currents in rod and cone bipolar cells was determined by using a tail-current protocol shown in Fig. 8A, in which cells were held at -80 mV and depolarized to a series of test pulses ranging from -60 to 20 mV. The duration of the test pulse was varied to match the maximal activation. Activation was measured by the tail-current amplitude evoked by repolarization at -80 mV after completion of the test pulse. The average values normalized to the test pulse at 20 mV for rod bipolar cells (; n = 11) and cone bipolar cells (; n = 10) were plotted versus the test potentials (Fig. 8C). Smooth curves fitted by Boltzmann function yielded the half-maximum activation potentials (V1/2) of -36.1 and -35.1 mV for rod and cone bipolar cells, respectively. The Boltzmann slope factor (k) was 9.7 mV for rod bipolar cells and 10.1 mV for cone bipolar cells. These two curves almost coincided, indicating that there is no significant difference in the voltage dependence of activation between rod and cone bipolar cells.



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Fig. 8. Comparison of activation and steady-state inactivation of LVA Ca2+ currents between rod and cone bipolar cells. A: activation protocol, in which cells were held at -80 mV and depolarized to a series of test pulses ranging from -60 to +20 mV. The duration of the test pulse was varied from 7 to 70 ms for rod bipolar cells and 4 to 50 ms for cone bipolar cells to match the maximal activation. Activation was determined by the amplitude of the tail-current evoked by repolarization at -80 mV after completion of the test pulse. B: inactivation protocol, in which a series of conditioning pulses ranging from -80 to 40 mV (3 s for rod bipolar cells and 1 s for cone bipolar cells) were applied before a brief (5 ms) repolarization to -80 mV, that preceded a 50-ms test pulse at -40 mV. Inactivation was determined by the amplitude of the peak-current evoked by the test pulse. C: voltage dependence of activation and steady-state inactivation for rod bipolar cells (; n = 11) and cone bipolar cells (; n = 9). Data were normalized at 20 mV for activation and at -80 mV for inactivation. The data points are mean and the error bars are standard deviation. Smooth curves are standard Boltzmann functions. Recordings were made in high-Ca2+ extracellular solution containing 5 µM nimodipine and 500 nM TTX.

The voltage dependence of inactivation was determined by applying conditioning pulses ranging from -80 to -40 mV followed by a test pulse at -40 mV (Fig. 8B). Inactivation was measured by the peak-current amplitude evoked by the test pulse. The average values normalized to the test pulse at -80 mV for rod bipolar cells (; n = 12) and cone bipolar cells (; n = 11) were plotted versus the conditioning potentials (Fig. 8C). The fitted curves for rod bipolar cells and cone bipolar cells also coincided. The half-maximum inactivation potentials for rod and cone bipolar cells were -58.8 and -60.4 mV, respectively. The slop factor was 5.2 mV for rod bipolar cells and 5.7 mV for cone bipolar cells. Again, these results indicate that the steady-state inactivation between rod and cone bipolar cells are similar.

Activation and inactivation kinetics

The activation and inactivation kinetics were compared by measuring the time-to-peak and the decay time constant at different membrane potentials. The decay time constant was obtained by fitting the decay current with a single exponential. The average values of time-to-peak and decay time constant for rod (n = 16) and cone (n = 23) bipolar cells were plotted versus the membrane potentials (Fig. 9).



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Fig. 9. Comparison of the time-to-peak and the decay time constant of LVA Ca2+ currents between rod and cone bipolar cells. A: voltage dependence of time-to-peak for rod bipolar cells (; n = 16) and cone bipolar cells (; n = 23). B: voltage dependence of decay time constant for rod bipolar cells (; n = 16) and cone bipolar cells (; n = 23). Recordings were made in high-Ca2+ extracellular solution containing 500 nM TTX. For some recordings, 5 µM nimodipine was included in the extracellular solution to block L-type Ca2+ currents. Because the decay time constants obtained with or without nimodipine did not differ, the data were combined. The decay time constant was obtained by fitting the current decay with a single exponential. Data points are mean and the error bars are standard deviation.

