Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Gao, Fan,
Bruce R. Maple, and
Samuel M. Wu.
I4AA-Sensitive Chloride Current Contributes to the Center Light
Responses of Bipolar Cells in the Tiger Salamander Retina.
J. Neurophysiol. 83: 3473-3482, 2000.
Light-evoked currents in depolarizing and hyperpolarizing bipolar cells
(DBCs and HBCs) were recorded under voltage-clamp conditions in living
retinal slices of the larval tiger salamander. Responses to
illumination at the center of the DBCs' and HBCs' receptive fields
were mediated by two postsynaptic currents:
IC, a glutamate-gated cation current with
a reversal potential near 0 mV, and
ICl,
a chloride current with a reversal potential near
60 mV. In DBCs
IC was suppressed by
L-2-amino-4-phosphonobutyric acid
(L-AP4), and in HBCs it was suppressed by
6,7-dinitroquinoxaline-2,3-dione (DNQX). In both DBCs and HBCs
ICl was suppressed by imidazole-4-acetic acid (I4AA), a GABA receptor agonist and GABAC receptor
antagonist. In all DBCs and HBCs examined, 10 µM I4AA eliminated
ICl and the light-evoked current became
predominately mediated by
IC. The
addition of 20 µM L-AP4 to the DBCs or 50 µM DNQX to
HBCs completely abolished
IC. Focal
application of glutamate at the inner plexiform layer elicited chloride
currents in bipolar cells by depolarizing amacrine cells that release
GABA at synapses on bipolar cell axon terminals, and such
glutamate-induced chloride currents in DBCs and HBCs could be
reversibly blocked by 10 µM I4AA. These experiments suggest that the
light-evoked, I4AA-sensitive chloride currents
(
ICl) in DBCs and HBCs are mediated by
narrow field GABAergic amacrine cells that activate GABAC
receptors on bipolar cell axon terminals. Picrotoxin (200 µM) or
(1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA)
(2 other GABAC receptor antagonists) did not block (but
enhanced and broadened) the light-evoked
ICl, although they decreased the chloride
current induced by puff application of GABA or glutamate. The light
response of narrow field amacrine cells were not affected by I4AA, but
were substantially enhanced and broadened by picrotoxin. These results
suggest that there are at least two types of GABAC
receptors in bipolar cells: one exhibits stronger I4AA sensitivity than
the other, but both can be partially blocked by picrotoxin. The GABA
receptors in narrow field amacrine cells are I4AA insensitive and
picrotoxin sensitive. The light-evoked
ICl in bipolar cells are mediated by the
more strongly I4AA-sensitive GABAC receptors. Picrotoxin,
although acting as a partial GABAC receptor antagonist in
bipolar cells, does not suppress
ICl
because its presynaptic effects on amacrine cell light responses
override its antagonistic postsynaptic actions.
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INTRODUCTION |
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Visual information is segregated into
ON and OFF pathways at the outer plexiform
layer of the retina, where photoreceptors make glutamatergic synapses
on bipolar cells. At photoreceptor synapses on hyperpolarizing bipolar
cells (HBCs), glutamate binds to
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and activates a cation conductance with a reversal potential near 0 mV (Attwell et al. 1987
; Maple and Wu
1996
; Slaughter and Miller 1983a
,b
; Wu
and Maple 1998
). In amphibian retinas, photoreceptor synapses
on depolarizing bipolar cells (DBCs) are mediated by L-2-amino-4-phosphonobutyric acid (L-AP4) type
glutamate receptors that close a cation conductance with a reversal
potential near
10 to 0 mV (Attwell et al. 1987
;
Maple and Wu 1996
; Nawy and Jahr 1990
;
Slaughter and Miller 1981
; Wu and Maple
1998
). In fish DBCs, the rod inputs are mediated by
L-AP4 receptors, whereas the cone inputs are mediated by a
glutamate-activated chloride conductance with a reversal potential near
60 mV (Grant and Dowling 1995
; Saito et al.
1979
).
In addition to photoreceptors, bipolar cells also receive synaptic
inputs from amacrine cells (ACs) in the inner retina (Wong-Riley 1974), and in some species from horizontal cells (HCs) in the outer retina (Naka 1972
). These inputs are commonly
referred as the lateral or "surround" synapses because HCs and ACs
have long lateral processes that convey visual signals to a bipolar
cell from cells in surrounding regions. Synapses onto bipolar cells from these lateral processes mediate light responses opposite in sign
to those mediated by photoreceptors, thus forming the center-surround
antagonistic receptive field (Kaneko 1970
;
Werblin and Dowling 1969
). In some species such as the
fish, the receptive fields of retinal HCs and ACs are very large, so
their responses to a small spot of light (center light stimulus) are
small compared with those elicited by a large light annulus (surround
light stimulus) (Kaneko 1971
). Consequently, the
lateral synapses mediate the "surround" light responses, but
contribute little to the "center" light responses in bipolar cells
(Kaneko 1970
). In other retinas such as the tiger
salamander, in addition to interneurons with large receptive fields,
there are HCs and ACs with narrow receptive fields, and thus they
exhibit large responses to small light spots (Lasansky and
Vallerga 1975
; Skrzypek and Werblin 1983
;
Vallerga 1981
; Werblin et al. 1988
). The
center light responses of bipolar cells in these retinas may be
mediated not only by the photoreceptor-bipolar cell synapses, but also
by lateral synapses from the narrow field HCs and ACs. It is important
to elucidate the synaptic mechanisms underlying the center light
responses of these bipolar cells.
