Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
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ABSTRACT |
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Koizumi, Amane, Shu-Ichi Watanabe, and Akimichi Kaneko. Persistent Na+ Current and Ca2+ Current Boost Graded Depolarization of Rat Retinal Amacrine Cells in Culture. J. Neurophysiol. 86: 1006-1016, 2001. Retinal amacrine cells are depolarized by the excitatory synaptic input from bipolar cells. When a graded depolarization exceeds the threshold level, trains of action potentials are generated. There have been several reports that both spikes and graded depolarization are sensitive to tetrodotoxin (TTX). In the present study, we investigated the contribution of voltage-gated currents to membrane depolarization by using rat GABAergic amacrine cells in culture recorded by the patch-clamp method. Injection of a negative current induced membrane hyperpolarization, the waveform of which can be well fitted by a single exponential function. Injection of positive current depolarized the cell, and the depolarization exceeded the amplitude expected from the passive properties of the membrane. The boosted depolarization sustained after the current was turned off. Either 1 µM TTX or 2 mM Co2+ suppressed the boosted depolarization, and co-application of TTX and Co2+ blocked it completely. Under the voltage clamp, we identified a transient Na+ current (fast INa), a TTX-sensitive persistent current that reversed the polarity near the equilibrium potential of Na+ (INaP), and three types of Ca2+ currents (ICa), L, N, and the pharmacological agent-resistant type (R type). These findings suggest that the INaP and ICa of amacrine cells boost depolarization evoked by the excitatory synaptic input, and they may aid the spread of electrical signals among dendritic arbors of amacrine cells.
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INTRODUCTION |
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Retinal amacrine cells are
axon-less interneurons that provide a lateral pathway between the
radial circuits of the vertebrate retina. Their rich dendrites spread
in the second synaptic layer, where the signal is relayed from bipolar
cells to ganglion cells. Since GABAergic cells make up the majority of
the amacrine cell population (Kolb 1997; Muller
and Marc 1990
; Yazulla 1986
), most amacrine
cells are believed to be inhibitory neurons that mediate lateral
inhibition (Euler and Masland 2000
; Watanabe et
al. 2000a
). The dendrites of amacrine cells are functioning as
both pre- and postsynaptic sites. In mammals, such as the cat and
rabbit, amacrine cells have extreme morphological diversity, and many
of them have dendritic arbors spanning over hundreds of micrometers
(Kolb 1997
; MacNeil and Masland 1998
).
Most dendrites of amacrine cells are quite long. Thus it is important
to understand the voltage-sensitive mechanisms through which electrical
signals are transmitted from one portion of the dendritic tree to another.
Physiologically, amacrine cells are classified into several types on
the basis of their light-evoked responses. In many amacrine cells,
action potentials are superimposed on light-evoked graded depolarization (mudpuppy, Werblin and Dowling 1969;
goldfish, Kaneko 1970
; tiger salamander, Barnes
and Werblin 1986
; Miller and Dacheux 1976
;
rabbit, Bloomfield 1992
; Dacheux and Raviola 1995
). The light-evoked graded depolarization of amacrine cells has been thought to be an excitatory postsynaptic potential (EPSP) evoked by the excitatory input from bipolar cells, but a
voltage-sensitive mechanism may be also involved. In fact, application
of tetrodotoxin (TTX) significantly suppresses the amplitude of the
light-evoked graded depolarization in fish (Watanabe et al.
2000b
) and rabbit (Bloomfield 1996
) amacrine
cells. Feigenspan et al. (1998)
have suggested that a
slowly inactivating Na+ current exists in
dopaminergic amacrine cell, but it is not known how the
voltage-sensitive mechanisms contribute to graded depolarization. It is
expected that such voltage-sensitive mechanisms play a significant role
in signal spread along the dendrites of amacrine cells.
The aim of the present study is to determine how the voltage-sensitive mechanisms contribute to the graded depolarization of amacrine cells. We recorded rat GABAergic amacrine cells in culture using a patch-clamp technique and identified two types of TTX-sensitive Na+ currents (transient and persistent) and three types of Ca2+ currents, L, N, and the pharmacological agent-resistant type (R type). We present evidence that these currents are found generally in GABAergic amacrine cells, where they may contribute to the spread of signals among dendritic arbors of amacrine cells.
