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The Journal of Neuroscience, September 15, 2000, 20(18):7087-7095
Origin of Transient and Sustained Responses in Ganglion Cells of
the Retina
Gautam B.
Awatramani and
Malcolm M.
Slaughter
Departments of Physiology and Biophysics and Ophthalmology, State
University of New York at Buffalo, Buffalo, New York 14214
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ABSTRACT |
Phasic and tonic light responses provide a fundamental division of
visual information that is thought to originate in the inner retina.
However, evidence presented here indicates that this duality originates
in the outer retina. In response to a steady light stimulus, the
temporal responses of On-bipolar cells fell into two groups. In one
group, the light response peaked and then rapidly declined ( ~ 400 msec) close to the resting membrane potential. At light offset,
these cells exhibited a transient afterhyperpolarization. In the second
group of On-bipolar cells, the light response declined 10-fold more
slowly and reached a steady depolarization that was ~40% of the peak
response. These neurons had a slowly decaying afterhyperpolarization at
light offset. A metabotropic glutamate antagonist,
(RS)- -cyclopropyl-4-phosphonophenylyglycine (CPPG),
blocked light responses in both types of On-bipolar cell. CPPG only
slightly depolarized transient On-bipolar cells, whereas sustained
On-bipolar cells were significantly depolarized. Inorganic calcium
channel blockers disclosed that these distinct On-bipolar responses
were inherent to the bipolar cell and not attributable to synaptic
feedback. CPPG had distinct effects on sustained and transient ganglion
cells, similar to its action on bipolar cells. The antagonist
depolarized and blocked the light responses of sustained ganglion
cells. In transient ganglion cells, CPPG suppressed the On light
response but did not depolarize the cell or block the Off light
response. These results suggest that transient and sustained light
responses in ganglion cells result from selective bipolar cell input
and that these two fundamental visual channels originate at the
dendritic terminals of bipolar cells.
Key words:
retina; metabotropic glutamate receptors; on-bipolar; ganglion cell; CPPG; visual neuroscience
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INTRODUCTION |
In the vertebrate retina,
photoreceptors (rods and cones) and second-order cells (bipolar and
horizontal cells) respond to light with graded potentials of complex
waveform (Werblin and Dowling, 1969 ; Kaneko, 1970). One of the striking
early observations in retinal physiology was that the graded potentials
of photoreceptors and bipolar cells have a distinct sustained component
that is present in some ganglion cells but disappears in On-Off
ganglion cells. This signifies that the visual information encoded by
graded potentials in the outer retina is decomposed and distributed
among ganglion cells. The initial response to change in illumination is
relayed to transient On-Off ganglion cells, whereas slower components
are conveyed to sustained On and Off ganglion cells (Kuffler, 1953;
Wunk and Werblin, 1979). Although observed for decades, the synaptic
mechanisms of the transformation have remained elusive.
Previous studies suggest that inhibitory GABAergic and glycinergic
feedback from amacrine cells truncates release from the bipolar cell
terminals to generate phasic output signals (Tachibana and Kaneko,
1988; Lukasiewicz et al., 1994; Zhang and Slaughter, 1995; Dong and
Werblin, 1998; Euler and Wässle, 1998; Luka-siewicz and
Shields, 1998a,b; Maple and Wu, 1998). Blocking this feedback prolongs
the responses of transient ganglion cells but does not convert them to
sustained responses (Lukasiewicz et al., 1995; Bieda and Copenhagen,
2000) (but see Dong and Werblin, 1998). Thus, feedback
inhibition from amacrine cells strongly modulates release from bipolar
cell terminals and contributes to the kinetics of transient responses
but is not the underlying mechanism generating these responses in
ganglion cells.
Glutamate receptor desensitization is another factor that contributes
to the dynamics of ganglion cell signals (Lukasiewicz et al., 1995).
Light responses in ganglion cells are mediated by both
N-methyl-D-aspartate (NMDA) and AMPA kainic acid (KA) glutamate receptors (Massey and Miller, 1988; Mittman et al., 1990;
Diamond and Copenhagen, 1993). AMPA/KA receptors are known to
participate in the rapid activation and inactivation of
glutamate-mediated synaptic transmission (Trussell et al., 1993), and
receptor desensitization might account for transient responses.