Most previous studies on bipolar cell light responses were carried out
with the intracellular recording techniques from eyecup or isolated
whole-mount retinas (Hare and Owen 1996; Thibos
and Werblin 1978
; Yang and Wu 1997
). One major
disadvantage of that technique is that it does not allow voltage-clamp
measurements of light responses at different membrane potentials to
separate ionic current components. In this article, we present a
systematic examination of light-evoked currents in DBCs and HBCs under
voltage-clamp conditions, and the effects of neurotransmitter agonists
and antagonists on various ionic currents evoked by center light
stimuli. Additionally, the synaptic pathways mediating light-evoked
excitatory and inhibitory inputs to DBCs and HBCs are discussed.
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METHODS |
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Larval tiger salamanders (Ambystoma tigrinum)
purchased from Charles E. Sullivan (Nashville, TN) and KON's Scientific
(Germantown, WI) were used in this study. The procedures of dissection
and making retinal slices were described in previous publications (Werblin 1978; Wu 1987
). For dark-adapted
experiments, dissection was done under infrared illumination with a
dual-unit Fine-R-Scope (FJW Industry, Mount Prospect, IL) or under dim
red light. Oxygenated Ringer solution was introduced continuously to
the superfusion chamber, and the control Ringer contained (in mM) 108 NaCl, 2.5 KCl, 1.2 MgCl2, 2 CaCl2, and 5 HEPES (adjusted at pH 7.7). All chemicals were dissolved in control Ringer solution. The retinal slices
were viewed with a Zeiss ×40 water immersion objective lens modified
for the Hoffman modulation contrast optics (Hoffman Modulation Optics,
Greenvale, NY). During the experiment, retinal cells as well as the
electrode were clearly observed, and for dark-adapted experiments, a TV
monitor connected to an infrared image converter (model 4415; COHU,
Palo Alto, CA) attached to the microscope was used.
A photostimulator whose intensity and wavelength could be adjusted by
neutral-density filters and interference filters, was used. The light
was transmitted to the preparation by way of the epi-illuminator and
the objective lens of the microscope, and the spot diameter on the
retina was adjusted by a diaphragm in the epi-illuminator. The
intensity of light sources was measured with a radiometric detector
(United Detector Technology, Santa Monica, CA). The intensity of
unattenuated 650 nm light (log I = 0) is 1.25 × 107 photons µm2
s
1.
Voltage-clamp recordings were made with an Axopatch 200A amplifier
connected to a DigiData 1200 interface and pClamp 6.1 software (Axon
Instruments, Foster City, CA). Patch electrodes of 5 M tip
resistance when filled with internal solution containing (in mM) 118 Cs
methanesulfonate, 12 CsCl, 5 EGTA, 0.5 CaCl2, 4 ATP, 0.3 GTP, 10 Tris, and 0.8 Lucifer yellow, adjusted to pH 7.2 with CsOH, were made with Narishige or Kopf patch electrode pullers. The
chloride equilibrium potential, ECl,
with this internal solution is about
60 mV. Estimates of the liquid
junction potential at the tip of the patch electrode prior to seal
formation varied from
9.2 to
9.6 mV. For simplicity, we corrected
all holding potentials in this paper by 10 mV. Bipolar cells were
identified by their morphology when filled with Lucifer yellow [DBCs
with axon terminals ramified in sublamina b and HBCs in sublamina a of
the inner plexiform layer (IPL)] and their characteristic
light responses (Wu and Maple 1998
).
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RESULTS |
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Light-evoked currents in DBCs
Figure 1A shows the
current records from a voltage-clamped DBC in a dark-adapted tiger
salamander retinal slice. Currents were recorded at various holding
potentials, and a 500-µm light spot, which covered the bipolar cell
receptive field center (Skrzypek and Werblin 1983), was
presented to the cell at each holding potential. The peak current
response reversed near
20 mV. Since the light response was composed
of excitatory and inhibitory components with similar time courses, the
two components tended to mask each other. To separate the two
components, light-evoked currents were measured at the reversal
potentials of the two postsynaptic currents. At
60 mV
(ECl, see METHODS), the
cell exhibited a sustained inward current
(
IC) mediated predominantly by
cation channels at glutamatergic synapses from photoreceptors
(Nawy and Jahr 1990
). At 0 mV [near EC, the equilibrium potential for
glutamate-gated cation channels (Wu and Maple 1998
)],
light onset and offset gave rise to transient outward currents
(
ICl), probably mediated by
chloride channels at synapses from amacrine [narrow field amacrine
cells exhibit large light responses with similar waveforms when
stimulated with small light spots (Vallerga 1981
)].