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METHODS |
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Culture
The experimental procedure conformed to the Guidelines for the Care and Use of Laboratory Animals, Keio University School of Medicine, and the University Animal Welfare Committee approved our experiments. After decapitating newborn rats (Wistar, P0 and P1), their retinas were isolated and incubated for 25 min in Ca2+-, Mg2+-free Hanks' balanced salt solution with HEPES (10 mM) supplemented with 1 mg/ml trypsin at 37°C. After incubation, they were rinsed with Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum and triturated with a fire-polished glass pipette in 10 ml of culture medium. Dissociated cells were seeded on poly-L-ornithine-coated glass cover slips at a density of 1.5 × 105 cells/ml and cultured for 10-14 days in DMEM supplemented with 14 mM NaHCO3, 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 5% heat-inactivated fetal bovine serum in a 5% CO2 environment at 37°C. Immediately after dissociation, the cells appeared round, and no dendrites were seen.
The voltage-gated currents developed with days in culture. Until day 5, no voltage-gated inward current was detectable, although morphologically the cell extended multiple dendrites by day 5. On day 7, a transient Na+ current (fast INa) and Ca2+ current (ICa) became detectable, but their amplitude was very small. After 10 days in culture, only large cells (soma diameter of >10 µm) survived. Dendrites from their soma extended 100 µm, as seen in the cell shown in Fig. 1D. The fast INa and ICa developed to the maximum amplitude and a persistent Na+ current (INaP) became detectable. This is a good indication that amacrine cells were fully matured by day 10 in culture. We carried out experiments on cells cultured for 10-14 days.
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Identification of amacrine cells by immunostaining
Amacrine cells in culture were identified by immunoreaction with
the anti-HPC-1/Syntaxin antibody, a marker for amacrine cells in the
retina (Akagawa and Barnstable 1986; Barnstable
et al. 1985
), and anti-GABA antibody (Gleason et al.
1993
; Wexler et al. 1998
). Each primary antibody
was diluted with 0.01 M phosphate buffer (PBS) containing 5% normal
goat serum, 0.05% saponin, and 1% bovine serum albumin. Cells were
fixed for 1 h with 4% paraformaldehyde and preincubated for 30 min in a solution containing 5% normal goat serum in PBS. After
preincubation, they were reacted in a humidified air chamber for 2 h with mouse anti-HPC-1/Syntaxin antibody (1:5000; Sigma, St. Louis,
MO) and for 2 h with rabbit anti-GABA antibody (1:2000; Sigma).
The secondary antibodies were diluted to 1:200 in the preceding
diluting solution, and the cells were then incubated for 1 h with
the secondary antibodies: Texas-Red-labeled anti-mouse IgG (H + L) to
anti-HPC-1/Syntaxin antibody and FITC-labeled anti-rabbit IgG (H + L)
to anti-GABA antibody (Vector Laboratories, Burlingame, CA). After each
process, the cells were washed with PBS.
The reacted preparations were examined with a confocal imaging system
(MRC600, Bio-Rad, Hercules, CA) equipped with a krypton-argon laser.
FITC was excited with 488 nm laser line and Texas Red with 568 nm line.
The barrier filters were BioRad 522DF35 and 585EFLP, respectively.
COMOS (Bio-Rad) and Adobe Photoshop (Adobe System, San Jose, CA)
softwares were used to process the image data (Satoh et al.
1998).
Of the 103 large multipolar cells (soma diameter of >10 µm)
examined, 99 cells (96.1%) were immunoreactive to HPC-1/Syntaxin, indicating that they were amacrine cells. Most of them (92 cells, 93%
of HPC-1/Syntaxin-positive cells) were also immunoreactive to GABA.
Examples are shown in Fig. 1. We therefore used cell size and
multipolar morphology as useful criteria for identifying GABAergic
amacrine cells. Wexler et al. (1998) reported that
virtually all large multipolar cells having a soma diameter >12 µm
among cultured retinal cells from newborn rats were immunoreactive to HPC-1/Syntaxin, whereas only 5% of large neurons expressed Thy1.1, a
ganglion cell marker. The present immunohistological data are all
consistent with those of Wexler et al. (1998)
. A
Nomarski photomicrograph of a recorded amacrine cell (Fig.
