Yale Vision Research Center, Yale School of Medicine, New Haven, Connecticut 06520-8061
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ABSTRACT |
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Cohen, Ethan D..
Light-Evoked Excitatory Synaptic Currents of X-Type Retinal
Ganglion Cells.
J. Neurophysiol. 83: 3217-3229, 2000.
The excitatory amino acid receptor (EAAR) types involved
in the generation of light-evoked excitatory postsynaptic currents (EPSCs) were examined in X-type retinal ganglion cells. Using isolated
and sliced preparations of cat and ferret retina, the light-evoked
EPSCs of X cells were isolated by adding picrotoxin and strychnine to
the bath to remove synaptic inhibition.
N-methyl-D-aspartate (NMDA) receptors contribute
significantly to the light-evoked EPSCs of ON- and
OFF-X cells at many different holding potentials. An NMDA
receptor contribution to the EPSCs was observable when retinal synaptic
inhibition was either normally present or pharmacologically blocked.
NMDA receptors formed 80% of the peak light-evoked EPSC at a holding
potential of 40 mV; however, even at
80 mV, 20% of the
light-evoked EPSC was NMDA-mediated. An
-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)
receptor-mediated component to the light-evoked EPSCs predominated at
a holding potential of
80 mV. The light-evoked EPSC was blocked by
the AMPA receptor-selective antagonist GYKI52466 (50-100 µM). The
AMPA receptor-mediated EPSC component had a linear current-voltage
relation. AMPA receptors form the main non-NMDA EAAR current on both
ON- and OFF- X ganglion cell dendrites. When synaptic transmission was blocked by the addition of
Cd2+ to the Ringer, application of kainate
directly to ganglion cells evoked excitatory currents that were
strongly blocked by GYKI52466. Experiments using selective EAAR
modulators showed the AMPA receptor-selective modulator cyclothiazide
potentiated glutamate-evoked currents on X cells, while the kainate
receptor-selective modulator concanavalin A (ConA) had no effect on
kainate-evoked currents. Whereas the present study confirms the general
notion that AMPA EAAR-mediated currents are transient and NMDA
receptor-mediated currents are sustained, current-voltage relations of
the light-evoked EPSC at different time points showed the contributions
of these two receptor types significantly overlap. Both NMDA and AMPA
EAARs can transmit transient and sustained visual signals in X ganglion cells, suggesting that much signal shaping occurs presynaptically in
bipolar cells.
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INTRODUCTION |
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In the vertebrate retina, the principle
neurotransmitter used to encode the excitatory visual signal to
ganglion cells is the excitatory amino acid (EAA) glutamate.
Photoreceptors release glutamate onto bipolar cells (Copenhagen
and Jahr 1989; Slaughter and Miller 1981
,
1983a
). Bipolar cells in turn release glutamate onto the
dendrites of ganglion cells (Slaughter and Miller 1983b
; Tachibana and Okada 1991
). Thus using glutamate,
light-evoked excitation to the ganglion cell is transduced through a
vertical pathway of EAA receptor (EAAR)-gated currents.
Glutamate, released by bipolar cells can potentially bind to a wide
variety of EAAR subunits known to be present on the retinal ganglion
cell. In situ hybridization studies of ionotropic EAARs in mammalian
retinae show that ganglion cells express the
N-methyl-D-aspartate (NMDA) receptor types, NR1,
and NR2A-C, the -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid
(AMPA) receptor types, GluR1, 2, and 4, and kainate receptor types KA1
and GluR7 (Brandstatter et al. 1994
;
Hammasaki-Britto et al. 1993
; Mishina et al.
1994
). Metabotropic G-protein-gated EAARs are also present on
ganglion cells (Brandstatter et al. 1994
; Rothe
et al. 1994
). Thus a number of potentially different glutamate receptor complexes could be involved in mediating the light-evoked current (LEC) signal at individual synapses on the ganglion cell dendrite (e.g., Puchalski et al. 1994
;
Takumi et al. 1999
).
Physiological studies of mammalian ganglion cells in dissociated and
sliced retinal preparations have shown that most cells respond to
exogenous application of several different EAAR agonists, including
NMDA, AMPA, and kainic acid (KA) (Aizenmann et al.
1988; Cohen et al. 1994
; Karschin et al.
1988
); however, little is known how the individual
EAAR-gated currents contribute to the light-evoked EPSC in mammalian
ganglion cells (Cohen 1998
). In addition, many early
pharmacological studies used the EAA agonist KA. KA also binds to AMPA
receptors, so it has been unclear what proportions of AMPA or kainate
type EAARs are involved in generating these ligand-gated currents.
Research on ON-OFF and ON-center
ganglion cells in amphibians has shown that both NMDA and AMPA/KA EAARs contribute to their light-evoked EPSCs (Diamond and Copenhagen 1993
; Mittman et al. 1990
). However, the EAARs
involved in generating the light-evoked EPSCs on the dendrites of
OFF-center ganglion cells remains unexamined in any species
to date (Cohen 1998
).
The "X" or "" ganglion cell is a well-known model cell type
in the mammalian visual system whose physiologic properties have been
extensively studied using extracellular recording (for review, see
Boycott and Wassle 1999
). X-type ganglion cells respond
with sustained firing to light, display linear receptive field
summation, have medium-large somas, and display a narrow bushy
dendritic arborization termed
. ON- and
OFF-X ganglion cells receive synaptic input from multiple
cone bipolar cell types (Cohen and Sterling 1991
,
1992
; Kolb 1979
; Kolb and Nelson
1993
; McGuire et al. 1986
). These cone bipolar
synaptic inputs to X ganglion cell dendrites could potentially use
different EAARs.