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To study the photoreceptor inputs to bipolar cells, pharmacological
methods were employed to block light-evoked chloride currents (ICl) mediated by inhibitory
synaptic inputs. It has been shown that horizontal cells, amacrine
cells, and interplexiform cells in the tiger salamander retina use GABA
or glycine as their neurotransmitter (Wu 1991
;
Yang et al. 1991
). GABA responses in bipolar cells are mediated predominately by GABAC receptors
(Lukasiewicz 1996
; Lukasiewicz et al.
1994
), and glycine responses in bipolar cells are strychnine sensitive (Maple and Wu 1998
). Among all antagonists
tested, we found that 5-20 µM imidazole-4-acetic acid (I4AA) was the
most effective in blocking light-evoked chloride currents in bipolar cells. I4AA is a GABAC receptor antagonist
(Qian and Dowling 1994
), and it blocked (or partially
blocked) the GABA-induced chloride currents in bipolar cells (see Figs.
6-8). It also acts as a GABA receptor agonist in salamander bipolar
cells (Lukasiewicz and Shields 1998a
), and in
all bipolar cells in normal Ringer, I4AA increased the chloride
conductance by variable amounts (at 0 mV, for example, I4AA induced an
outward baseline current of 10-60 pA). Therefore the blockade of
light-evoked chloride currents by I4AA presented throughout this paper
is likely to be mediated by a mixture of antagonistic and agonistic
actions on GABA receptors. Figure 1B shows that in the
presence of 10 µM I4AA,
ICl
disappeared and the light-evoked current became sustained at all
voltages and reversed near 0 mV, indicating that it was predominately
mediated by
IC. The addition of
1-2 µM strychnine (not shown) did not exert further effects on the
light-evoked current.
IC was
completely abolished by subsequent addition of 20 µM
L-AP4, a specific glutamate analogue for the DBC glutamate
receptors (Nawy and Jahr 1990
; Slaughter and
Miller 1981
), to the bath (Fig. 1C). The light
responses recovered after washing with normal Ringer (Fig.
1D), indicating that the disappearance of light responses in
I4AA + L-AP4 was not due to cell rundown. This experiment
was repeated with 12 other DBCs, and all cells exhibited similar
responses to I4AA and L-AP4.
Similar experiments were conducted with the order of I4AA and
L-AP4 application reversed. Figure
2 shows the current traces of a DBC with
the same voltage clamp protocol and light stimuli as the DBC shown in
Fig. 1. In normal Ringer (Fig. 2A), the light-evoked current
reversed near 30 mV, indicative of a mixture of
IC and
ICl. In the presence of 20 µM
L-AP4 (Fig. 2B), the peak light-evoked current
reversed near
60 mV, suggesting that it was mediated predominately by
ICl. With subsequent addition of 10 µM I4AA (Fig. 2C), the light-evoked currents were
completely abolished. The light responses recovered after washing with
normal Ringer (Fig. 2D), indicating that the disappearance
of the light responses in I4AA + L-AP4 was not due to cell
rundown. This experiment was repeated on 10 other DBCs, and all cells
exhibited similar responses to L-AP4 and I4AA.
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These results suggest that the current responses of DBCs to center
illumination (500-µm light spots) are mediated by two current components: a L-AP4-sensitive
IC and an I4AA-sensitive
ICl.
Light-evoked currents in HBCs
Figure 3A shows the
responses of a HBC recorded with the same voltage-clamp protocol and
light stimulus as the DBC shown in Fig. 1. The peak current response
() did not show a reversal potential, but the brief initial response
(
) reversed near 0 mV. At the light offset, a large transient
current reversed near 0 mV. Since HBCs also receive both excitatory
synaptic inputs from photoreceptors and inhibitory synaptic inputs from
amacrine cells, we again measured the light-evoked current at the
reversal potentials of the two postsynaptic currents. At
60 mV
(ECl), the cell exhibited a sustained
outward current (
IC) mediated predominantly by photoreceptor inputs (Slaughter and Miller
1983a
,b
). At 0 mV [near EC,
the equilibrium potential for glutamate-gated cation channels
(Wu and Maple 1998
)] the cell gave rise to an outward
current (
ICl) during the light step
and a transient outward current at light offset, both probably mediated
by amacrine cells.
