Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
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ABSTRACT |
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Watanabe, Shu-Ichi,
Amane Koizumi,
Shinya Matsunaga,
Jonathan W. Stocker, and
Akimichi Kaneko.
GABA-Mediated Inhibition Between Amacrine Cells in the
Goldfish Retina.
J. Neurophysiol. 84: 1826-1834, 2000.
Retinal amacrine cells have abundant dendro-dendritic
synapses between neighboring amacrine cells. Therefore an amacrine cell has both presynaptic and postsynaptic aspects. To understand these synaptic interactions in the amacrine cell, we recorded from amacrine cells in the goldfish retinal slice preparation with perforated- and
whole cell-patch clamp techniques. As the presynaptic element, 19% of
the cells recorded (15 of 78 cells) showed spontaneous tetrodotoxin
(TTX)-sensitive action potentials. As the postsynaptic element, all
amacrine cells (n = 9) were found to have GABA-evoked responses and, under perforated patch clamp, 50 µM GABA
hyperpolarized amacrine cells by activating a
Cl conductance. Bicuculline-sensitive
spontaneous postsynaptic currents, carried by
Cl
, were observed in 82% of the cells (64 of
78 cells). Since the source of GABA in the inner plexiform layer is
amacrine cells alone, these events are likely to be inhibitory
postsynaptic currents (IPSCs) caused by GABA spontaneously released
from neighboring amacrine cells. IPSCs were classified into three
groups. Large amplitude IPSCs were suppressed by TTX (1 µM),
indicating that presynaptic action potentials triggered GABA release.
Medium amplitude IPSCs were also TTX sensitive. Small amplitude IPSCs
were TTX insensitive (miniature IPSCs; n = 26). All of
spike-induced, medium amplitude, and miniature IPSCs were
Ca2+ dependent and blocked by
Co2+. Blocking of glutamatergic inputs by
DL-2-amino-phosphonoheptanoate (AP7; 30 µM) and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 2 µM) had almost no
effect on spontaneous GABA release from presynaptic amacrine cells. We
suggest that these dendro-dendrotic inhibitory networks contribute to
receptive field spatiotemporal properties.
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INTRODUCTION |
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The amacrine cells are a group
of third-order neurons that exhibit a wide variety of light-evoked
responses and morphological subtypes (Chan and Naka
1976; Hidaka et al. 1993
; Kaneko
1973
; Kolb 1997
; Kujiraoka et al.
1988
; MacNeil and Masland 1998
; Murakami and Shimoda 1977
; Teranishi et al. 1987
;
Vaney 1990
; Wagner and Wagner 1988
;
Watanabe and Murakami 1985
). GABAergic cells constitute the majority of the amacrine cell population (Marc 1992
,
1997
; Muller and Marc 1990
;
Yazulla 1986
) and are generally believed to mediate
lateral inhibitory interactions between vertical excitatory channels
composed of bipolar and ganglion cells. In fact, different GABA
receptor types have been identified in axon terminals of bipolar cells
(Greferath et al. 1993
; Lin and Yazulla
1994
) and in ganglion cell dendrites (Lin and Yazulla
1994
; Wässle et al. 1998
), and
GABA-mediated inhibition has been demonstrated in bipolar cells
(Euler and Wässle 1998
; Karschin and
Wässle 1990
; Kondo and Toyoda 1983
;
Qian et al. 1997
; Suzuki et al. 1990
;
Tachibana and Kaneko 1988
) and ganglion cells
(Cohen et al. 1989
; Gao and Wu 1998
;
Protti et al. 1997
).
A unique feature of amacrine cells is the abundance of serial
conventional synapses found between the dendrites of amacrine cells
(Dowling 1987). In spite of their numerical dominance in the inner plexiform layer, there have been no direct systematic physiological assessments of synapses between amacrine cells. There
have been some suggestions from recordings of ganglion cells that
GABAergic inhibitory synapses between amacrine cells operate (Roska et al. 1998
; Zhang et al. 1997
).
However, direct recording of GABAergic input to amacrine cells was
reported only from a type of amacrine cells in the tiger salamander,
the wide-field amacrine cell (Roska et al. 1998
).