This paper examines the pharmacology of the light-evoked EAAR-gated synaptic current components driving ON- and OFF-X ganglion cell types in cat and ferret retinae. The results of this study show that in mammals, both AMPA and NMDA EAARs strongly contribute to the time course of the light-evoked excitatory postsynaptic current (EPSC) of ON- and OFF-sustained (X) type ganglion cells. In contrast, kainate receptors contribute little direct synaptic current to the light-evoked EPSC of X-type ganglion cells.
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METHODS |
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The preparation of slices and isolated retina has been described
in detail in a previous paper (Cohen 1998) and will only be summarized here. The eyes of cats and ferrets were obtained using
university approved protocols. Both adult and young cats were used for
these experiments. Some adult cats (age >1 yr) were used in the
experiments using pentobarbital sodium anesthesia (25 mg/kg,
n = 20). The remaining 70% of the feline experiments used kittens or young cats (averaging 74 days of age), whose eyes were
typically obtained after a series of physiology experiments using
halothane anesthesia. The animals were euthanized after eye removal
using pentobarbital. Ferret eyes were obtained from animals 2-4 mo of
age, anesthetized with intraperitoneal ketamine/pentobarbital, following decapitation. No significant differences were noted between
the light-evoked conductance mechanisms among the animal ages used in
this study.
The EAARs involved in the LECs of cat and ferret ganglion cells were
studied using thick retinal slice and isolated retina preparations.
However, results using these two preparations were found to be
virtually identical and have been pooled in this study (see
Cohen 1998). Using retinal slice preparations, the LECs
of 73 X ganglion cells were examined using whole cell recording. Most
recordings were from cat retinal ganglion cells; however, a limited
number of ferret X ganglion cells (10) have been included (e.g.,
Cohen 1998
, Fig. 1D). In the isolated retina,
the LEC pharmacology of a series of 81 X ganglion cells were recorded,
mainly from cats but also including 7 ferret retinal units.
Retinal slice preparation
Retinal slices were prepared similar to the methods of
(Cohen 1998; Cohen et al. 1994
). The
retina was isolated in the dark using dim far red wavelength lamps for
dissection purposes. Small pieces of central retina were placed on
nitrocellulose filter paper disks and cut into slices (300 µm
thickness) in a chilled low sodium Ringer. The slices were placed in a
covered incubation chamber containing oxygenated (95%
O2-5% CO2) Ames Ringer
solution that contained (in mM) 120 NaCl, 3.1 KCl, 0.5 KH2PO4, 23 NaHCO3, 1.2 Mg2SO4, 1.15 CaCl2, and 6 dextrose, with 26 vitamins and amino acids (including 7 µM glycine) as described by Ames and
Nesbett (1981)
. Slices were held in the gassed incubation
chamber for 2-30 h before recording.
Isolated retina preparation
Under dim deep red illumination, peripheral portions of the retina were isolated, mounted on a holed nitrocellulose filter, and superfused with Ames Ringer. Large bodied (>15 µm diam) X ganglion cells were targeted for whole cell recording. The cell body was exposed by using thinly tapered patch electrodes to dissect off the nerve fiber layer and Muller cell endfeet. For cell identification, internal solutions contained 0.05% Lucifer yellow and neurobiotin/biocytin (0.5%; Molecular Probes, Eugene, OR). After recording, the Lucifer-filled cells were photographed, fixed in 4% paraformaldehyde in 0.12 M phosphate buffer, and processed for neurobiotin histochemistry.
Drugs and perfusion
Light-evoked currents were studied in retinal slices and
isolated retina superfused with Ames Ringer. The Ringer flowed by gravity at a rate of 4-5 ml/min., and was heated to 35-37°C by an
in line heater just before entering the recording chamber. Drugs were
stored as pH 7.4 stocks at 10°C. NS102 (6,7,8,9 tetrahydro-5-nitro-1Hbenz(g)indole-2,3,dione-3-oxime), GYKI 52466 (1,4-Aminophenyl-4-methyl-7,8-methylenedioxy,2,3,benzodiazepine), and
cyclothiazide were purchased from Research Biochemicals (Natick, MA).
DAP5 (D-amino-5-phosphono-heptanoic acid) and AP7
(±amino-7-phosphono heptanoic acid) were purchased from Tocris Cookson
(St. Louis, MO). ATP Na+ salt was purchased from
P-L Biochemicals (Milwaukee, WI). All other drugs were purchased from
Sigma (St. Louis, MO). Drug solutions were held in Ames Ringer at
37°C in a series of continuously gassed wells, and exchanged with the
bath Ringer under manual control.
Recording
Patch electrodes (3-5 M) were drawn from borosilicate glass
(1.5 mm OD, 0.86 mm ID) on a Brown Flaming P-87 electrode puller (Sutter Instruments, San Rafael, CA). Cells were voltage clamped using
a DAGAN 3900A amplifier (DAGAN Corp., Minneapolis, MN) and PCLAMP
software (Axon Instruments, Foster City, CA) and held at the standard
holding potential of
80 mV. Current records were Bessel filtered at 2 kHz and voltage records at 10 kHz. All data were simultaneously
recorded on VCR tape at a sampling rate of 18.5 kHz using a VR10B data
recorder (Instrutech, Great Neck, NY). On formation of a gigaseal with
the patch electrode, the seal resistance before rupture was typically
around 3-6 G
. The center light-evoked response of the ganglion cell
was examined from the cell-attached capacitative spike currents. On
rupture of the patch, cellular charging currents to a 5-ms calibration pulse were compensated using the DAGAN 3911A whole cell expander controls. The settings for series resistance compensation, and cell
capacitance were noted for each cell, and series resistance compensation was used for all whole cell recordings (typically 40-60%, fast setting). The standard recording pipette (internal) solutions were methanesulphonate salt-based and contained low concentrations of Cl
to distinguish inhibitory
postsynaptic currents (IPSCs) from EPSCs. The calculated
ECl of the internal solutions was
67 to
70 mV
(Table 1). The reference electrode was a
chlorided wire or agar bridge in the bath located out of the light
path. Holding potentials were corrected for liquid junction potentials
(Barry 1994
; Neher 1992
).