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In addition to light-evoked currents, discrete spontaneous excitatory
postsynaptic currents (sEPSCs) were observed in all HBCs
(n = 75). The sEPSCs (small arrows in Fig. 3,
A and D), mediated by photoreceptors, reversed
near 0 mV, and they were blocked by 6,7-dinitroquinoxaline-2,3-dione
(DNQX) (Maple et al. 1994). The sEPSC frequency in
darkness was high, and it was substantially reduced by light,
consistent with the idea that they are mediated by vesicular release of
glutamate from photoreceptors in the dark (Maple et al.
1994
; Wu and Maple 1998
). In some bipolar cells (both DBCs and HBCs), spontaneous inhibitory currents (sIPSCs, large
arrow in Fig. 3A), reversing near
60 mV were observed. These inhibitory currents, thought to represent glycinergic inputs from
amacrine cells and interplexiform cells, were always abolished by 1-2
µM strychnine (Maple and Wu 1998
).
In the presence of 10 µM I4AA (Fig. 3B),
ICl disappeared and the
light-evoked current reversed near 0-20 mV, indicating that it was
predominately mediated by
IC. Both
the sustained
IC during light-ON and the transient OFF response became
smaller, and the frequency of sEPSCs decreased. The addition of 1-2
µM strychnine (not shown) did not exert further effects on the
light-evoked currents. With subsequent addition of 50 µM DNQX, a
specific AMPA/kainate receptor antagonist known to block glutamate
receptors in HBCs, HCs, and amacrine cells (Hensley et al.
1993
; Yang and Wu 1991a
),
IC was completely abolished (Fig.
3C). The light responses recovered after washing with normal
Ringer (Fig. 3D), indicating that the disappearance of light
responses in I4AA + DNQX was not due to cell rundown. We repeated this
experiment on 11 other HBCs, and all cells exhibited similar responses
to I4AA and DNQX.
We then reversed the order of I4AA and DNQX application on HBCs.
Figure 4 shows responses of a HBC
recorded using the same voltage-clamp and light stimulus protocols as
for the DBC in Fig. 1. In normal Ringer (Fig. 4A), the
light-evoked current did not exhibit an observable reversal potential,
and this is consistent with a current generated by a simultaneous
conductance decrease (IC) and
conductance increase (
ICl). In the
presence of 50 µM DNQX (Fig. 4B), the light-evoked current
almost completely disappeared (12 of 15 HBCs). In 3 of 15 HBCs, about
30% of the light-evoked current persisted in 50 µM DNQX. We then
added 10 µM I4AA (Fig. 4C); the light-evoked currents (of
all 15 HBCs) were completely abolished. The light responses recovered
after washing with normal Ringer (Fig. 4D), indicating that
the disappearance of the light responses in I4AA + DNQX was not due to
cell rundown.
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These results suggest that the current responses of HBCs to center
illumination (500-µm light spots) are mediated by two current components: a DNQX-sensitive IC and
an I4AA-sensitive
ICl. Since DNQX
blocks most of the glutamatergic synapses in the salamander retina
(Wu and Maple 1998
), the DNQX-sensitive
IC may be mediated not only by the
photoreceptors, but also by other synapses that gate a current with
reversal potential near 0 mV.
Effects of picrotoxin, bicuculline, TPMPA, CACA, and I4AA
on GABA-induced chloride current and light-induced ICl
in bipolar cells
We also examined the effects of picrotoxin, another
GABAC receptor antagonist (Lukasiewicz et
al. 1994), on light-evoked chloride currents in bipolar cells.
Since picrotoxin has been shown to suppress the chloride current
elicited by puff application of GABA in salamander bipolar cells
(Lukasiewicz et al. 1994
; Maple and Wu
1996
), we combined experiment protocols of light stimuli and
puff application of GABA on the same cells. Figure
5 shows that at 0 mV (near
EC), 200 µM picrotoxin greatly
suppressed GABA-induced chloride currents in both DBCs (Fig.
5A) and HBCs (Fig. 5B), but failed to block the
light-evoked
ICl. It instead
enhanced and prolonged the
ICl
(picrotoxin exerted little effects on the light-evoked
IC at
60 mV). We observed similar
results in seven other DBCs and six other HBCs, and only in one DBC was
the light-evoked
ICl reduced by
picrotoxin. These results confirm the previous findings that chloride
currents induced by puff application of GABA on bipolar cells are
mediated largely by picrotoxin-sensitive GABAC receptors (Lukasiewicz et al. 1994
; Lukasiewicz
and Shields 1998b
). However, light-evoked
ICl appeared to persist in the
presence of picrotoxin. We also performed the same experiments testing the effects of (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid
(TPMPA) [another GABAC receptor antagonist
(Lukasiewicz and Shields 1998a
) 50-150 µM],
cis-4-aminocrotonic acid (CACA) [GABAC receptor
agonist (Qian et al. 1998
) 100 µM], bicuculline (a
GABAA receptor antagonist, 100-200 µM), and
strychnine (glycine receptor antagonist, 1-2 µM) on chloride
currents elicited by light and puff application of GABA or glycine
(results not shown). TPMPA exerted effects similar to those of
picrotoxin, and bicuculline had little effect on either the light- or
GABA-induced chloride currents. CACA caused a substantial baseline
current (at 0 mV, for example, an outward baseline current of 30-100
pA was observed). Its effects on light-evoked
ICl in bipolar cells were highly variable, and it appeared to have stronger actions in reducing
ICl in DBCs (by 0-60% of the peak
response) than in HBCs (by 0-20% of the peak response). Strychnine
almost completely suppressed the glycine-induced chloride current
[consistent with our previous finding (Maple and Wu
1998
)], but exerted little effect on the light-evoked
ICl.