Another feature of some amacrine cells is generation of action
potentials superimposed on light-evoked slow potentials (Barnes
and Werblin 1986
; Feigenspan et al. 1998
; Miller and Dacheux 1976
; Murakami and Shimoda
1977
). The action potentials are shown to contribute to the
transmitter release from amacrine cells, but its contribution is
reported to be only partial (Bieda and Copenhagen 1999
;
Hartveit 1999
; Protti et al. 1997
).
Cook and McReynolds (1998)
suggested that surround
inhibition observed in ganglion cells was mediated by action potentials
generated in amacrine cells. It is the aim of the present study to
obtain direct evidence on the physiological properties of GABAergic
serial synapses between retinal amacrine cells and contribution of
action potentials in release of GABA from amacrine cells. We studied amacrine cells in the slice preparation of the goldfish retina using
patch-clamp techniques, and found 1) an extensive inhibitory network mediated by GABAergic synapses, 2) a large release
of GABA triggered by action potentials generated in presynaptic
amacrine cells, and 3) spontaneous release of GABA from
presynaptic amacrine cells independent of the glutamatergic input to them.
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METHODS |
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Preparation
All experiments were performed on retinal slices prepared from goldfish. The care of the animals was in accordance with Guideline for the Care and Use of Laboratory Animals of Keio University School of Medicine, and our experiments have been approved by the University Animal Welfare Committee. Each goldfish was dark adapted for more than 2 h, rapidly decapitated, double pithed, and eyes enucleated and hemisected. To liquify the vitreous humor the eyecup was soaked for 10 min in hyaluronidase (0.07 mg/ml, Type I-S, 300 units/mg, Sigma)-containing Ringer solution composed of (in mM) 125 NaCl, 2.6 KCl, 2.5 CaCl2, 1.0 MgCl2, 10 HEPES, and 16 glucose, pH 7.8. The eyecup was then rinsed with the Ringer solution without hyaluronidase. The retina was carefully removed from the pigment epithelium and placed photoreceptor side up on a round filter paper (cellulose nitrate, pore size, 0.2 µm, 13 mm diam, Advantec Toyo, Tokyo, Japan). The liquified vitreous humor was first absorbed from the back of the filter paper with tissue paper, then the filter paper with the retina was placed on a syringe filter holder (SX0001300, Millipore) with the retina side up. Suction was applied so that the remaining vitreous humor was more thoroughly removed and the retina became firmly attached to the filter paper. The retina was cut together with the filter paper in 150-µm slices by a home-made slicer. Slices were fixed to the glass bottom of the recording chamber (cut surface parallel to the bottom) with a small amount of silicone grease (Dow Corning) at both ends. All experiments were performed under normal room illumination and room temperature of 20-25°C.
Recording procedure
Slices were continuously perfused with solutions by gravity feed
and viewed using a microscope (Optiphot-2 Nikon or BHS-RFC Olympus)
equipped with a water-immersion lens (WI-40 Nikon or WPlanFL40XUV
Olympus), epifluorescence illumination and a camera (FX-35AUFX-II Nikon
or PM-20 Olympus). Patch and pressure ejection pipettes were pulled
(P-87 Sutter, or PP-83 Narishige) from borosilicate filament tubing
(1.5 mm OD; 0.87 mm ID; Hilgenberg, Malsfeld, Germany). The tip
diameter of the patch pipette was 1-2 µm giving a resistance of
approximately 3-7 M when filled with the pipette solution and
measured in Ringer solution. The tip of the patch pipette was coated
with Apiezon wax (Apiezon Products) or dental wax (GC corporation,
Tokyo, Japan) to reduce stray capacitance. Several kinds of pipette
solution were used according to the type of experiment. The pipette
solution for perforated patch clamp was composed of (in mM) 120 KCl, 10 NaCl, 5 EGTA, and 10 HEPES, pH 7.3, and contained 15 µg/ml gramicidin
D. The gramicidin D ionophore is thought to be impermeable to
Cl
(Akaike 1996
). Gramicidin was
first dissolved in dimethyl sulfoxide at 37.5 mg/ml and then dissolved
into the pipette solution to make the final concentration. The tip of
the patch pipette was filled with gramicidin D-free pipette solution,
and gramicidin D pipette solution was then back filled immediately
before use. The pipette solution for the whole cell clamp
(Hamill et al. 1981
) contained equimolar CsCl instead of
KCl to block outward currents. A part of Cl
of
the pipette solution was replaced with equimolar gluconate or
methansulfonate ions when used in experiments to measure the reversal
potential of the GABA-induced current and the spontaneous postsynaptic
current. All pipette solutions contained 0.2% Lucifer yellow to
visualize cells under the epifluorescence microscope.