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Light-evoked currents of X ganglion cells were examined at holding
potentials between 100 mV and +40 mV in 20-mV steps, and were leak
subtracted. For isolation of the light-evoked EPSCs, 120 µM
picrotoxin (40-80 µM in a few early experiments) and 1-2 µM
strychnine were added to the tetrodotoxin (TTX) Ringer (termed "PST
Ringer"). The average holding current during the 100 ms prior to
onset/offset of the stimulus was subtracted from the light-evoked response components, depending on the polarity of the ganglion cell
receptive field center. For displaying cumulative data on a sample of
ganglion cells, normalized current-voltage (I-V) curves were
employed (see Cohen 1998
for details). The magnitude of
the current at each holding potential reflects the average value of the
conductance estimate for all cells in the sample.
After recording, the Lucifer yellow-stained cells were examined with
epifluorescence illumination, and the position and branching pattern of
the stained cell's dendritic arborization was observed with
transmitted illumination in the slice (for examples, see Cohen
1998; Cohen et al. 1994
). In the isolated
retina, the LEC response polarities of cells were verified in some
cases by application of L-2-amino-4-phosphonobutyric acid
(L-APB) (50-100 µM) to the bath and on
anatomical analysis of the stratification of the neurobiotin-stained cell with respect to the capillary border in the inner plexiform layer
(IPL). The stained cells were then photographed at ×130.
Light stimuli
The retina was viewed for recording using a fixed stage Zeiss Axioskop microscope (Carl Zeiss, Thornwood, NY) equipped with a ×40 fluor water immersion lens, and a high numerical aperature condenser whose iris was held fixed in position. Retinal slices and isolated retina were manipulated and viewed under dim red light (>650 nm filter) or using infrared (IR) illumination and a charge-coupled device (CCD) camera. White light stimulus spots, imaged on the slice through the microscope condenser, were used to stimulate the ganglion cell receptive field center. The unattenuated illuminance at the ganglion cells was 140 lux. In most experiments in the isolated retina, a dim white background light was used in the optical path of the microscope or projected on the preparation (~0.01 lux) to prevent complete dark adaptation, as noted in RESULTS.
Direct effects of EAA agonists
The effects of AMPA and kainate receptor agonists were studied on ganglion cells in retinal slices using a Cd2+-containing Ringer to block synaptic transmission. Recordings were made at room temperature. Slices were perfused with an oxygenated HEPES-buffered Ringer containing 200 µM Cd2+, 120 µM picrotoxin, 1 µM strychnine, 100 µM DAP5, and 200 nM TTX. The Ringer contained the following salts (in mM): 135 NaCl, 4.3 KCl, 1.2 MgCl2, 0.1 CaCl2, 10 HEPES, and 15 glucose, pH 7.4. Glucose was reduced to 6 mM in some experiments using Concanavalin A (ConA). An EAA agonist-containing puffer pipette (1 µm diam) was positioned next to the IPL directly above the recorded ganglion cell. Under computer control, a series of short-duration puffs (typically 40-80 ms duration) were applied to the IPL, in the control condition, and in the presence of the test agent in the bath (Picospritzer, General Valve, Fairfield, NJ).
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RESULTS |
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Using K+-based electrodes, current-clamp
recordings monitored the light-evoked firing of X ganglion cells in the
intact retina. The cells were then switched into voltage-clamp mode to
examine the corresponding light-evoked generator currents. Figure
1 shows examples of this experiment on
cat ON- and OFF-center X ganglion cells
recorded in Ames Ringer (right and left panel,
respectively). Figure 1, A and C, shows examples
of the change in action potentials elicited by a spot stimulus.
ON-X ganglion cells are depolarized by center spot
stimulation, while OFF-X ganglion cells are hyperpolarized. Excluding voltage spikes to form action potentials, the ganglion cell's membrane potential is limited to an excursion of about 12 mV
even at high sustained firing rates (see also Baylor and Fettiplace 1979; Diamond and Copenhagen 1995
).
Figure 1, B and D, shows the LECs generating
these firing patterns at a holding potential of
80 mV. For the
ON-X cell, center spot stimulation evoked a large transient
inward current with a smaller sustained component that declined at spot
offset. For the OFF-X cell, center spot stimulation evoked
a small net outward current (see also Cohen 1998
, Fig.
1, C and D). At spot offset an inward current was
evoked with larger transient and smaller sustained kinetic components.
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Figure 2 shows the voltage dependance of
the light-evoked conductances involved in exciting ON- and
OFF-X ganglion cells in the presence of normal retinal
synaptic inhibition. Center spot stimulation of the receptive field
center of ON-center X ganglion cells activated a prominent
transient-sustained excitatory conductance at light-ON that
reversed positive to 20 mV, while at light-OFF a smaller
inhibitory conductance was activated on many cells (Fig. 2, top
right) (see Belgum et al. 1982
; Cohen
1998
; Freed and Nelson 1994
). This inhibitory
conductance often declined with continued perfusion of TTX Ringer,
suggesting that it may be amacrine mediated (see also Cohen
1998
; Cook et al. 1998
). Center spot stimulation of the receptive field center of OFF-center X ganglion
cells activated a transient-sustained inhibitory conductance. This
conductance reversed near or slightly negative to the calculated
ECl of the internal solution. At
light-OFF, an excitatory conductance predominated; reversing positive to
20 mV (Fig. 2, bottom right). In
this fashion, the LECs of X ganglion cells are composed of both
inhibitory and excitatory current components. These light-evoked
currents are superimposed over a resting level of glutamate release in
the dark (Cohen 1998
).