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We next examined the effects of I4AA on GABA-induced chloride current
using the same protocol in Fig. 5. Figure
6 show that at 0 mV (near
EC), 20 µM I4AA almost completely suppressed
the light-evoked ICl (similar to the results
shown in Figs. 1 and 3), but partially reduced the GABA-induced
chloride currents in both DBCs (Fig. 6A) and HBCs (Fig.
6B). While the effects of I4AA on light-evoked
ICl were consistent, the effect on
GABA-induced chloride current varied greatly from cell to cell. For the
GABA concentration (500 µM in puff pipette) used in Fig. 6, 10-20
µM I4AA reduced the GABA-induced chloride current to 5-85% of their control amplitudes (7 DBCs and 6 HBCs), and in one DBC, 20 µM I4AA
enhanced the GABA-induced current by about 10%. In view of the fact
that I4AA is a competitive GABA receptor antagonist, we also observed
the effects of I4AA on bipolar cell responses to lower concentrations
of GABA. Responses to 5 µM GABA in the puff pipette (near the lowest
concentrations for which puff responses could be observed) also
displayed highly varying sensitivity to I4AA (not shown). In these low
concentration experiments, the effects of 10 µM I4AA on GABA-induced
chloride currents varied from a complete block to no effect whatsoever.
This suggests that multiple types of GABA receptors may be present in
different proportions in different types of bipolar cells.
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To compare the actions of I4AA and picrotoxin on light-evoked and
GABA-induced currents on bipolar cells, we combined the experiments in
Figs. 5 and 6. Similar to the results shown in Fig. 5, Fig.
7 shows that 200 µM picrotoxin enhanced
and prolonged ICl and reduced the
GABA-induced chloride current in both DBCs and HBCs. Subsequent
addition of 20 µM I4AA substantially reduced both
ICl and the GABA-induced current.
However,
ICl in I4AA + picrotoxin
was larger and more prolonged than that in I4AA alone (Fig. 6). We
obtained similar results from four other DBCs and three other HBCs.
These results suggest that although both I4AA and picrotoxin suppressed
the GABA-induced chloride current in bipolar cells, their actions on
ICl appeared to oppose each other. It is important to note that
ICl is
mediated by light responses of interneurons that gate chloride
conductance in bipolar cells, and thus it can be altered by
augmentation of presynaptic light responses as well as blockade of
postsynaptic receptors. We next studied what types of interneurons are
likely to mediate
ICl and how I4AA
and picrotoxin alter light responses of these cells.
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I4AA and picrotoxin suppress glutamate-induced synaptic transmission between amacrine cells and bipolar cells
To determine the synaptic sites and interneurons mediating
ICl in bipolar cells, we studied
the actions of I4AA and picrotoxin on bipolar cells by focal
application of neurotransmitters. In a previous study, we found that
focal application of glutamate to the IPL induces large chloride
currents in DBCs and HBCs (Maple and Wu 1996
),
presumably by depolarizing GABAergic or glycinergic neurons in the
inner retina. It is unlikely that glutamate directly activates chloride
channels on bipolar cell axon terminals, because the IPL glutamate
response is abolished by substitution of Co2+ for
Ca2+ in the bath medium (Maple and Wu
1996
). The glutamate response therefore appears to involve
Ca2+-dependent release of an inhibitory
transmitter in the IPL. In Fig. 8 we show
that such glutamate-induced chloride currents in DBCs (Fig.
8A) and HBCs (Fig. 8B) can be reversibly blocked
by 5-10 µM I4AA. I4AA (10 µM) completely abolished the glutamate response in all seven DBCs examined and in two of three HBCs studied. In one HBC the glutamate-elicited chloride current was only partially blocked by 10 µM I4AA. This result is consistent with the idea that
the light-evoked, I4AA-sensitive chloride currents
(
ICl) in both DBCs and HBCs are
mediated by neurons in the inner retina that release a neurotransmitter
that gates chloride channels associated with I4AA-sensitive receptors
on bipolar cell axon terminals. We also tested the effects of
picrotoxin on glutamate-induced chloride currents in DBCs (Fig.