The recording pipette was connected to a patch-clamp amplifier (L/M
EPC7 List, Darmstadt, Germany or CEZ-2300 Nihon Kohden, Tokyo, Japan).
An Ag-AgCl electrode was connected to the superfusate via ceramic- or
agarose-bridge and served as an indifferent electrode. Signals were
filtered with a bandwidth of 0-2 or 3 kHz by a built-in filter of the
patch-clamp amplifier, recorded on a thermal array recorder (WR7600 or
WR7700 Graphtec, Yokohama, Japan; a bandwidth, 0-5 kHz), stored on
magnetic tape (A-45 or PC204A Sony, Tokyo, Japan; bandwidth, 0-10
kHz), and digitized by a 12-bit A/D converter. Command voltages were
generated by a personal computer (DeskproXE466 Compaq or PC-9801RX NEC,
Tokyo, Japan). In whole cell voltage-clamp recordings, capacitance
(approximately 10-50 pF) was electrically compensated as much as
possible, but complete elimination was unachievable. The series
resistance varied from about 10 to 50 M. As the voltage error
produced by the series resistance was <10%, it was not compensated.
Pharmacology
Sources of chemicals were as follows: gramicidin D, bicuculline methochloride, and strychnine were purchased from Sigma; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-phosphonoheptanoate (AP7) from Research Biochemicals; tetrodotoxin (TTX) from Sankyo (Tokyo, Japan); and Bay K8644 from Funakoshi (Tokyo, Japan). Solution containing Co2+ did not contain Ca2+. CNQX and AP7 were mixed directly into the Ringer solution. Pharmacologic agents were applied either through a puffer pipette (2-5 µm diam) by pressure or Y-tube by gravity.
Cell identification
Cells were classified as ON, OFF, or
ON-OFF type by dendritic structure
(ON type, dendrite in sublamina b; OFF type,
dendrite in sublamina a; ON-OFF type, dendrite
in both sublamina a and b). As we used the slice preparation, our
recordings were exclusively from pyriform amacrine cells and not from
fusiform amacrine cells according to the morphological classification
of the carp amacrine cells by Teranishi et al. (1987).
Although Teranishi et al. (1987)
reported Lucifer yellow
coupling within the same type of the carp amacrine cell, dye coupling
between amacrine cells was not observed in the present study.
Data analysis
As the properties of GABA-induced currents and spontaneous postsynaptic currents were almost identical in all amacrine cells of different morphological subtypes, we pooled all data without subtype classification in the following analyses.
AMPLITUDE OF GABA-INDUCED CURRENT. The current-voltage relation of GABA-induced current was obtained by measuring the response peak amplitudes. Then, the reversal potential was determined.
QUANTITATIVE ANALYSIS OF THE SPONTANEOUS POSTSYNAPTIC CURRENTS.
As the spontaneous events occurred randomly, we used mean current for
quantitative analyses. The mean current was calculated by one of the
following two methods. In method 1, an amplitude histogram
(to be referred to as an ensemble histogram) was calculated for a 5- to
20-s segment of current record (usually 10 to 100 or more events
occurred within 1 s) using pCLAMP software (Axon Instruments) at
10 kHz. Next, the distribution of the baseline noise was subtracted
from the ensemble histogram to yield the amplitude histogram of the
spontaneous events. To do this, a Gaussian curve that fit to the silent
side of the ensemble histogram (where the spontaneous currents were not
contributing) was generated by a software (DeltaGraph, DeltaPoint or
Igor, WaveMetrics), and the full Gaussian curve was subtracted from the
ensemble histogram. The amount of charge carried by the spontaneous
currents was calculated by integrating the difference histogram, and
the mean current of the postsynaptic current was obtained by dividing
the charge by the time of the recording segment. Method 2 was the time integration of the current in the raw data. The zero
current level was determined by eye on the computer monitor. The
current integral was divided by the time of the recording segment. Both
methods gave very similar values (mostly method 1 was used
and method 2 was applied to relatively short records). Then,
the current-voltage relation of the mean current was obtained for each
cell and the reversal potential was determined. For analysis of
reversal potentials, ionic activity was used instead of concentration
and liquid junction potential was corrected.