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NMDA receptors contribute to X cell light-evoked currents in the presence of synaptic inhibition
To compare the relative synaptic contributions from NMDA and
AMPA/KA EAARs in the presence of synaptic inhibition, the peak excitatory LECs of individual ON- and OFF-X
ganglion cells were measured at holding potentials of 80 and
40 mV.
The ratio of the peak LEC at
40 to
80 mV in TTX Ringer was
calculated for 10 ON- and OFF-X ganglion cells.
In Ringer containing physiological levels of
Mg2+, most studies indicate that NMDA receptor
conductances are largely blocked at a holding potential of
80 mV
(e.g., Ascher and Nowak 1987
; Cohen et al.
1994
; Mittman et al. 1990
). However, at
40 mV,
NMDA receptors begin to enter their Mg2+ ion
unblocked current-voltage region, and their conductance increases. A
purely NMDA receptor-mediated light-evoked conductance would give a
40 mV/
80 mV ratio around 3.0 (see also Fig. 4B). In
contrast AMPA/KA EAARs have a linear I-V relation, reversing
near 0 mV. The current contribution from a purely AMPA/KA
receptor-mediated conductance at
40 mV would be half of its value at
80 mV, giving a ratio of 0.5. The distribution histogram of the
40
mV/
80 mV ratios for the ten ON- and OFF-X
cells measured is shown in the left panels of Fig. 2. For
ON-X ganglion cells, the ratios averaged 0.76 ± 0.26 (mean ± SD), while the average ratio for OFF-center X
cells was higher: 1.15 ± 0.54. These values were both
significantly >0.5. This implies that on average, light-evoked NMDA
receptor-mediated conductance components were present on cat X
ganglion cell dendrites under near normal physiological conditions.
Light-evoked excitatory current properties of X cells in the absence of synaptic inhibition
The light-evoked EPSCs of X ganglion cells were pharmacologically isolated by adding picrotoxin and strychnine to the bath Ringer to remove synaptic inhibition. In the absence of synaptic inhibition, center spot stimulation of the ganglion cell evoked an EPSC with a prominent fast initial transient and smaller sustained current components similar to those seen in TTX Ringer alone. Figure 3A shows an example of the raw light-evoked EPSCs of an ON-X ganglion cell taken at a series of different holding potentials. At negative holding potentials, the sustained component of the light-evoked EPSC was noisier and smaller in magnitude when compared with the corresponding sustained EPSC component at positive potentials (n = 8 cells).
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When synaptic inhibition was removed, I-V relations of the
light-evoked EPSCs of both ON- and OFF-X
ganglion cells showed a prominent L-shape at negative potentials. An
example of the raw EPSCs of an ON-X cell is shown in Fig.
3A at different holding potentials. At both positive and
negative holding potentials, the light-evoked EPSC to the spot stimulus
had a peak and a sustained component. I-V relations of this
cell, taken at the peak current showed an L-shaped relation. Similar
L-shaped curves could be seen on individual ON-X cells
taken at their peak currents (Fig. 3B). The L-shape is
indicative of a "mixed" receptor synapse where both NMDA and
AMPA/KA EAARs contribute to the light-evoked synaptic current (see
Cohen 1998; Diamond and Copenhagen 1995
;
Edmonds et al. 1995
). However, the light-evoked EPSCs of
the ON-X ganglion cell did not decline to zero at holding
potentials of
80 to
100 mV, a region where NMDA receptors are
thought to be normally inactive. This implies that an AMPA/KA
EAAR-mediated EPSC component is also present. Furthermore, at nearly
all time points during the sustained and also at the peak portion of
the EPSC, the EPSC showed significant current components at these
negative holding potentials (Fig. 3A). Figure 3C
shows the I-V relation of the light-evoked EPSCs of an
OFF-X ganglion cell. An L-shaped I-V
relationship was also observed at all times during the EPSC. L-shaped
I-V relations could be seen on the peak light-evoked EPSCs
of many OFF-X cells examined (Fig. 3D).
Examination of the 40 to
80 mV ratio of the peak light-evoked EPSCs
of ON- and OFF-X ganglion cells in PST Ringer
gave ratios averaging significantly above 0.5 (0.94 ± 0.42, n = 8 cells and 1.56 ± 1.03, n = 6 cells), respectively. These ratios were similar to the values found
in TTX Ringer alone, again with the ratio for OFF-X cells
averaging slightly larger than their ON-X cell counterparts. Thus NMDA receptors contribute significantly to the LECs
of ON- and OFF-X ganglion cells either in the
presence or absence of retinal synaptic inhibition and at many time
points overlap with their non-NMDA receptor counterparts.
Effects of NMDA receptor antagonists on X cell light-evoked currents
The contributions of NMDA EAAR receptors to forming the X ganglion
cell light-evoked EPSC were examined on 21 X ganglion cells by bath
applying a series of selective NMDA receptor antagonists. The
antagonists DAP5 (100 µM), and AP7 (200 µM) were both used at
concentrations that strongly blocked exogenously applied NMDA (see
Cohen et al. 1994). Using extracellular recording
techniques, previous EAAR studies of mammalian ganglion cells have
reported that NMDA antagonists had moderate depressant effects on their light-evoked firing rates (Boos et al. 1990
;
Cohen and Miller 1994
; Massey and Miller
1990
). The results of this study indicate that NMDA receptors
contribute a significant fraction of the light-evoked EPSC at the X
ganglion cell.
Application of NMDA receptor antagonists had significant depressant
effects on the light-evoked EPSCs and noise of most ganglion cells even
at a holding potentials of 80 mV. On average, the peak EPSC of both
ON- and OFF-center X ganglion cells were
reduced 20-30% in NMDA antagonists at
80 mV, and averaged of
81 ± 18% and 67 ± 45%, of control values
(n = 7 and n = 14 cells, respectively). However, the contributions of NMDA receptor-mediated EPSCs are better
observed at more positive holding potentials where their conductance increases.