9A) and HBCs (Fig.
9B) in the presence of 1 µM strychnine. In contrast to its
effects on light-evoked
ICl, 100 µM picrotoxin substantially suppressed the chloride currents induced
by focal application of glutamate at the IPL in both DBCs
(n = 4) and HBCs (n = 3), suggesting
the existence of picrotoxin-sensitive GABAergic synaptic pathways in
these bipolar cells.
|
|
Light responses of amacrine cells are not significantly altered by I4AA, but they are enhanced and prolonged by picrotoxin
Results from the last section suggest that the I4AA-sensitive
ICl in bipolar cells is likely to
be mediated by interneurons in the inner retina. Since narrow field
amacrine cells are largely GABAergic (Yang and Yazulla
1988
) and exhibit large responses to the 500-µm center light
spots (Vallerga 1981
), they are the primary candidates
for the interneurons mediating
ICl
in bipolar cells. I4AA acts as a GABA receptor agonist [in addition to
its antagonistic action on GABAC receptors
(Lukasiewicz and Shields 1998b
)], it is possible that
it suppresses light responses of GABAergic amacrine cells by inhibiting
bipolar cell inputs. This may contribute to the I4AA-induced
suppression of light-evoked
ICl in
bipolar cells. We therefore studied effects of I4AA and picrotoxin on
light responses of narrow field amacrine cells. Figure
10 shows current responses (under
voltage-clamp conditions at
60 mV) of a narrow field amacrine cell
(morphology revealed by Lucifer yellow fluorescence, processes spread
about 200 µm in IPL, not shown) to a 500-nm light spot in normal
Ringer (Fig. 10A), in 20 µM I4AA (Fig. 10B) and
in 200 µM picrotoxin (Fig. 10C). I4AA did not
significantly alter either the light-evoked current response or the
baseline current. On the other hand, 200 µM picrotoxin substantially
enhanced and prolonged the light-evoked current, possibly by
suppressing GABA-mediated inhibition between amacrine cells
(Yang and Yazulla 1988
). We obtained similar results
from eight other narrow field amacrine cells. These results suggest that the I4AA-induced suppression of
ICl in bipolar cells is unlikely to
be mediated by suppression of presynaptic light responses in GABAergic
amacrine cells. It instead probably resulted from the antagonistic (by
receptor blockade) and/or agonistic (by receptor saturation) actions of
I4AA on GABA receptors in bipolar cells. The picrotoxin-induced
enhancement and prolongation of
ICl
in bipolar cells (Fig. 5), however, are probably mediated by its presynaptic action on amacrine cell light responses. The larger and
more prolonged amacrine cell light responses may override the partial
antagonistic action (on GABA receptors in bipolar cells) of picrotoxin
and result in larger and more prolonged
ICl in bipolar cells.
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DISCUSSION |
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Bipolar cell responses to center illumination are mediated by two postsynaptic currents
Our results demonstrate that responses of DBCs and HBCs in the
tiger salamander retina to illumination at the center of their receptive fields are mediated by two postsynaptic currents:
IC, a glutamate-gated cation
current with a reversal potential near 0 mV mediated by photoreceptor
inputs, and
ICl, an I4AA-sensitive chloride current with a reversal potential near
ECl
(ECl =
60 mV in our experiments)
mediated by cells in the inner retina.
IC in DBCs is suppressed by
L-AP4, and in HBCs is suppressed by DNQX, indicative that
the former current is mediated by L-AP4 receptors
(Nawy and Jahr 1990
; Slaughter and Miller
1981
), and the latter current is mediated by AMPA/kainate
receptors (Slaughter and Miller 1983a
,b
).
Under physiological conditions, the dark membrane potential of
bipolar cells [ranging from 40 to
60 mV (Lasansky
1992
; Yang and Wu 1997
)] is close to
ECl (Miller and Dacheux
1983
), and thus the light-evoked voltage responses are mediated
predominately by
IC, similar to the
situation when bipolar cells are voltage clamped at
60 mV
(ECl) in our experiments. Near the
dark membrane potential, the light-evoked chloride conductance
increase,
gCl, in bipolar cells,
may elicit little chloride current due to a lack of driving force;
nevertheless, it reduces the voltage response amplitudes by shunting
the photoreceptor inputs. In the presence of background light the
driving force for
gCl is
substantially increased in the DBCs, which can depolarize to potentials
far from ECl. Under this condition
ICl may contribute greatly to the
light-evoked voltage responses. Therefore
ICl activated by amacrine cell
inputs on bipolar cell axon terminals may influence bipolar cell
responses differently under different adaptational conditions.
