Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 M
. Series resistance ranged from 12 to 40 M
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,
-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).
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RESULTS |
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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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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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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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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 -conotoxin-MVIIC (n = 3;
not illustrated).
-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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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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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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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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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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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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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 (
).
|
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).
|
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).
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DISCUSSION |
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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.
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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