At more positive holding potentials, NMDA receptor antagonists
selectively reduced a slow rise time, long duration kinetic component
of the light-evoked EPSC of ON-X ganglion cells. Figure 4A shows detailed comparisons
of the light-evoked EPSCs of an ON-X ganglion cell in the
control condition and in the presence of the NMDA receptor antagonist
DAP5 at different holding potentials (left panels).
Subtraction of the two curves yields the EPSC components reduced in
DAP5 (right panels). In the presence of NMDA antagonists, slower sustained kinetic components of the light-evoked EPSC were preferentially blocked. What remained in DAP5 were fast onset light-evoked EPSCs. These EPSCs are typical of those thought to be
mediated by AMPA/KA receptors (Forsythe and Westbrook
1988; Mittman et al. 1990
). However, even in the
presence of the NMDA antagonist DAP5, small sustained current
components of the EPSC persisted during the spot stimulus.
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I-V relations of the light-evoked EPSC in the presence of
NMDA antagonists showed a linear I-V relation for a series
of seven ON- and OFF-X ganglion cells (Fig.
4B). In the control condition ( line), an L-shaped
I-V relation was observed. When NMDA receptors were blocked,
the I-V relation became linear in form (- - - - - line),
as expected for the remaining AMPA/KA receptor-mediated currents
(Forsythe and Westbrook 1988
; Mittman et al.
1990
). The inset graph shows the I-V
relation of the EPSC component blocked in NMDA antagonists, obtained by
subtracting the two curves. This difference current showed a
characteristic J-shaped I-V relation, as expected for NMDA
receptors. At
60 mV, near the normal resting potential of X ganglion
cells, the NMDA receptor conductance contribution to the light-evoked
EPSC of X cells averaged ~35% of the total conductance, and
increased with more positive holding potentials.
Reductions in the LEC by NMDA receptor antagonists could also be
observed in the presence of normal retinal synaptic inhibition. In TTX
Ringer, the light-evoked excitatory currents of ON- and OFF-X cells averaged, respectively, 85 and 68% of their
peak control LECs at 80 mV (n = 7, 4 cells). Figure
4C shows the effects of an NMDA antagonist on an
ON-X ganglion cell at
80 mV in TTX Ringer. In the
presence of DAP5, the light-evoked inward current changed, becoming
more transient at spot onset with a smaller sustained component that
lasted for the spot duration. The current blocked by DAP5 had a slow
onset and long duration typical of NMDA receptor-mediated processes
(bottom trace). However, in DAP5, the light-evoked EPSC remaining still shows a sustained current component. This explains how
ON-sustained ganglion cells recorded extracellularly in
NMDA antagonists retained their sustained firing patterns in the
absence of NMDA receptor input (e.g., Cohen and Miller
1994
; Massey and Miller 1990
).
Effects of AMPA and kainate receptor antagonists on X cell light-evoked currents
The effects of selective AMPA or kainate receptor antagonists were
tested on the light-evoked EPSCs of X ganglion cells. The benzodiazepine antagonist GYKI52466 has been reported to be a selective
antagonist for AMPA EAARs (Donevan and Rowgawski 1993; Paternain et al. 1995
). The antagonist NS102
(Verdoorn et al. 1994
) has been reported to block
kainate receptor-mediated release of GABA in hippocampal interneurons
(Chittajallu et al. 1996
; Cunha et al.
1997
). To examine these EAAR contributions to the light-evoked
EPSC, X ganglion cells were held at
80 mV, where the conductance
contributions of these non-NMDA receptor types predominate.
AMPA receptor antagonists
ON-Center ganglion cells possess both NMDA and AMPA/KA
EAARs on their dendrites (Cohen 1998; Cohen et
al. 1994
). Since the LECs of their presynaptic
ON-bipolar cell inputs use a metabotropic glutamate
receptor, one would expect that the changes observed in the LECs of
ON-center X ganglion cells in AMPA receptor antagonists would predominately reflect effects of these agents at receptors located directly on the ganglion cell.
Application of the AMPA-selective antagonist GYKI52466 (50-100 µM)
produced massive reductions in the magnitude of the light-evoked EPSC
at 80 mV. The effects of GYKI were measured in the presence of 100 µM DAP5 to block NMDA responses and reveal any small residual kainate
receptor-mediated components to the light-evoked EPSC. GYKI strongly
reduced the light-evoked EPSC of ON-X cells, and EPSCs
averaged 8.2 ± 7.3% of the control response, n = 5 cells (Fig. 5A). Similar
strong LEC reductions were observed with GYKI alone for
ON-X cells in PST Ringer (13 ± 18% control,
n = 7 cells), or in TTX Ringer (n = 5 cells). These results for ON-X cells all imply that a large
component of the non-NMDA receptor-mediated light-evoked EPSC at the
ganglion cell was mediated by AMPA receptors.
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OFF-Center X ganglion cells are excited by EAARs operating
through a disynaptic ligand-gated EAAR-mediated circuit. Cones release
glutamate onto OFF-center cone bipolar cells activating non-NMDA type EAARs (Sasaki and Kaneko 1996;
Slaughter and Miller 1983a
; Vardi et al.
1998
), which in some species appear to be kainate receptors
(DeVries and Schwarz 1999
). OFF-Center cone bipolar cells in turn release glutamate onto OFF-center
ganglion cells (Cohen 1998
; Cohen et al.
1994
). This makes localization of the effects of AMPA or KA
receptor antagonists on OFF-X cell light-evoked EPSCs more
complex to interpret.