AMPLITUDE HISTOGRAM OF THE SPONTANEOUS POSTSYNAPTIC CURRENTS. From the digitized record each spontaneous postsynaptic event was identified by eye and peak amplitude (from the onset to peak) was measured on the computer monitor, using pCLAMP software (Axon Instruments). Records from cells that did not show a clear onset and peak of the event were not used for amplitude histogram analysis.
Data are reported as means ± SE. ![]() |
RESULTS |
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Effect of GABA is inhibitory on amacrine cells
Application of exogenous GABA (50 µM, applied by a puffer
pipette) induced hyperpolarization in all amacrine cells
(n = 9; 4 ON-type cells, 2 OFF-type cells, 3 ON-OFF-type cells) when
recorded by a gramicidin perforated patch, indicating that GABA is
functioning as an inhibitory transmitter in all amacrine cell types
(Fig. 1A). The resting
potential was 57 ± 3 (SE) mV, almost the same voltage as
previously reported for light-responding amacrine cells in the intact
retina recorded by intracellular microelectrode (Djamgoz et al.
1996
; Watanabe and Murakami 1985
).
Hyperpolarization induced by 50 µM GABA measured 9 ± 2 mV (Fig.
1E). Spontaneous action potentials (see the next section)
were suppressed by the GABA-induced hyperpolarization (Fig.
1A). The reversal potential of the GABA-induced current
estimated under voltage clamp was
76 ± 4 mV (n = 6; 4 ON-type cells, 1 OFF-type cell, 1 ON-OFF-type cell, Fig. 1B).
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The reversal potential of the GABA-induced current was dependent on the
intracellular Cl concentration. When the whole
cell configuration was established in the cell shown in Fig.
1A by breaking the perforated patch membrane, GABA induced a
tonic depolarization in accordance with an increase of intracellular
Cl
concentration by the intracellular dialysis
with the pipette solution
([Cl
]i, 32 mM, Fig.
1C). Under this condition, GABA-induced current reversed its
polarity at about
30 mV (Fig. 1D; calculated equilibrium potential for Cl
,
ECl, was
32 mV). The relation
between GABA-induced current and the voltage was almost linear (Fig.
1F), and the reversal potential was nearly identical to
ECl calculated by the Nernst equation
(Fig. 1G). These observations indicate that GABA selectively opens a Cl
-permeable channel. The intracellular
Cl
concentration in amacrine cells was
estimated to be lower than 10 mM from the reversal potential of the
GABA-induced current obtained in the perforated patch recordings. The
GABA-induced current was suppressed by 100 µM bicuculline (data not
shown), suggesting that it was mediated by GABAA receptors.
The effect of GABA was identical for all amacrine cells, regardless of morphological subtype. Similar results were obtained from all cells that showed fluorescence only from the soma, possibly their dendrites lost in the preparation process (n = 3, Fig. 1F, inset). These findings indicate that GABA receptors are widely distributed over the cell surface, the soma, and dendrite, of various types of amacrine cells.
Action potential of amacrine cells
The spontaneous action potentials seen in current-clamp recordings
(see Fig. 1A) were also seen even when the cell was
"voltage clamped" (Figs. 1, B and D, and 2).
These activities were identified as the current due to regenerative
action potentials ("spike currents") because 1) the
amplitudes were large (>100 pA) and almost constant, and they had a
rapid time course (Fig. 2A,
top and bottom traces with expanded time scale,
*), 2) the current was inward and seen at the holding
potential between approximately 60 and
40 mV (Fig. 1, B
and D; see also Fig. 2B). They were observed in
19% of cells recorded (15/78) including all morphological subtypes (ON-type cell, 5/17; OFF-type cell, 5/23;
ON-OFF-type cell, 5/38). Furthermore, under the
voltage-clamp condition, depolarization evoked a transient inward
current in all spiking and some nonspiking amacrine cells
(ON-type cell, 9/17; OFF-type cell, 13/23;
ON-OFF-type cell, 8/38). Both spike currents and the
transient inward currents evoked by depolarization were suppressed by 1 µM TTX (not shown). Under voltage clamp, the action potential might
be generated somewhere remote from the soma where the membrane voltage
escaped from the holding voltage due to an incomplete space clamp.