Synaptic pathways mediating light-evoked chloride currents
(ICl) in bipolar cells
Our data show that in both DBCs and HBCs the light-evoked chloride
currents, ICl, can be reversibly
blocked by 10 µM I4AA. I4AA is known to be a
GABAC receptor antagonist (Qian and
Dowling 1994
; Tunnicliff 1998
) and a partial
GABA receptor agonist (Lukasiewicz and Shields 1998a
),
and therefore the neurons that mediate
ICl are likely to be GABAergic
[I4AA is also known as an imidazoline receptor agonist
(Tunnicliff 1998
), but imidazoline receptors have not
been localized in the salamander retina]. In the tiger salamander
retina, subpopulations of HCs and amacrine cells contain GABA
(Wu 1991
; Yang et al. 1991
), and focal
application of GABA in Co2+ Ringer (which blocks
synaptic transmission) has shown that GABA receptors are localized on
the axon terminals but not the dendrites of bipolar cells (Maple
and Wu 1996
). In Fig. 8 we show that the chloride current
elicited by focal application of glutamate at the IPL is I4AA
sensitive. Glutamate delivered at the IPL depolarizes ACs and causes
GABA release, leading to activation of I4AA-sensitive GABAC receptors that open chloride channels at
bipolar cell axon terminals. We propose that the light-evoked chloride
currents,
ICl, in DBCs and HBCs are
also predominately mediated by GABAergic ACs that activate
I4AA-sensitive GABAC receptors at bipolar cell axon terminals.
In Fig. 2 we show that when the photoreceptor-DBC synapses are
suppressed by 20 µM L-AP4, the light-evoked current in
DBCs is predominately ICl with a
reversal potential near ECl (
60 mV).
This
ICl in L-AP4 can
be completely abolished by I4AA, suggesting that it is mediated by
GABAergic ACs that activate GABAC receptors on
bipolar cell axon terminals. Results from previous reports reveal that
20 µM L-AP4 suppresses the depolarizing responses of DBCs
and converts it into a hyperpolarizing response. It does not affect the
HBC or HC light responses. Additionally, L-AP4 suppresses
the AC responses driven by DBCs but not that driven by HBCs
(Yang and Wu 1991b
). Based on these results, the
ICl in L-AP4 in DBCs
(Fig. 2B) is likely to be mediated by GABAergic ACs whose
light responses are driven by HBCs. Although we cannot rule out the
possibility that HCs may mediate
ICl in DBCs (Yang and Wu
1991b
), the fact that focal application of glutamate at the
outer plexiform layer (which depolarizes HCs) elicits little or
no chloride current in bipolar cells (Maple and Wu
1996
), and the localization of I4AA-sensitive
GABAC receptors in IPL (Fig. 5) suggests that the
primary synaptic sites mediating
ICl are in the inner retina.
When 50 µM DNQX was applied to HBCs (Fig. 4), the light-evoked
currents were abolished in 12 of 15 HBCs. This is because DNQX blocks
not only the photoreceptor-HBC synapses (thus suppressing IC in HBCs), but also the
photoreceptor-HC and DBC-AC and HBC-AC synapses (Hensley et al.
1993
; Yang and Wu 1991a
). Consequently, HCs and
ACs lose their light responses, and thus
ICl in HBCs is suppressed. In three
HBCs, about 30-50% of the light response persisted in 50 µM DNQX,
and the residual currents were blocked by I4AA. Since the predominate
DNQX-resistant second-order retinal neurons are the DBCs, there may be
an I4AA-sensitive synaptic pathway between DBCs and these HBCs. A
recent report on GABAergic bipolar cells in the salamander retina
provides a possible anatomical basis for a direct connection between
DBCs and HBCs (Yang 1998
), but it is also possible that
the I4AA-sensitive pathway involves amacrine cells that are excited via
N-methyl-D-aspartate (NMDA) receptors at
synapses from DBCs (Maple and Wu, unpublished observations).
Our results suggest that the contribution of glycinergic synapses to
the light-evoked ICl in bipolar
cells is relatively minor in most bipolar cells, although previous
studies have suggested that glycinergic amacrine cells and
interplexiform cells can exert significant synaptic inputs to bipolar
cells (Maple and Wu 1998
). The function of glycinergic
synaptic inputs in bipolar cells warrants further investigation.
The surround responses of bipolar cells in isolated salamander retinas
are not affected by picrotoxin and strychnine (Hare and Owen
1996), and this may imply that the horizontal cell contribution to the surround response is mediated by picrotoxin- and
strychnine-resistent synaptic pathways [feedback or feed-forward
synapses (Wu 1992
)] in the outer retina. In light of
the results presented in this study, however, it is possible that at
least part of the surround response is generated by amacrine cell
inputs at I4AA-sensitive chloride-mediated synapses on bipolar cell
axon terminals. Due to its geometry, the retinal slice preparation used
in this study is not well suited to the investigation of classical
"surround" inputs. It will be interesting to examine whether, in
intact retinas, I4AA suppresses the surround responses of bipolar cells.