Similar to ON-X cells, the light-evoked EPSCs of most OFF-X cells were either blocked, or strongly reduced by the AMPA receptor antagonist GYKI52466 in PST Ringer. An example of GYKI's effects on the EPSC of an OFF-center X cell is shown in Fig. 5B. Addition of GYKI strongly reduced the light-evoked EPSC at light-OFF. Light-evoked EPSCs of OFF-X cells in GYKI (50-100 µM, all in the presence of DAP5) averaged 8.2 ± 5.8% of control values (n = 6 cells). These results also imply that AMPA-type EAARs form the main non-NMDA receptor-mediated component of the light-evoked EPSC of OFF-X ganglion cells. However, it was also important to examine the effects of kainate receptor-selective antagonists on the LECs of X ganglion cells.
Kainate receptor antagonists
NS-102 (5-10 µM), a reported kainate receptor selective
antagonist, was tested on the LECs of a limited sample of X cells (n = 12 cells) at a holding potential of 80 mV
(Chittajallu et al. 1996
; Cunha et al.
1996
). Application of NS102 had only small effects on their
LECs. An example of NS102's effects is shown on an ON-X
cell in the inset of Fig. 5. For ON-cells, LECs
in NS102 averaged near control levels (96.8 ± 17.5%,
n = 5 cells; TTX or PST Ringer). The LECs of
OFF-X ganglion cells showed only a small reduction in
NS102, averaging 84.9 ± 32.1% of controls in PST Ringer
(n = 5 cells) and 106.3 ± 11.0% of controls
(n = 3 cells) in TTX Ringer. While this result could
imply that OFF-bipolar cells may have AMPA receptors on
their dendrites, the selectivity of NS102 antagonism against all
kainate receptor types is currently unclear (see
DISCUSSION).
The experimental results above with selective AMPA and KA receptor
antagonists suggested that the LECs at cone bipolar to X ganglion cell
synapses use AMPA type EAAR-mediated mechanisms. However, as previously
stated, AMPA or KA receptor antagonists can potentially also exert
presynaptic effects on the retinal network, which could distort the
EPSC observed at the ganglion cell (e.g., Cohen and Miller
1999). This question was particularly important given the known
synaptic circuitry for OFF-center X ganglion cells.
Therefore it was necessary to reexamine the pharmacology of these
non-NMDA EAARs directly on the X ganglion cell dendrite.
Tests of direct effects of AMPA and kainate receptor antagonists on X cells
Previous EAAR studies in mammalian ganglion cells have not
distinguished whether distinct AMPA or KA receptor-mediated
conductances were present on ganglion cell dendrites (e.g.,
Aizenmann et al. 1988; Cohen et al.
1994
). To examine this question, synaptic transmission was
blocked in the retinal network using a
Cd2+-containing Ringer, and the nonselective
agonist KA was applied to six X ganglion cells in retinal slices (2 ON, 4 OFF) using a puffer pipette (see
METHODS for details). Short-duration puffs of kainate were
applied directly to ganglion cell dendrites at a holding potential of
80 mV. These puffs evoked a series of large, rapidly rising inward
currents in X cells that slowly decayed, as shown in Fig.
6A. The kainate puff currents
were strongly reduced by the addition of the selective AMPA receptor
antagonist GYKI52466 to the Ringer. Dose-response curves to KA puffs
with GYKI revealed an IC50 of 4.5 µM (Fig.
6B). Thus using concentrations of GYKI52466 > 30 µM,
the inward current evoked by kainate application was strongly blocked.
These results imply that AMPA type non-NMDA EAAR conductances
predominate directly on X ganglion cell dendrites.
|
Tests of selective AMPA and kainate receptor modulators
This finding was reinforced by a series of experiments examining
the effects of AMPA- and kainate-selective receptor modulators on X
ganglion cells. Several different drugs can selectively potentiate the
action of AMPA or kainate receptors, by preventing receptor desensitization. For AMPA receptors, these drugs include aniracetam, cyclothiazide, and its derivatives (Wong and Mayer
1993; Yamada and Tang 1993
) Cyclothiazide (30 or
100 µM) was tested on X ganglion cell EAAR currents (4 ON-, 3 OFF-X cells) elicited by short puffs of
L-glutamate (500 µM) in the
Cd2+-containing Ringer. Glutamate application
normally evokes a rapidly desensitizing inward current at AMPA
receptors (Fig. 6C). Bath application of cyclothiazide (30 µM) potentiated the peak glutamate current on all cells tested by an
average of 5.9 ± 2.7 fold (n = 5 cells). Thus
AMPA receptors are present on X ganglion cell dendrites.
Cloned kainate receptors rapidly desensitize to application of
glutamate or its agonists. Kainate receptor desensitization can be
slowed irreversibly by preapplication of the high molecular weight
lectin ConA (Huettner 1990). To examine the role of
kainate receptor desensitization at the ganglion cell dendrite, retinal slices were preincubated in the ConA for >1 h prior to recording (see
METHODS for details). Kainate (500 µM) was applied to X
ganglion cell dendrites using a puffer pipette in the same
Cd2+-based Ringer. In ConA treated slices, this
resulted in a series of transient inward currents (Fig. 6D).
However, the kainate-selective antagonist NS-102 (10 µM) still caused
only a slight reduction in the kainate-evoked puff current. In
contrast, the AMPA-selective EAAR antagonist GYKI 52466 (100 µM)
blocked nearly all of the kainate-evoked current under these
conditions. Similar results were seen with the two antagonists on all
ON- and OFF-X ganglion cells tested (Fig.
6D, left panel histogram). ConA pretreatment did
not potentiate a kainate receptor-mediated current component on X
ganglion cells. These results reinforce the conclusion that while
kainate EAARs contribute at best to only a minor fraction of the
kainate-evoked current, AMPA-type EAARs account for the bulk of the
kainate-induced current tested directly on X ganglion cell dendrites.