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GABAA receptor-mediated spontaneous postsynaptic currents in amacrine cells
In addition to the spike current, other spontaneous events were seen in whole cell voltage-clamp recordings. These events were smaller in amplitude and slower in the time course than the spike current. The amplitude varied from one event to another, and they were reversibly suppressed by 100 µM bicuculline (Fig. 2A, top trace and bottom traces with expanded time scale), suggesting that they were mediated by GABAA receptors. These events, interpreted as spontaneous postsynaptic currents, were observed in 82% of cells recorded (64/78) including all morphological subtypes (ON type, 14/17; OFF type, 21/23; ON-OFF type, 29/38).
We also tested strychnine (2 µM), CNQX (5 µM), and AP7 (50 µM) to
determine whether glycine,
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate
(KA) or N-methyl-D-aspartate (NMDA) receptors significantly contributed to the generation of the spontaneous postsynaptic currents (cf. Dixon and Copenhagen 1992
).
The spontaneous postsynaptic currents (determined as mean currents, see
METHODS) were largely suppressed by bicuculline (by 78 ± 7%, n = 8), but slightly by other antagonists
(7 ± 3% by strychnine, n = 5; 13 ± 7% by
CNQX, n = 9; 8 ± 3% by AP7, n = 9, Fig. 3). Despite nonuniform coverage
of dendritic fields by puffer-applied antagonists, there was an obvious
difference among the potencies.
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The reversal potentials of spontaneous postsynaptic currents were
dependent on [Cl]i
(Fig. 4). The current was inward at
membrane voltages more negative than
ECl (about
30 mV,
[Cl
]i, 32 mM), and
outward at membrane voltages more positive than ECl in a cell shown in Fig.
4A. At the voltage slightly more positive than
ECl, spontaneous inward currents (
27
mV, Fig. 4A,
) were seen together with the reversed
outward currents (
). It seems likely that these inward currents
represent cation-mediated postsynaptic events, most likely evoked by
spontaneous glutamate release from bipolar cells (Gao and Wu
1999
; Taylor et al. 1995
; Tian et al. 1998
). Actually, application of 50 µM glutamate evoked
currents in all amacrine cells (data not shown). The current-voltage
relation of the mean current was almost linear (Fig. 4B),
and the reversal potential measured with various
[Cl
]i was close to
ECl (Fig. 4C). Slight
deviations from ECl (solid line) to
the depolarizing direction may reflect a small contamination of cation
currents, possibly glutamatergic inputs from bipolar cells. The
reversal potential followed well when
[Cl
]o was changed (Fig.
4, D and E). All these data strongly suggest that
the spontaneous postsynaptic currents in amacrine cells are mostly
mediated by GABAA receptors, and that they
represent IPSCs. Spontaneous postsynaptic currents with identical
properties were also observed in some cells that seemed to have lost
their dendrites (3 of 5 cells recorded; Fig. 4B,
inset), suggesting that GABAergic synapses are also located
on the soma (see Fig. 1F, inset).
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Since amacrine cells are the primary and perhaps only sources of GABA in the inner plexiform layer, it is reasonable to assume that most of the spontaneous postsynaptic currents were IPSCs evoked by GABA released from amacrine cells. We further conclude that there is a physiologically relevant network within the inner plexiform layer based on GABA-mediated inhibition between amacrine cells.
Control of GABA release in presynaptic amacrine cells
CONTRIBUTIONS OF SPONTANEOUS ACTION POTENTIALS TO GABA RELEASE FROM
AMACRINE CELLS.
Spontaneous postsynaptic currents consisted of two groups: a
TTX-sensitive group and a TTX-insensitive group (Fig.
5). The amplitude of TTX-sensitive
spontaneous postsynaptic current was large (approximately >50 pA
at holding potential of 61 or +59 mV, or >67 pA at
81 or +79 mV,
[Cl
]i, 130 mM; Figs. 5,
6, and 8). When 1 µM TTX was perfused, postsynaptic events larger
than 50 pA were suppressed (Fig. 5). In addition, medium-sized events
(amplitude about 20-50 pA) seemed to be suppressed slightly. Small
events remained relatively unaffected. These results suggest
1) that the TTX-sensitive large spontaneous postsynaptic current was generated by the release of a large amount of GABA, driven
by an action potential in the presynaptic amacrine cells, 2)
that the TTX-sensitive medium spontaneous postsynaptic current was
generated by spontaneous GABA release independent of action potentials
from the presynaptic amacrine cell, but by mechanism sensitive to TTX
(Watanabe et al. 2000
), or by dendritic small amplitude
action potential (Miller and Dacheux 1976
), and
3) that the TTX-insensitive spontaneous postsynaptic current
(miniature IPSCs) was generated by spontaneous GABA release (cf.