Overall, both activation and inactivation kinetics of the LVA Ca2+ currents for rod and cone bipolar cells are voltage dependent, consistent with the early report of Kaneko and co-workers (1989), but the kinetics of the LVA Ca2+ currents between rod and cone bipolar cells are noticeably different. As shown in Fig. 9A, the activation of the LVA Ca2+ currents for cone bipolar cells () is faster than that of rod bipolar cells () at all potentials. At the test potential of -50 mV, the times-to-peak for the LVA Ca2+ currents of rod bipolar cells were >200 ms but could not be accurately determined (data points not shown). The voltage dependence of the decay time constant of LVA Ca2+ currents between rod and cone bipolar cells also differs (Fig. 9B). At negative potentials, such as at -50 and -40 mV, the current decay constant for rod bipolar cells () is larger than that for cone bipolar cells (). Particularly, at the test potential of -50 mV, the currents of rod bipolar cells did not show any inactivation (data points not shown). At more depolarized potentials, greater than -30 mV, the current decay constants of the LVA Ca2+ currents of rod bipolar cells become smaller than those of cone bipolar cells. That is, the inactivation of Ca2+ currents for cone bipolar cells is less voltage dependent than that for rod bipolar cells. Furthermore, it should be pointed out that the decay time constants of the LVA Ca2+ currents for cone bipolar cells display an unusual spread at more depolarized potentials (greater than -30 mV), evident by the large deviation of the data.

Deactivation

The voltage dependence of deactivation was compared by their tail currents. LVA Ca2+ currents were evoked by a depolarization from -80 to -40 mV for 50 ms for rod bipolar cells (Fig. 10A) or 20 ms for cone bipolar cells (Fig. 10B). Tail currents were then evoked by repolarizing to a series of potentials ranging from -120 to -60 mV. The decay time constant was obtained by fitting the tail current decay with a single exponential. The average decay time constant for rod and cone bipolar cells was plotted versus repolarization potential (Fig. 10C). As shown in Fig. 10C, the deactivation of the LVA Ca2+ currents for both rod and cone bipolar cells is voltage dependent and faster at more negative potentials. On the other hand, at all potentials, the deactivation of the LVA Ca2+ currents of cone bipolar cells () is faster than that of rod bipolar cells ().



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Fig. 10. Comparison of the deactivation kinetics of LVA Ca2+ currents between rod and cone bipolar cells. LVA Ca2+ currents were evoked by depolarization to -40 mV for 50 ms for rod bipolar cells (A) or 20 ms for cone bipolar cells (B) from the holding potential of -80 mV. Tail currents were generated by a series of repolarization potentials ranging from -120 to -60 mV. C: the decay time constant was obtained by fitting the tail current decay with a single exponential. The decay time constants for the tail currents for rod (; n = 9) and cone (; n = 15) bipolar cells were plotted vs. repolarization potentials. Most recordings were made in high-Ca2+ extracellular solution. Some recordings were made in extracellular solutions with Ca2+ replaced by Ba2+. Because no significant differences were observed in the tail currents between Ca2+ and Ba2+, the data were combined. Data points are mean and the error bars are standard deviation.

Divalent ion permeation

The permeation of Ba2+ and Ca2+ was also compared. The currents were evoked by a depolarizing pulse at -40 mV from a holding potential of -80 mV, which did not significantly activate L-type Ca2+ currents. Recordings were made in normal Hanks' solution. Solutions containing 10 mM Ca2+ or 10 mM Ba2+ were applied by local perfusion. For the LVA Ca2+ currents of rod bipolar cells, perfusion of 10 mM Ba2+ solution produced a larger inward current than that of 10 mM Ca2+ (Fig. 11A). In contrast, for cone bipolar cells, perfusion of Ba2+ solution produced a current either similar or smaller than the Ca2+ currents (Fig. 11B). For both types of LVA Ca2+ channels, the peaks of the inward currents did not shift significantly in Ba2+ and Ca2+, as confirmed by ramp stimulation (not illustrated). The average ratio of Ba2+/Ca2+ currents is 1.51 ± 0.17 (n = 10) for rod bipolar cells and 0.89 ± 0.13 (n = 10) for cone bipolar cells (Fig. 11C). These two values are significantly different (P < 0.05; by t-test).