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DISCUSSION |
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I have examined the EAAR pharmacology of the LECs of X ganglion
cells in voltage clamp using isolated and sliced preparations of the
cat and ferret retina. Previous EAAR studies on the light responses of
mammalian ganglion cells have relied mainly on extracellular recording
techniques (e.g., Boos et al. 1990; Cohen and
Miller 1994
; Massey and Miller 1988
). The
results of these pharmacology studies in the intact retinal network
have been difficult to interpret in relation to direct effects on EAARs
localized directly on the ganglion cell. This was particularly true for
the light responses of OFF-center ganglion cells in some
retinas. In the presence of the quinoxalines or kynurenic acid,
OFF-center rabbit ganglion cells often paradoxically
increased their firing rates (e.g., Cohen and Miller
1999
; Massey and Miller 1988
).
The light-evoked EPSCs of ON- and OFF-X
ganglion cells ganglion cells showed a significant NMDA-mediated EPSC
component in addition to those mediated by AMPA receptors. These
results for ON- and particularly for the lesser known
OFF-center ganglion cells resemble pharmacological studies
of the light-evoked EPSCs of ON- and
ON-OFF-center ganglion cells in the retinas of
the larval tiger salamander (Diamond and Copenhagen
1993, 1995
; Hensley et al. 1993
;
Mittman et al. 1990
; Velte et al. 1997
).
Although the light-evoked EPSCs of X ganglion cells were often
increased in the presence of PST Ringer (Cohen 1998
),
NMDA receptor-mediated EPSC components could still be observed on X
cells in TTX Ringer, where many forms of retinal synaptic inhibition
persist. This was evident by the finding that the
40 to
80 mV LEC
ratios of ON- and particularly OFF-X ganglion
cells were on average significantly >0.5 in TTX Ringer. Thus the LECs
on X ganglion cells are normally composed of NMDA EAAR-mediated
components that appear to summate with their AMPA receptor counterparts
to excite X type ganglion cells.
The light-evoked EPSC kinetics of sustained X ganglion cell types to
bright spot stimuli show that they are composed of a fast initial
transient current followed by slower sustained current components.
Their kinetics, recorded under light-adapted conditions, differ from
the slower sustained light-evoked EPSCs found in
ON-sustained larval tiger salamander ganglion cells under
dark-adapted conditions (Diamond and Copenhagen 1993).
The I-V relation of the X cell light-evoked EPSCs showed a
prominent L-shaped relation (Cohen 1998
). The form of
this I-V relation is typical for cells receiving mixed
excitatory amino acid receptor synaptic input (Diamond and Copenhagen 1993
, 1995
; Edmonds et al.
1995
; McBain and Mayer 1994
). Other
potential excitatory neurotransmitter contributors to ganglion cell
LECs, such as from cholinergic neurons could also produce rectifying
I-V relations. However, these neurons appear to play only a
minor role in the light-evoked EPSCs of X ganglion cells in the
presence of PST Ringer, as application of the nicotinic antagonist
dihydro-
-erythroidine had little effect on the light-evoked EPSC
(n = 3 cells) (unpublished observations).
Given that AMPA and KA receptor-mediated currents have a faster rise
time than those of NMDA receptors, it might be expected that these
receptors would play a more critical role in the generation of the
initial peak current transients of the light-evoked EPSCs of X
(sustained type) ganglion cells. However, careful examination of the
I-V relations of the light-evoked currents of X ganglion cells at many different time points showed this was only partially true. Even at the peak of the light-evoked EPSC, the NMDA and AMPA EAAR
contributions to the light-evoked EPSC appear to significantly overlap
(Fig. 4) (see also Cohen 1998, Fig.
7). The light-evoked EPSC of X cells also
show an initial transient to the EPSC at
100 mV; a potential where
NMDA receptor EPSC contributions are largely absent, and the same
initial transient persists at +40 mV, a potential where the NMDA
receptor-mediated EPSC contribution becomes especially large.
Conversely, the faster AMPA EAARs can mediate a portion of the
sustained EPSC, such as that found on ON-X cells at
80
mV. However, the limited excursion range of the light-evoked
depolarizations generating ganglion cell spiking (given a typical
resting potential of
58 to
60 mV) will constrain the region of the
I-V relation where the light-evoked EAAR-mediated conductances operate so that both receptor types can excite the ganglion cell (see also Cohen 1998
; Diamond and
Copenhagen 1995
). This result explains why extracellular
recordings of sustained firing ganglion cell types in rabbit and
primate retinas had persistent sustained firing patterns in the
presence of NMDA antagonists (Cohen and Miller 1994
;
Massey and Miller 1990
). In addition, the similarity of
the waveform of the light-evoked EPSC at X ganglion cells at both
positive and negative holding potentials suggests that the EPSC form
strongly reflects in part the time course of glutamate release by an X
cell's presynaptic cone bipolar cell inputs (e.g., Matsui et
al. 1998
). The time course of this glutamate release could
reflect kinetic contributions from several different synaptic vesicle
stores in the bipolar cell (e.g., Mennerick and Matthews
1996
).
|
In the presence of blockers of NMDA receptors, the principal LEC
kinetic components lost at negative holding potentials were slow onset
components of long duration. In NMDA antagonists, I-V relations of the light-evoked EPSC of ON- and
OFF-X ganglion cells lost their normal L-shaped relation
and became linear, reversing near 0 mV in PST Ringer. These linear
I-V relations reflect the properties of the remaining
AMPA/KA EAAR-mediated EPSCs (Forsythe and Westbrook
1988; Mittman et al. 1990
). Similar losses of
slow components could be seen by NMDA antagonists on the LECs of X cells in the presence of normal synaptic inhibition. Since cat X
ganglion cells, like many other ganglion cells, possess direct conductances for NMDA receptors (Aizenmann et al.