Bieda and Copenhagen 1999
). Similar results were
obtained in 10 other cells.
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RELEASE OF GABA BY THE PRESYNAPTIC AMACRINE CELL IS CA2+ DEPENDENT. All of the TTX-sensitive large spontaneous postsynaptic currents, medium-sized TTX-sensitive spontaneous postsynaptic currents, and the TTX-insensitive spontaneous postsynaptic currents were Ca2+ dependent. Application of 4 mM Co2+ to the bath suppressed spontaneous postsynaptic currents almost completely (the mean postsynaptic current was suppressed by 91 ± 3%, n = 5, Fig. 6). On the other hand, the frequency and amplitude of the spontaneous postsynaptic currents were enhanced by an application of 10 µM Bay K8644 (data not shown). Thus it is likely that GABA is released by presynaptic amacrine cells in a Ca2+-dependent manner. Perhaps GABA release is triggered by Ca2+ entering into the presynaptic amacrine cell via L-type Ca2+ channels.
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SPONTANEOUS RELEASE OF GABA FROM THE PRESYNAPTIC AMACRINE CELLS IS
INDEPENDENT OF GLUTAMATERGIC INPUT TO THE PRESYNAPTIC CELL.
As a small fraction of the spontaneous postsynaptic currents are
thought to be carried by cation (Fig. 4A), we examined
whether spontaneous release of GABA is driven by depolarization of
presynaptic amacrine cells induced by the spontaneous glutamatergic
input from bipolar cells to the presynaptic cell. In the following
experiment, no clear hint suggesting the glutamatergic drive was
obtained. Bath application of AP7 (30 µM) and CNQX (2 µM), together
with strychnine (2 µM) did not affect the current significantly
(n = 8, Fig. 7). It is
therefore likely that the membrane potential of the presynaptic
GABAergic amacrine cell fluctuates spontaneously without glutamatergic
influence. Spontaneous generation of action potentials in the absence
of synaptic inputs was reported in dopaminergic interplexiform amacrine
cells dissociated from the mouse retina, and this activity was
abolished by GABA or glycine (Feigenspan et al. 1998;
Gustincich et al. 1997
).
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DISCUSSION |
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Postsynaptic inhibition in the amacrine cell
Anatomical studies have shown that GABAergic cells dominate the
amacrine cell population in the goldfish retina (Marc
1992; Marc et al. 1978
; Yazulla
1986
). Presence of GABAA receptors in amacrine cells has been also shown by immunocytochemistry (Lin and Yazulla 1994
). Inhibitory GABAergic synapses between
amacrine cells have been suggested indirectly by recordings from
ganglion cells and directly in some type of amacrine cells
(Roska et al. 1998
; Zhang et al. 1997
).
We have demonstrated directly that goldfish amacrine cells not only use
GABA as a transmitter, but that most and perhaps all also receive
GABAergic input. In the present study we obtained evidence that GABA
mediates inhibitory signals between amacrine cells. We recorded
GABA-induced currents in amacrine cells of the goldfish retinal slice,
and a spontaneous postsynaptic activity that is also mediated by GABA.
We infer that both of these responses were mediated by
GABAA receptors as the currents, carried by
Cl
, were inhibited by bicuculline. Perforated
patch experiments demonstrated that GABA produces hyperpolarizing
voltage changes in amacrine cells. In fact, GABA suppressed spontaneous
action potentials. Our results demonstrate that GABAergic synapses
between amacrine cells likely function in key circuit processes in the goldfish retina, providing a physiological role for anatomically ubiquitous serial synapses.