1D) and its fluorescent (Fig. 1E) image are shown
as an example.
Recording procedure
A coverslip to which cultured cells had adhered was placed in a
recording chamber, and the chamber was mounted on the stage of an
inverted microscope equipped with Nomarski optics (IX-70, Olympus,
Japan) and a ×60 objective lens. The chamber was continuously superfused with solutions gravity-fed at a rate of ~1 ml/min at room
temperature (~25°C). Membrane voltages and currents were recorded
by a patch-clamp method in the whole cell configuration (Hamill
et al. 1981). The patch pipette was made of Pyrex tubing pulled
on a micropipette puller (P-87, Sutter Instrument, Novato, CA). The
recording pipette was connected to the input stage of a patch clamp
amplifier (CEZ-2400, Nihon Kohden, Japan, and Axopatch 200B, Axon
Instruments, Foster City, CA). An Ag-AgCl wire connected to the bath
via a ceramic bridge served as an indifferent electrode. The pipette
resistance was ~10 M
when filled with pipette solution. The input
capacitance (~50 pF) and the series resistance (~20 M
) were
measured by the built-in circuit of the patch-clamp amplifier and
electrically compensated as much as possible (series resistance
60%). The junction potential was measured under each recording condition and the membrane voltages were corrected for the junction potential. Recorded signals were low-pass filtered (Bessel filter, cutoff frequency 5 kHz) and sampled at 10 kHz with a DigiData 1200 interface and pCLAMP 7 software (Axon Instruments). Data were analyzed
with Igor Pro software (WaveMetrics, Lake Oswego, OR).
Solutions
The standard external solution for the current-clamp experiments
contained (in mM) 135 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4), and the
standard pipette solution contained (in mM) 10 NaCl, 130 K gluconate, 1 CaCl2, 1.1 EGTA, 10 HEPES, and 2 ATP-Na2 (pH 7.2). When measuring
Na+ currents under the voltage clamp, we used an
external solution containing (in mM) 135 NaCl, 2.5 CsCl, 4 CoCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4), and a pipette solution containing (in mM) 110 N-methyl-D-glucamine (NMDG)-Cl, 20 NaCl, 20 TEA-Cl, 10 BAPTA, 10 HEPES, and 5 ATP-Tris (pH 7.2) to suppress both
K+ and Ca2+ currents. When
measuring Ca2+ currents, we used an external
solution ("Ca2+-containing solution")
containing (in mM) 115 NaCl, 2.5 CsCl, 20 TEA-Cl, 2 CaCl2, 1 MgCl2, 10 HEPES,
and 10 glucose (pH 7.4) and 1 µM TTX, and a pipette solution
containing (in mM) 10 Na gluconate, 130 Cs methanesulfonate, 10 TEA-Cl,
1 CaCl2, 1.1 EGTA, 10 HEPES, and 2 ATP-Na2 (pH 7.2) to suppress both
Na+ and K+ currents. To
suppress Ca2+ currents, external
Ca2+ of Ca2+-containing
solution was substituted by equimolar Co2+
("Co2+-containing solution"). TTX (Sankyo,
Japan), nifedipine (Sigma), diltiazem (Sigma), -conotoxin GVIA
(Alomone, Israel) and
-agatoxin IVA (Alomone) were dissolved into
the external solution and applied by pressure from a puffer pipette or
by a gravity feeding system.
Verification of space clamp and estimation of membrane parameters
It seems hard to voltage-clamp the long and narrow dendrites of cultured amacrine cells uniformly (Fig. 1, D and E), but we have evidence that at least the soma was clamped satisfactorily (Fig. 2). Here, two recording pipettes were placed on the soma of the same cell and made in the whole cell configuration. A command voltage was given by the first pipette connected to an amplifier that was set in the voltage clamp mode, and the membrane voltage was recorded by the second pipette connected to another amplifier set in the current-clamp mode. The amplitude of the recorded signal (Fig. 2B) was almost identical (>95%) to that of the command signal (Fig. 2A). The rising and falling phases of the recorded signals were delayed by ~5 ms due to the remaining stray capacitance.
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Membrane parameters were determined from the voltage change evoked by
injecting a 20 pA negative current from a patch pipette (Fig.