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Fig. 11. Ba2+ and Ca2+ ion selectivity for the LVA Ca2+ currents of rod and cone bipolar cells. Currents were evoked by depolarizing to -40 mV from the holding potential of -80 mV. A: a typical example of rod bipolar cells, which displayed larger current in Ba2+ than in Ca2+. B: a typical example of cone bipolar cells, which displayed smaller current in Ba2+ than in Ca2+. C: the average ratios of Ba2+/Ca2+ currents were obtained from 10 rod bipolar cells and 10 cone bipolar cells, respectively. Error bars are standard deviation. The difference between these 2 ratios is significant (P < 0.05, by t-test). Recordings were conducted in normal Hanks' extracellular solution. Solutions containing 10 mM Ba2+ or 10 mM Ca2+ were applied by local perfusion.

Dihydropyridine and inorganic calcium channel blockers

The pharmacological properties of the LVA Ca2+ currents in rod and cone bipolar cells were compared. First, both LVA Ca2+ currents were found to be antagonized by high concentrations of dihydropyridine antagonists, such as nimodipine. The IC50 and Hill coefficient values of nimodipine for the LVA Ca2+ currents of rod bipolar cells are 17 µM and 1.4 (n = 12), whereas for cone bipolar cells are 23 µM and 1.4 (n = 11), respectively. When nimodipine was applied by bath application, complete blockade of LVA Ca2+ currents in both rod and cone bipolar cells were observed around 30- to 50-µM concentrations. Dihydropyridine components at high concentrations have been reported to be no longer specific for L-type Ca2+ channels (Akaike et al. 1989; Jones and Jacobs 1990; Randall and Tsien 1997).

The effect of Cd2+ and Ni2+ on the LVA Ca2+ currents of rod and cone bipolar cells was also tested. Applications of 100 µM Cd2+ and Ni2+ by local perfusion or in bath application did not produce significant blockade of either type of LVA Ca2+ currents (not illustrated), whereas only high concentrations (>500 µM) of Cd2+ and Ni2+ produced substantial blockade of both types. For example, Cd2+ at 1 mM reduced the Ca2+ currents of rod and cone bipolar cells to 21 ± 1.0% (mean ± SE; n = 6) and 12 ± 1.8% (mean ± SE, n = 4) of the control, respectively. Similarly, Ni2+ at 1 mM reduced the Ca2+ currents of rod and cone bipolar cells to 20 ± 2.3% (mean ± SE; n = 5) and 15 ± 1.4% (mean ± SE; n = 4) of the control, respectively. Therefore LVA Ca2+ currents of both bipolar cells are highly resistant to Cd2+ and Ni2+ ions, consistent with the finding in isolated mouse bipolar cells (Kaneko et al. 1989).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of isolated rod and cone bipolar cells

In the present study, two groups of morphologically different bipolar cells isolated from rat retinas were identified. Results of this study indicated that these two groups of bipolar cells were rod and cone bipolar cells, respectively. First of all, cells of both groups had the characteristic bipolar cell morphology. In addition, they were found to display GABAC responses. Previous studies have shown that only bipolar cells but not third-order neurons in rats display significant GABAC responses (Euler and Wässle 1998; Feigenspan et al. 1993). Furthermore, bipolar cells of the first group were immunoreactive to PKC, whereas those of the second group were not. PKC has been a well-established marker for rod bipolar cells in rats (Greferath et al. 1990; Wood et al. 1988). Finally, bipolar cells of the first group were found to represent the majority of the isolated bipolar cells, consistent with the early findings that almost all of the isolated bipolar cells in rats were rod bipolar cells (Greferath et al. 1990).