To understand the role of this postsynaptic inhibition of the amacrine
cells in information processing, it will be important to know the
distribution of GABAA receptors on amacrine cell
dendrites and somas. Although the slice preparation is inappropriate to examine the spatial distribution of GABA receptors, we obtained direct
evidence that GABAA receptors are present not
only on the dendritic membrane, but also in the region close to the
soma as has been reported in the salamander (Cook and Werblin
1994; Maguire 1999
). We recorded GABA-induced
and spontaneous postsynaptic currents from cells that had apparently
lost their dendrites in the slice preparation. One of the imaginable
functions of GABA receptors in the primary dendritic stem or on the
soma is to shunt currents generated in the peripheral dendrites, thus
isolating every dendrite as an independent site of signal processing.
Functioning GABAergic synapses between amacrine cells have been
suggested by recordings from ganglion cells in the retina (Roska
et al. 1998
; Zhang et al. 1997
) and reported in
a wide-field amacrine cells of the tiger salamander (Roska et
al. 1998
). Such synapses have been also reported by
Gleason et al. (1993)
in cultured amacrine cells of the
chick retina. We show here that functioning GABAergic synapses are
common in the intact retina.
Control of GABA release from the amacrine cells
GABA release from presynaptic amacrine cells, as assayed both
TTX-sensitive and TTX-insensitive IPSCs, is dependent on
Ca2+ entry, possibly through L-type
Ca2+ channels, as shown for cultured chick
amacrine cells (Gleason et al. 1994). Spontaneous
release might occur when membrane potential of the presynaptic amacrine
cell is depolarized to the activation level of the L-type
Ca2+ channel. In the chick amacrine cell, GABA
release was observed only when the presynaptic voltage was depolarized
to
40 mV or more under voltage clamp (Gleason et al.
1993
). Threshold voltage of L-type Ca2+
channel is between
60 and
50 mV both in the chick (Fig.
1A of Gleason et al. 1994
) and goldfish
amacrine cells (unpublished data). As the resting membrane potential of
the goldfish amacrine cell was about
60 mV in the present study,
depolarization of only a few millivolts would activate L-type
Ca2+ channels. Recently, we reported that
goldfish amacrine cells also possess TTX-sensitive persistent current
that seemed to be activated about
50 mV (Watanabe et al.
2000
). The TTX-sensitive persistent current might contribute to
TTX-sensitive medium-sized, action potential-independent spontaneous
postsynaptic currents. There was a CNQX-sensitive component in the
spontaneous postsynaptic current (Fig. 3) that might be mediated by
cations (inward currents at
27 mV in Fig. 4A). However, it
is likely that these spontaneous glutamatergic inputs do not trigger
spontaneous release of GABA from presynaptic amacrine cells, because
blocking of glutamatergic inputs did not suppress the spontaneous
GABA-mediated postsynaptic currents significantly (Fig. 7). Therefore
some intrinsic depolarizing mechanism should exist in the presynaptic
amacrine cell.
Role of the action potential in the amacrine cell
It has been suggested that amacrine cells produce dendritic spikes
(Miller and Dacheux 1976) and that the properties of
action potentials of amacrine cells are different from those of
ganglion cells (Barnes and Werblin 1986
). Recently
action potential propagation in dendrites has been reported in
neocortical pyramidal cells (Stuart and Sakmann 1994
),
hippocampal pyramidal neurons (Colbert et al. 1997
),
mitral cells of the olfactory bulb (Bischofberger and Jonas
1997
), and rabbit ganglion cells (Velte and Masland 1999
). However, the functional significance of dendritic spikes in amacrine cells has not been identified, although there have been
several reports that used simulation-based models (Smith and
Vardi 1995
; Velte and Miller 1997
). A recent
report by Cook and McReynolds (1998)
demonstrated that
TTX-suppressible action potentials contribute long distance lateral
inhibition. We demonstrated here that spontaneous action potentials
contributed to the release of large quantities of GABA from presynaptic
amacrine cells to postsynaptic amacrine cells. The release of large
quantities of GABA from amacrine cells has also been suggested by
responses recorded from ganglion cells in the rat retinal slice
(Protti et al. 1997
; Tian et al. 1998
).
Spontaneous action potentials in presynaptic amacrine cells might be
evoked by spontaneous depolarization of presynaptic amacrine cells as
reported in dopaminergic interplexiform amacrine cells
(Feigenspan et al. 1998
; Gustincich et al.
1997
) and in some case by spontaneous glutamatergic inputs from
bipolar cells (Gao and Wu 1999
; Taylor et al.