1988
; Cohen et al. 1994
; Mittman
et al. 1990
). The above data suggest that these NMDA receptors
appear to be located at or near bipolar synapses (Matsui et al.
1998
) and actively participate in forming part of the
transient, and a large fraction of the sustained LEC in X ganglion
cells under normal physiological conditions.
AMPA type glutamate receptors appear to mediate the major non-NMDA EAAR
component of the LEC on X ganglion cell dendrites. At a holding
potential of 80 mV, where NMDA receptors are largely inactive, the
AMPA-selective antagonist GYKI52466 blocked the light-evoked EPSCs of
both ON- and OFF-X cells. A similar pattern of
pharmacology was observed in a series of experiments applying EAA
agonists directly at the ganglion cell. When synaptic transmission was
blocked in the presence of Cd2+, bath application
of GYKI52466 blocked virtually all of the kainate or glutamate
puff-evoked currents at the ganglion cell. Glutamate puff-evoked
currents could be strongly potentiated by the selective AMPA receptor
modulator cyclothiazide. In contrast, application of a reported kainate
receptor selective antagonist, NS-102 (Chittajallu et al.
1996
) had little effect on kainate puff-evoked currents. Finally, application of a kainate receptor-selective modulator ConA
did not potentiate the kainate-evoked currents, even with prolonged
preincubation of the retinal slice in the lectin. These results all
suggest a model where AMPA receptors predominantly mediate the non-NMDA
receptor LEC component on both ON- and
OFF-center X type ganglion cells (Fig. 7). These results
are similar to the conclusions of a previous study of ON-
and ON-OFF type retinal ganglion cells in the
larval tiger salamander by Lukasiewicz et al. (1997)
.
What function is conferred by the kainate receptors thought to be
present on retinal ganglion cells is unclear. In situ hybridization and
immunocytochemical studies for these receptors show they are clearly
present in the ganglion cells of mammals (Brandstatter et al.
1994; Hammasaki-Britto et al. 1993
; Qin
and Pourcho 1996
; Vardi et al. 1998
). While
kainate receptors could conceivably be transported to the axon
terminals of X cells in the lateral geniculate nucleus, kainate
receptors could also play several other roles in the ganglion cell.
Physiological evidence shows that kainate receptors may act by either
metabotropic or ionotropic mechanisms. In the hippocampus, kainate
receptors act indirectly by a pertussis toxin-sensitive
G-protein-mediated mechanism; presynaptically inhibiting GABAergic
synaptic release onto pyramidal cells (Rodriguez-Moreno and
Lerma 1998
). Kainate receptors also appear to be involved in
the generation of the slow excitatory currents activated by multiple
tetanic stimuli on pyramidal cells (e.g., Vignes et al. 1997
). For X-type retinal ganglion cells, the residual LECs and agonist-induced currents observed in the noncompetitive AMPA antagonist GYKI52466 (in the presence of DAP5) were quite small. This suggests that while kainate receptors contribute at best to only a minor current
fraction at cone bipolar-X ganglion cell synapses, they could influence
ganglion cell firing through metabotropic receptor-mediated mechanisms.
However, the currently developed kainate receptor antagonists, such as
NS102 can exhibit poor selectivity in some brain regions (Paternain et al. 1996), making isolation of AMPA
receptor-mediated EPSC components more difficult. In the ground
squirrel retina, it has been proposed that cone to
OFF-bipolar cell neurotransmission uses kainate receptors
exclusively (DeVries and Schwartz 1999
). However, it
remains to be determined whether kainate receptors play a similar role
on OFF-bipolar cells in the retinae of other species, such
as the cat. Further insights into the role of kainate receptors will
require the development of more selective and potent kainate receptor antagonists.
Thus in summary, the light-evoked synaptic currents on ON-
and OFF-"X/sustained" type mammalian ganglion cell
dendrites show overlapping contributions from NMDA and AMPA EAAR types.
The contribution of NMDA receptors to the light-evoked EPSC of X
ganglion cells is substantial. Given the slow nature of the light
transduction mechanisms in retinal networks, an NMDA receptor
contribution to the "faster" components of the LEC at the ganglion
cell would not be unexpected. The polysynaptic delay from photoreceptor
stimulation to generation of the LEC in retinal ganglion cells is much
longer than the monosynaptic delay of most single synapses that have been previously studied (i.e., Forsythe and Westbrook
1988; Lester et al. 1990
; Rossi et al.
1995
). The time constant of activation of mature NMDA receptors
(~10 ms) is entirely within the response rise time of the peak EPSC
of a ganglion cell to a light stimulus (~35 ms) (see McBain
and Mayer 1994
for review). Thus this study supports a
substantial contribution from NMDA receptors to the light-evoked EPSCs
of sustained ganglion cell types. However, during periods of sustained
spiking in ganglion cells, activation of voltage-dependent
Na+ channels and subsequent activation of delayed
rectifier K+ channels will limit the NMDA
receptor contributions at more positive potentials (Diamond and
Copenhagen 1995
) (Fig. 1, this paper). In addition, AMPA
EAAR-mediated mechanisms also play a significant role in generating X
cell LECs; as some 60-70% of the light-evoked EPSC at the ganglion
cell's resting potential (approximately
60 mV) is mediated by AMPA receptors.
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ACKNOWLEDGMENTS |
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I thank M. Slaughter, D. McCormick, the Bristol-Myers Corporation, N. Daw, A. Roe, and J. Morgan for generous assistance and J. Huettner for technical comments.
This research was supported by National Eye Institute Grant EY-10617 to E. D. Cohen and by the Zeigler Foundation for the Blind.
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FOOTNOTES |
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Present address and address for reprint requests: Dept. of Cellular and Molecular Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138.
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 10 September 1999; accepted in final form 22 February 2000.
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REFERENCES |
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