Multiple types of GABAC receptors in bipolar cells
Our results are consistent with previous reports that GABA
responses in salamander bipolar cells are mediated by
GABAC receptors, and probably not by
GABAA receptors (due to lack of bicuculline sensitivity) (Lukasiewicz et al. 1994). The large
variability we have observed for the effects of I4AA and picrotoxin on
bipolar cell GABA responses suggests that multiple types of
GABAC receptors may be present in bipolar cells,
and that these receptors may occur in different proportions on
different types of bipolar cells. It is possible that bipolar cells
contain at least two different subtypes of GABAC
receptors: one is more strongly I4AA sensitive than the other, but both
can be partially blocked by picrotoxin (Lukasiewicz and Shields
1998
). Puff application of GABA and glutamate to the IPL favors
the activation of both types of GABAC receptors (Figs. 5-8), while the light stimuli primarily elicit transmitter release at synapses using the more strongly I4AA-sensitive
GABAC receptors (Figs. 1-6). This is not
surprising, since molecular cloning studies have shown that multiple
subtypes of GABAC receptors, with varying
sensitivity to I4AA and picrotoxin, exist in the retina (Qian et
al. 1998
). It is worth noting that although I4AA is effective
in suppressing light and glutamate-induced chloride currents in bipolar
cells, it does not always completely block them. In
agreement with a previous report (Lukasiewicz and Shields 1998a
), I4AA acts as a GABA receptor agonist (in addition to a GABAC receptor antagonist) in bipolar cells since
it caused baseline current changes in almost all DBCs and HBCs in this
study. Therefore the suppressive actions of I4AA on the light-, GABA-,
or glutamate-induced chloride currents in bipolar cells can be caused
either by receptor antagonism and/or receptor saturation. I4AA does
not, on the other hand, affect the light-evoked current responses of
amacrine cells, suggesting that the I4AA-induced suppression of
ICl in bipolar cells is not due to
presynaptic inhibition of amacrine cell light responses. Although I4AA
did not alter the light response in amacrine cells, it blocked the
ICl in bipolar cells. The reason
for this discrepancy is not totally clear. A possible explanation is
that I4AA enhanced the light responses of most DBCs (see Fig. 1,
A and B), but decreased the light responses of
most HBCs (see Fig. 3, A and B), and thus the
postsynaptic sum (may or may not be linear) of the two changes may
cancel each other. Further experiments are needed to clarify this issue.
At the first glance it is somewhat surprising to find that 200 µM
picrotoxin did not block, but enhanced and broadened, the light-evoked
chloride currents in DBCs and HBCs, since the same or even lower doses
of this antagonists decreased bipolar cell responses both to focal
application of GABA and to glutamate-elicited synaptic transmission
from amacrine cells (Figs. 5 and 7) (Lukasiewicz et al.
1994; Maple and Wu 1998
). This apparent
discrepancy may be resolved, however, by considering the presynaptic
effects of picrotoxin. While the GABAC receptors
of bipolar cells are partially picrotoxin sensitive, they are not
completely blocked by picrotoxin, at least at the concentration range
used in our experiments (100-500 µM). At the same time, the GABA
receptors on amacrine cells are also picrotoxin sensitive [conceivably
more so, due to a larger contingent of GABAA
receptors (Dong and Werblin 1998
)]. Our results in Fig.
10 show that picrotoxin results in larger and broader amacrine cell
light responses, presumably by disinhibiting the inhibitory GABAergic
synapses between amacrine cells. This suggests that the
picrotoxin-induced enhancement and broadening of
ICl in bipolar cells is probably
mediated by presynaptic actions in amacrine cells. Picrotoxin partially
blocks the GABAC receptors in bipolar cells, but
such antagonistic action is out-weighted by the increase of presynaptic
light responses in amacrine cells. Consequently, picrotoxin results in
a larger and broader
ICl (but not
IC at
60 mV) in bipolar cells. In
Fig. 7 we show that in the presence of picrotoxin, I4AA only partially
blocked
ICl in bipolar cells (instead of almost completely blocking
ICl in the absence of picrotoxin,
Figs. 1-6), suggesting that the enhancement and broadening of amacrine
cell light responses also partially overcomes the antagonistic action
of I4AA on the GABAC receptors on bipolar cells.
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ACKNOWLEDGMENTS |
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We thank Prof. X. L. Yang and Dr. Roy Jacoby for reading the manuscript.
This work was supported by National Eye Institute Grants EY-04446 and EY-02520, the Retina Research Foundation (Houston), and the Research to Prevent Blindness, Inc.
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FOOTNOTES |
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Address for reprint requests: S. M. Wu, Cullen Eye Institute, Baylor College of Medicine, 6565 Fannin St., NC-205, Houston, TX 77030.
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 27 July 1999; accepted in final form 2 March 2000.
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REFERENCES |
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