1995
) (see also Fig. 4A).
Gap junctions between amacrine cells
Amacrine cells of the identical subtype are shown to be dye
coupled (Hidaka et al. 1993; Teranishi et al.
1987
; Vaney 1991
). Gap junctions and reciprocal
GABAergic synapses are also shown between amacrine cells cultured from
the chick embryo (Gleason et al. 1993
). In our
experiments, dye coupling was not observed. Lack of detectable dye
coupling suggests that coupling may be weak, although it is likely that
a smaller probe such as neurobiotin will diffuse into neighboring
amacrine cells as reported by Vaney (1991)
. Through the
putative gap junction, action potentials generated in neighboring
amacrine cells may invade the recorded cell. This does not necessarily
mean that the action potential we recorded in an amacrine cell spread
from its neighbors. It is possible, however, that the tonic spread of
neighboring action potential helps triggering spontaneous action
potentials in the recorded cell.
Another effect of putative gap junctions is to make space clamp incomplete, resulting in incorrect measurement of the reversal potential. In our recording condition, the frequency and amplitude of spontaneous postsynaptic currents were almost the same at the voltages either positive or negative from the reversal voltage in equal amount (cf. Fig. 4A). This finding indicates that most of the postsynaptic membrane was nearly equally polarized from the reversal voltage. We therefore did not account a significant contribution of gap junctions in the present study.
Functional significance of the inhibitory network among amacrine cells
GABAergic input was observed in most amacrine cells recorded,
regardless of their morphology. This kind of GABAergic interaction was
also observed in mouse amacrine cells in the slice preparation (Kaneda and Kaneko 1998). Therefore it is likely that
the GABAergic inhibition system is a common motif in amacrine cell
circuits. Similar inhibitory interactions between neurons have been
reported in perigeniculate neurons in the dorsal lateral geniculate
nucleus (Sanchez-Vives et al. 1997
). There are several
functional aspects of inhibitory interactions between amacrine cells,
possibly common features of interneurons in general, which should be
further investigated. 1) It should be determined whether
GABAergic inputs between amacrine cells restrict the spread of
excitatory information from the bipolar cell, perhaps attenuating
propagation of dendritic action potentials in amacrine cells.
2) Interneuronal reciprocal inhibition mediated by GABA is
reported to be involved in oscillation mechanisms (Zhang et al.
1998
). In the frog retina, Ishikane et al.
(1999)
reported that oscillation and synchronization of the
ganglion cell firings were affected by bicuculline. 3)
Amacrine cell inhibitory networks may be subject to modulation, perhaps
by activation of metabotropic glutamate receptors (mGluR) as reported
for olfactory neurons (Hayashi et al. 1993
;
Isaacson and Strowbridge 1998
), because several types of
mGluR have been reported to be located on amacrine cells
(Brandstätter et al. 1998
).
To fully understand the functional roles of amacrine cells, we have to accumulate further information on the distributions of excitatory and inhibitory inputs, sodium channels, and other ion channels within dendrites and the soma, as well as defining the release sites of various transmitters. However, it is clear that GABAergic serial synapses could have a profound influence on the operations of amacrine cells as lateral interneurons.
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ACKNOWLEDGMENTS |
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We thank Dr. R. Marc for commenting on the manuscript.
This work was supported in part by a grant from Keio Gijuku Academic Development Funds and a Research Grant for Sciences and Medicine from Keio University Medical Fund to S.-I. Watanabe, by a grant from Keio Health Counseling Center Foundation and a Keio University Grant-in-Aid for Encouragement of Young Medical Scientists to A. Koizumi, and by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture to A. Kaneko (09268232 and 08458272). J. W. Stocker was supported by fellowships from the Japan Society for Promotion of Science and the Uehara Memorial Foundation.
Present address of J. W. Stocker: ICAgen, Inc., PO Box 14447, Research Triangle Park, NC 27709.
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FOOTNOTES |
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Present address and address for reprint requests: S.-I. Watanabe, Dept. of Physiology, Saitama Medical School, 38 Morohongo, Moroyama-machi, Saitama 350-0495, Japan (E-mail: siwata{at}saitama-med.ac.jp).
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 29 July 1999; accepted in final form 26 June 2000.
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REFERENCES |
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