So far, only one type of rod bipolar cell had been described in mammals (Boycott and Dowling 1969; Boycott and Kolb 1973; Dacheux and Raviola 1986; Greferath et al. 1990). Thus rod bipolar cells are commonly believed to be homogeneous. Interestingly, several different dendrite branching patterns among rod bipolar cells were observed in this study.

The cone bipolar cells observed in this study displayed a greater heterogeneity in their morphology, consistent with the findings of multiple subtypes of cone bipolar cells in mammals (Euler and Wässle 1995; Famiglietti 1981; Kolb et al. 1981; Pourcho and Goebel 1987). In the rat, at least nine subtypes of cone bipolar cells have been described (Euler and Wässle 1995; Hartveit 1997).

Differential expression of voltage-activated Ca2+ channels in rod and cone bipolar cells

Results of this study show that, in many respects, the property and expression level of voltage-activated Ca2+ channels between rod and cone bipolar cells are different. First, Ca2+ current density in cone bipolar cells is much larger than that of rod bipolar cells. Second, although both rod and cone bipolar cells express LVA Ca2+ channels, the LVA Ca2+ channels in rod and cone bipolar cells appear to be distinct (more discussion below). Third, the L-type Ca2+ currents were almost exclusively located at the rod axon terminals, whereas the L-type Ca2+ channels were observed in the soma of cone bipolar cells. Thus results of this study indicate that there is a differential expression of voltage-activated Ca2+ channels between rod and cone bipolar cells of mammals.

Two distinct LVA Ca2+ channels in rod and cone bipolar cells

The present study demonstrates that the LVA Ca2+ channels in rod and cone bipolar cells are distinct. First, the activation and inactivation kinetics of the LVA Ca2+ currents between these two groups of bipolar cells are different. Particularly, the LVA Ca2+ currents in rod bipolar cells display a noticeable sustained component in addition to a transient one. In contrast, the LVA Ca2+ currents of the cone bipolar cells are mainly transient. The activation and inactivation properties of the LVA Ca2+ channels in cone bipolar cells are similar to those of LVA Ca2+ channels reported in rat hypothalamic neurons (Akaike et al. 1989). Results of this study suggest that the sustained currents in rod bipolar cells are unlikely to be contributed from other currents, such as outward K+, Ca2+-activated K+, or Cl- currents (see Fig. 4). On the other hand, it is possible that the inactivation course of the LVA Ca2+ currents of rod bipolar cells involves two phases. LVA Ca2+ currents showing two phases of inactivation have been previously reported (Bossu and Feltz 1986; Herrington and Lingle 1992; Huguenard et al. 1993).

It should be emphasized that the properties of LVA Ca2+ currents among rod bipolar cells were found to be quite homogeneous, consistent with the homogeneous population of rod bipolar cells. In contrast, the properties of LVA Ca2+ currents among cone bipolar cells were rather variable. This is exemplified by a larger spread in the inactivation and deactivation decay constants. It is unlikely that such variation is due to the presence of different subtypes of HVA Ca2+ currents. This is because HVA Ca2+ currents in cone bipolar cells were sustained and largely blocked by low concentrations of nimodipine. On the other hand, it may imply a further heterogeneity in LVA Ca2+ channels among cone bipolar cells.