2C, a). The current-induced graded voltage change was fitted by a single exponential function (Fig. 2C, c). The good
agreement with a single exponential function indicates that in the
hyperpolarizing voltage range, no voltage-gated mechanism was
activated. In fact, the waveform of the hyperpolarizing voltage was not
affected by the application of 1 µM TTX and 2 mM
Co2+ (Fig. 2C, b). Similar
observations were made on six cells. The membrane capacitance, input
resistance and time constant were 40 ± 1 pF, 917 ± 16 M
and 38 ± 1 ms (means ± SE, n = 6). In
analyzing the depolarizing wave form induced by a brief current pulse,
a mirror image of the waveform induced by a negative current pulse of
the same current amplitude and duration was used as a control "passive waveform," because no "active" components were evoked by hyperpolarization.
The leakage current was measured by giving voltage commands in the
Co2+-containg solution (see
Solutions). The solutions contained
Co2+ to block Ca2+ current,
TTX to block Na+ current,
TEA+ and Cs+ to block
K+ current. The current-voltage (I-V)
relationship is shown in Fig. 2D. It was linear between
100 and
10 mV. The leak conductance was estimated as 0.8 ± 0.02 nS (n = 17) and the reversal potential was
66 ± 1 mV.
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RESULTS |
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Depolarization induced by positive current injection
To mimic the synaptic currents generated in amacrine cells,
extrinsic step currents were injected from a patch pipette into the
cultured amacrine cell recorded under the current-clamp condition (resting potential 65 ± 1 mV, n = 54). As shown
in Fig.
3A,
injection of a positive current of >12 pA to an amacrine cell with a
resting potential of
64 mV induced a depolarization (red line) that
exceeded the passive waveform (yellow line) in amplitude. The enhanced depolarization was seen on the rising phase, and it persisted after the
current was terminated. The deviation from the passive waveform
disappeared when 1 µM TTX and 2 mM Co2+ were
added to the medium (blue line), indicating that
Na+ and Ca2+ currents are
contributing to the amplitude enhancement. Figure 3B
illustrates the relation between the absolute peak potential and the
amount of injected current. Enhanced depolarization was noted by
positive current injection of more than +5 pA (membrane voltage more
positive than
55 mV; Fig. 3B, filled red circle). The
enhanced depolarization was absent in the presence of TTX and
Co2+ (Fig. 3B, open blue square). To
evaluate the effect of TTX on the enhanced depolarization, we applied
TTX (1 µM) alone (Fig. 3C). TTX dramatically reduced the
enhanced depolarization (Fig. 3C). The application of
Co2+ (2 mM) alone also blocked the enhanced
depolarization significantly (data not shown). These observations
suggested that TTX- and Co2+-sensitive components
in concert contributed to the enhanced depolarization. Figure
3D illustrates the relation between the absolute peak
potential and the amount of injected current in nine cells. The
enhanced depolarization appeared at membrane voltages more positive
than
55 mV. Simultaneous application of TTX and
Co2+ blocked the response enhancement completely,
and the relation became linear (Fig. 3D).
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Deviation from the passive waveform was more pronounced when more current was injected (Fig. 3E, +30 pA, red line). Plateau depolarization lasted long after the termination of the positive current injection and action potentials of various amplitudes were superimposed on the sustained depolarization. During the sustained depolarization, injection of a brief hyperpolarizing current terminated the plateau depolarization and the membrane voltage returned close to the resting level (data not shown). Suprathreshold sustained depolarization triggered by the brief positive current was sensitive to both TTX (Fig. 3E) and Co2+ (Fig. 3F). TTX suppressed the action potentials as well as the sustained depolarization (Fig. 3E, green line). Action potentials were blocked by TTX in all recorded cells (n = 17), and the sustained depolarization was significantly suppressed (16 of the 17 cells). The sustained depolarization was also suppressed partially by 2 mM Co2+ (Fig. 3F, purple line, n = 3). Co-application of TTX and Co2+ suppressed the sustained depolarization nearly completely (Fig. 3E, blue line, n = 11), and the waveform was fitted by the passive waveform (Fig. 3F, yellow line). These findings strongly suggest that both the TTX-sensitive current and Ca2+ current (ICa) contribute to the sustained depolarization.