Furthermore, the deactivation of the LVA Ca2+ currents of cone bipolar cells is faster than that of rod bipolar cells. Slow deactivation is one of the marked properties of LVA Ca2+ channels. The deactivation time constant for cone bipolar cells is similar to that of the LVA Ca2+ currents in rat clonal pituitary cells (Herrington and Lingle 1992) but slightly slower than that in chick and rat sensory neurons (Carbone and Lux 1987; Todorovic and Lingle 1998). Interestingly, the tail currents evoked in rod bipolar cells were unusually large and slow, especially by prolonged depolarization pulses (see Figs. 3A and 4). Results of this study suggest that the large tail currents in rod bipolar cells should not be significantly contaminated by other currents. This is because the tail currents were not significantly altered by blocking or minimizing other potential currents, such as outward K+, Ca2+-activated K+, or Cl- currents (see Fig. 4). In addition, no significant tail currents were observed once the LVA Ca2+ currents were inactivated or blocked (see Figs. 3E and 4D), suggesting that the tail currents were originated by LVA Ca2+ currents. Furthermore, similar tail currents were present in rod bipolar cells without axon terminals (see Fig. 4B). This might argue the possibility that the large tail currents of rod bipolar cells were due to inadequate clamping of the axon terminals. The slow kinetics of the tail current may be the intrinsic property of LVA Ca2+ currents of rod bipolar cells. On the other hand, it is also possibility that the combination of these other currents amplified by the prolonged test-pulse contributes to the tail current. Large and slow tail currents also have been noticed in rod bipolar cells of rat retinal slice preparations (Protti and Llano 1998).

Finally, the LVA Ca2+ channels of rod bipolar cells are more permeable to Ba2+ than to Ca2+, whereas the LVA Ca2+ channels for cone bipolar cells are equally or less permeable to Ba2+ than to Ca2+. Taken together, results of this study indicate that the rod and cone bipolar cells express at least two distinct types of LVA Ca2+ channels.

Fast inactivation and slow deactivation are the hallmark of LVA Ca2+ channels (Carbone and Lux 1984; Huguenard 1996; Nowycky et al. 1985). LVA Ca2+ channels are also called T-type Ca2+ channels. T-type Ca2+ channels usually display equal permeation by Ba2+ and Ca2+ (Huguenard 1996). Interestingly, in many respects, the LVA Ca2+ channels of rod bipolar cells do not share the common properties of the T-type Ca2+ channels observed in other brain areas. The LVA Ca2+ channels in rod bipolar cells appear to be different from the conventional T-type Ca2+ channels.

Comparison with earlier studies of Ca2+ currents in rodent bipolar cells

Results of this study are consistent with previous findings that mouse bipolar cells in isolated or retinal slice preparations display LVA Ca2+ channels (de la Villa et al. 1998; Kaneko et al. 1989; Satoh et al. 1998). It is also consistent with the reports of Protti and Llano (1998) that cone bipolar cells display transient Ca2+ channels. However, this study indicates that LVA Ca2+ channels in rod and cone bipolar cells are distinct. Furthermore, this study further confirms that the L-type Ca2+ channels are almost exclusively expressed at the axon terminals of rod bipolar cells (de la Villa et al. 1998; Pan and Lipton 1995; Protti and Llano 1998; Satoh et al. 1998). But such property does not hold in cone bipolar cells.

Results of the present study are different from that of Protti and Llano (1998), who reported that rod bipolar cells in rats displayed only L-type but no LVA Ca2+ currents. It is not clear why they failed to observe the LVA Ca2+ currents.

Both transient and sustained Ca2+ currents were reported in mouse bipolar cells of slice preparations. However, the sustained currents were reported to be exclusively derived from the L-type Ca2+ channels at the axon terminals (de la Villa et al. 1998). The T-type Ca2+ currents reported in isolated mouse bipolar cells appeared also to be transient (de la Villa et al. 1998; Kaneko et al. 1989; Satoh et al. 1998). Such property is different from that of rod bipolar cells in rats described in this study. This study shows that the LVA Ca2+ currents of rod bipolar cells contain a sustained component. The reason for such discrepancy is not clear. Nevertheless, two distinct LVA Ca2+ currents with properties similar to that of rat bipolar cells were observed in isolated mouse bipolar cells in the present study, although the possibility of a slight difference in the Ca2+ channel property between rat and mouse retinal bipolar cells could not be totally excluded.

Functional implications of multiple types of Ca2+ channels in bipolar cells

It has been demonstrated that transmitter release in goldfish bipolar cells is triggered by Ca2+ influx through dihydropyridine-sensitive L-type Ca2+ channels (Heidelberger et al. 1994; Tachibana et al. 1993). Results of this study show that both rod and cone bipolar cells express L-type Ca2+ channels. Particularly, the L-type Ca2+ channels in rod bipolar cells are almost exclusively located at axon terminals. The latter implies that transmitter release in mammalian bipolar cells, especially in rod bipolar cells, might also be triggered by Ca2+ influx through L-type Ca2+ channels.

What, then, are the functional implications of the expression of multiple subtypes of LVA Ca2+ channels in retinal bipolar cells? As previously suggested by Kaneko and co-workers (1989), the LVA Ca2+ channels are likely to be activated in the physiological range of bipolar cell membrane potentials, which were reported to range from -70 to -20 mV (Ashmore and Copenhagen 1983; Ashmore and Falk 1980; Saito and Kaneko 1983; Simon et al. 1975). On the other hand, the activation of other voltage-activated currents was reported to be largely outside the bipolar cell membrane potential (Tessier-Lavigne et al. 1988). Therefore the activation of LVA Ca2+ channels may affect the light response waveform of bipolar cells. Furthermore, the LVA Ca2+ channels in rod and cone bipolar cells differ both in their kinetic and current density. Thus the activation of LVA Ca2+ channels in rod and cone bipolar cells could differently affect their light response waveform and, in turn, the activation of voltage-activated Ca2+ channels at the axon terminals.

Furthermore, the activation of LVA Ca2+ channels in bipolar cells may directly trigger bipolar cell transmitter release as previously proposed by Kaneko and co-workers (1989). Currently, it is commonly thought that transmitter release of bipolar cells is triggered by the activation of L-type Ca2+ channels. The evidence is in part based on the blockade of dihydropyridine antagonists. However, results of this study showed that dihydropyridine antagonists, at relatively high concentrations, would effectively block LVA Ca2+ channels. Moreover, results of this study show that the physiological range of bipolar cell membrane potentials appears to fit better to the activation voltage range of LVA Ca2+ channels than that of L-type Ca2+ channels. This could be especially true for OFF bipolar cells because the dark membrane potential of OFF bipolar cells was reported to be around -45 mV (Ashmore and Copenhagen 1983; Saito and Kaneko 1983), which seems too low to effectively activate L-type Ca2+ channels. Therefore it is possible that transmitter release of OFF bipolar cells may be triggered mainly by the activation of LVA Ca2+ channels.

How the diversified light response of third-order neurons is derived from the sustained response of bipolar cells is still not clear. It has been proposed that the transient response in third-order neurons may be generated by the activation of T-type Ca2+ currents at bipolar cell terminals (Kaneko et al. 1989). The modulation of Ca2+ influx at bipolar cell terminals by GABA through multiple GABA receptors has also been proposed to be, in part, the mechanism (Maguire et al. 1989; Pan and Lipton 1995; Tachibana and Kaneko 1988). Furthermore, different types of Ca2+ channels in other retinal neurons have been reported to be differentially modulated by neurotransmitters or neuromodulators (Pfeiffer-Linn and Lasater 1993; Shen and Slaughter 1999; Zhang et al. 1997). The expression of multiple subtypes of Ca2+ channels in rod and cone bipolar cells may provide additional mechanisms to generate diversified synaptic transmission among different subtypes of bipolar cells and third-order neurons in the retina.


    ACKNOWLEDGMENTS

The author thanks S. Winfield for preparing the figures, Dr. Hui-Juan Hu for technical assistance, Dr. Guang Bai for helpful advice on the immunocytochemistry, Dr. Stuart A. Lipton for valuable advice and support, and Dr. Malcolm M. Slaughter for valuable comments on the manuscript.

This research was supported by National Eye Institute Grant EY-12180.


    FOOTNOTES

Address for reprint requests: Dept. of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 20 April 1999; accepted in final form 17 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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