Similar voltage responses to current injection were observed in 47 of the 54 cells examined, and the duration of the sustained depolarization was 170 ± 10 ms (ranging between 150 ms and 2 s). The remaining seven cells did not show the enhanced and sustained depolarization.
Persistent inward currents
TTX-SENSITIVE PERSISTENT INWARD CURRENT.
To estimate the effect of TTX on the current of amacrine cells, they
were voltage clamped by the whole cell patch-clamp technique in the
presence and absence of TTX (Fig.
4A). TTX
(1 µM) application not only blocked transient inward current but also
increased the sustained outward current (Fig. 4A,
left). Figure 4A, right, illustrates the relation
between the membrane potential and the persistent current amplitude,
measured at 200 ms after the onset of the command pulse, in the
presence and the absence of TTX (n = 6). TTX
application increased the amplitude of net outward current obviously
between 27 and +23 mV. These observations strongly suggest the
presence of a TTX-sensitive persistent inward current.
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TTX-SENSITIVE PERSISTENT INWARD CURRENT WAS CARRIED BY NA+. The TTX-sensitive persistent inward current reversed its polarity near the equilibrium potential of Na+ (ENa). The reversal potential of the persistent current (+24 ± 2 mV, n = 8) measured with a pipette solution containing 50 mM Na+ was close to ENa (+25 mV). When the pipette solution containing 20 mM Na+ (ENa = +48 mV) was used, the reversal potential shifted to +42 ± 2 mV (n = 7, Fig. 5). These results clearly indicate that Na+ carried the persistent current (INaP).
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CA2+ CURRENTS.
As shown in Fig. 3F, Co2+-sensitive
components also contributed to the enhanced and the sustained
depolarization. To isolate Ca2+ currents,
K+ currents were blocked by
TEA+ and Cs+, and
Na+ currents were blocked by TTX (1 µM). As
shown in Fig. 6A, the persistent inward current was observed in the
Ca2+ (2 mM)-containing solution; in contrast, the
persistent inward current was completely blocked in the
Co2+ (2 mM)-containing solution and only the
passive leak current (cf. Fig. 2D) remained. The
I-V relationship of the cell in Fig. 6A is shown
in Fig. 6B. To characterize
ICa, we isolated it by computer
subtraction of the current recorded in
Co2+-containing solution from the current
recorded in the Ca2+-containing standard solution
in the following experiments. The average I-V relationship
of ICa is shown in Fig. 6C
(n = 9). ICa was
activated at voltages more positive than 50 or
40 mV, similar to
the voltages at which INaP was
activated. Peak current amplitude was
178 ± 17 pA.
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DISCUSSION |
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The present study demonstrates that amacrine cells from the
newborn rat retina, cultured for 10-14 days in vitro, possess INaP and several types of
ICa as well as fast
INa.
INaP and
ICa significantly contribute to the
enhanced and the sustained depolarization of amacrine cells. They are
activated by membrane depolarization to voltages more positive than
55 mV and, once activated, are maintained as long as depolarization
lasted. Because of the preceding properties, depolarization produced by
injection of an extrinsic current is boosted both in length and
amplitude, and generation of the action potentials is facilitated.
Under the current-clamp conditions,
INaP and
ICa work in concert. Therefore
application of a blocker selective to either one of the two current
components results in suppressing not only its target but also another current.
The enhanced depolarization was observed at membrane voltages more
positive than 55 mV, but neither
INaP nor
ICa was not clearly detected at
55
mV. Under our voltage-clamp recording conditions, the noise level was
at a few picoamperes. Because the input resistance of amacrine cells
was ~1 G
, inward current of 1 pA depolarizes the cell by 1 mV. In
this sense, measurement of the membrane voltage under the current clamp
is a more sensitive method to detect a small amount of membrane
current. Thus it is highly likely that
INaP or
ICa of a few picoamperes are activated at about
55 mV, which induces enhanced depolarization in a cell under
current clamp.
Significance of the enhanced and the sustained depolarization in signal propagation in the inner plexiform layers
In amacrine cells having dendritic arbors spanning hundreds of
micrometers, propagation of excitatory synaptic signals along the
dendrite is important for processing input signals and forming the
receptive field of neurons in the inner retina. Cook et al. (1998) showed that long-distance lateral inhibition (spreading 250 µm) in the large-field amacrine cells of the mudpuppy is
mediated by a TTX-sensitive mechanism and that a local transient
inhibition of ganglion cells does not require a voltage-sensitive
mechanism. Cook and McReynolds (1998)
suggested that
lateral inhibition in the inner retina is mediated by a sustained
spiking activity in GABAergic amacrine cells. Taylor
(1999)
also showed that TTX attenuates surround inhibition in
rabbit retinal ganglion cells. They proposed that TTX suppressed the
propagating action potentials, and the spread of signals within a
large-field amacrine cell was limited.
In amacrine cells, it has been shown that TTX and
N(2,6-dimethylphenyl carbamoyl methyl) triethylammonium
(QX314) significantly reduce the amplitude of both
light-evoked graded potentials and action potentials
(Bloomfield 1996; Watanabe et al. 2000b
).
Our results show that TTX suppressed
INaP and as a consequence reduced the
amplitude of graded depolarization. Thus it seems reasonable to
conclude that amplification of small potential changes, such as EPSPs,
to the enhanced and the sustained depolarization by INaP and
ICa plays an important role in signal
propagation and assists in generating action potentials in the
large-field amacrine cells of the rabbit and in some goldfish amacrine
cells. It is reasonable to speculate that the suppression of
INaP narrows lateral inhibition
mediated by GABAergic amacrine cells. In the rat, amplification should
be operating at least in GABAergic amacrine cells because all GABAergic
amacrine cells in our culture possessed
INaP and ICa.
Currents recorded in the present study perhaps originated mainly in the soma and nearby dendritic stems since it is unlikely that dendrites were uniformly voltage clamped judging from their thin and long structure. By extrapolating the present observation, it is tempting to speculate that INaP and ICa of similar characteristics are also present in the dendritic membrane. If they really exist in the dendrites, they should contribute to the spread of subthreshold depolarization over a wide dendritic field of an amacrine cell. In conclusion, INaP and ICa play an important role in the lateral spread of signals in the inner plexiform layer.
Contribution of INaP and ICa to the enhanced and the sustained depolarization
In the present study, we showed that
INaP and
ICa boost a small and brief
depolarization produced in cultured amacrine cells. The boosting action
of the TTX-sensitive sustained current has been demonstrated in several
preparations. Llinás and Sugimori (1980) have
demonstrated an example in the cerebellar Purkinje cell. A positive
extrinsic current injected into a cerebellar Purkinje cell evoked a
plateau depolarization that outlasted the duration of current injection
by several hundred milliseconds. The plateau potential was not blocked
by Cd2+, but it was abolished by removing the
extracellular Na+ or adding TTX. They proposed
that the action potential was generated by the Hodgkin-Huxley-type fast
Na+ conductance that inactivates rapidly and that
the plateau response was generated by the persistent
Na+ conductance. In fact, Raman and Bean
(1999)
showed that a TTX-sensitive persistent current
contributed to spontaneous activities of action potentials in mouse
cerebellar Purkinje cells. Stuart and Sakmann (1995)
have shown that the TTX-sensitive current generated in the soma and the
axon amplifies a subthreshold EPSP of neocortical pyramidal neurons.
The fact that the amplification mechanism was sensitive to TTX and had
a slow time course suggests that the underlying mechanism is
INaP. The enhanced and the sustained
depolarization we recorded in amacrine cells in the present study was
very similar to that described by Llinás and Sugimori
(1980)
in Purkinje cells at a supra-threshold level and by
Stuart and Sakmann (1995)
in cortical pyramidal neurons
at a sub-threshold level. In addition to
INaP,
ICa also contributed to the enhanced
and the sustained depolarization of amacrine cells in our study.
Morisset and Nagy (1999) reported that L-type
Ca2+ currents contribute to TTX-resistant
voltage-dependent plateau potentials recorded in the deep dorsal horn
neurons (DHNs) of the rat spinal cord in a slice preparation.
As the plateau potential was highly sensitive to dihydropyridine (DHP),
they concluded that the plateau potential of deep DHNs is supported
mainly by Ca2+ influx through the L-type
Ca2+ channels. The contribution of
INaP to the plateau potential of DHNs
was not examined because their solution always contained TTX to block
presynaptic action potentials.
We showed that INaP and ICa contribute to the enhanced and the sustained depolarization in cultured rat amacrine cells and that both of them are activated at near the resting membrane potential. These two components in concert contribute to the boosting mechanism of the graded depolarization.
Identification of INaP in amacrine cells
Feigenspan et al. (1998) reported a
TTX-sensitive slowly inactivating INa
as contributing to the interspike slow depolarization in acutely
dissociated mouse dopaminergic amacrine cells. This slowly inactivating
INa may be identical to the
INaP we recorded in the present study.
INaP has been identified in neurons of
the mammalian neocortex, thalamus, entorhinal cortex, hippocampus, and
cerebellum (Crill 1996
; French et al.
1990
).
In the present experiments, we demonstrated that INaP was activated at more negative voltages than the fast INa. However, we cannot tell whether INaP and fast INa flow through the same channel or not. Although there are still ambiguous interpretations of Na+ channel itself, difference in the kinetics and the activation voltage suggests that INaP effectively plays a different role from fast INa.
It might be argued that INaP is an
immature type of INa. As stated in
METHODS, we also followed the developmental changes of
cultured amacrine cells, and we used cells cultured >10 days in which
INa had fully developed. At least in
fish retina, INaP has been recorded in
mature neurons. Hidaka and Ishida (1998) reported
INaP as distinct from the fast
INa in the mature ganglion cells
acutely isolated from the goldfish. Watanabe et al.
(2000b)
also detected INaP in
amacrine cells of the mature goldfish slice preparation.
Another argument on the identity of the prolonged inward
Na+ current is whether it is the current carried
by a Na+-Ca2+ exchanger.
The Na+-Ca2+ exchanger was
shown to exist in amacrine cells cultured from chick embryos
(Gleason et al. 1995). It is activated by the
intracellular Ca2+ that flowed into the cell
through the voltage-activated Ca2+ channel. In
our preparation, however, this possibility is unlikely since the
sustained inward current was recorded in the presence of
Ca2+ channel blockers and was TTX sensitive.
Identification of ICa in amacrine cells
ICa was also detected in our
cultured amacrine cells. Pharmacological experiments revealed that
ICa consisted of several subtypes. Gleason et al. (1994) reported that the L-type current
is the major component of ICa in chick
amacrine cells in culture. However, they reported that nifedipine
reduced Cd2+-suppressible
Ca2+ currents by an average of 59%, and the
remaining current was not sensitive to other antagonists. In our own
preparation, the Cd2+-suppressible
ICa was not completely blocked by
nifedipine and
-conotoxin GVIA. It is known that the R-type
ICa shows resistance to any
pharmacological agent (Randall and Tsien 1995
;
Tottene et al. 1996
). Thus the DHP-,
-conotoxin GVIA-
and
-agatoxin IVA-resistant Ca2+ current in
the present study is likely to be the R-type
ICa. ICa of amacrine cells had an unusually
low activation voltage of approximately
50 mV, which is close to the
resting potential and more negative than the activation voltage of the
fast INa [see also, rat AII amacrine
cells (Boos et al. 1993
) and cultured chick amacrine
cells (Gleason et al. 1994
)]. The low activation voltage suggests a strong possibility that
ICa is also contributing to the
enhancement of graded depolarization at subthreshold level.
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ACKNOWLEDGMENTS |
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We are grateful to H. Satoh, N. Mukainaka, and Y. Yamada for technical assistance.
This work was supported in part by a grant from the Keio Health Counseling Center Foundation, by the Keio University Grant-in-Aid for Encouragement of Young Medical Scientists (A. Koizumi), by Research Grants for Life Sciences and Medicine from the Keio University Medical Fund and Keio Gijuku Academic Development Funds (S.-I. Watanabe), by a grant-in-aid for scientific research from the Japanese Ministry of Education, Science and Culture (06454715), and by a grant from Research for the Future Program of Japan Society for the Promotion of Science under the Project "Cell Signaling" (JSPS-RFTF97L00301) (A. Kaneko).
Present address of S.-I. Watanabe: Dept. of Physiology, Saitama Medical School, 38 Morohongo, Moroyama, Saitama 350-0495, Japan.
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FOOTNOTES |
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Address for reprint requests: A. Koizumi, Dept. of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (E-mail: amane{at}physiol.med.keio.ac.jp).
Received 17 August 2000; accepted in final form 6 April 2001.
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