However, when AMPA/KA receptor desensitization was blocked with
cyclothiazide, transient responses were prolonged but still distinct
from sustained responses. Moreover, in the presence of both picrotoxin
and cyclothiazide, light responses in ganglion cells remained transient
(Lukasiewicz et al., 1995). Thus, although desensitization and
inhibition play important roles in shaping light responses in ganglion
cells, factors determining the sustained or transient nature of the
response are still unclear.
Here we report that there are two groups of bipolar cells, one with
transient light responses and the other with sustained responses. This
suggests that the transient and sustained signals in ganglion cells
originate in the light responses of bipolar cells. We also identify a
specific antagonist,
(RS)--cyclopropyl-4-phospho-nophenylyglycine (CPPG),
for the synaptic metabotropic glutamate receptor (mGluR) at On-bipolar
cell dendrites. CPPG had a differential effect on transient and
sustained bipolar cells, in each case mimicking the effect of steady
light. This indicates that transient and sustained signals originate at
the dendritic inputs to bipolar cells. Using this information, we
assessed the contribution of these two groups of bipolar cells to
ganglion cell light responses. The results indicate that sustained and
transient signals in ganglion cells arise from distinct populations of
bipolar cells.
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MATERIALS AND METHODS |
Slice preparation. Larval tiger salamanders
(Ambystoma tigrinum) were obtained from Kons Scientific
(Germantown, WI) and Charles Sullivan (Nashville, TN) and were kept in
tanks maintained at 4°C on a 12 hr light/dark cycle. Retinal slices
were prepared as described by Wu (1987). All procedures were performed
in accordance with the US Animal Welfare Act and the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals (publication 85-23) and were approved by the university
Animal Care Committee. In dim red light, animals were decapitated and
double-pithed, and the eyes were enucleated. Under these conditions rod
responses are greatly compromised (Hensley et al., 1993). The retina
was removed from the eyecup, placed on a 0.45 µm membrane filter
(Millipore, Bedford, MA) with the vitreal side down, and then sliced at
150-250 µm intervals using a tissue slicer (Stoelting, Woods Lane,
IL). A single slice was then transferred to the recording chamber and viewed under infrared light using a CCD camera attached to an upright
Olympus Optical (Tokyo, Japan) IMT2 fluorescent microscope, equipped
with a 40× water immersion lens (Zeiss, Thornwood, NY). This slice was
continually bathed with control Ringer's solution containing (in
mM): 111 NaCl, 2.5 KCl, 1.8 CaCl2, 1 MgCl2, 10 dextrose, and 5 HEPES, buffered to pH 7.8. Using a gravity-fed perfusion system Ringer's solution could be exchanged with
drug-containing solutions within 5-10 sec. CPPG and
L-2-amino-4-phosphonobutyrate (AP4), obtained
from Tocris (Ballwin, MO), were prepared as 10 mM
stock solutions and diluted to the desired concentration before the
experiment. All other chemicals were obtained from Sigma (St. Louis, MO).
Whole-cell and perforated patch-clamp recordings. Most of
the recordings were made using the whole-cell patch technique.
Recordings from On-bipolar cells were obtained within 2 min of
establishing whole-cell configuration. After this period the responses
began to run down, and the cell was discarded. In the other cell types, whole-cell recordings were stable for longer periods (30-120 min). Cells were identified based on their appearance after staining with
Lucifer yellow. To ensure that cell dialysis did not affect our
observations, a few recordings were done in each cell type using the
nystatin perforated patch technique. Whole-cell recordings were made
using ~5M electrodes containing (in mM): 105 K-gluconate, 5 KCl, 1 MgCl2, 0.2 BAPTA, 10 HEPES, 4 ATP-Na2, 0.5 GTP-Na3 and 1% Lucifer yellow. The pH was
adjusted to 7.4 with KOH. The reversal potential for chloride
(ECl) was calculated to be  70 mV.
The voltage- and current-clamp recordings were made with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Analog signals were
filtered at 1 kHz and sampled at 2 kHz with the Digitadata 1200 analog-to-digital board (Axon Instruments). Clampex8 (PClamp8; Axon
instruments) was used to control the voltage command outputs, acquire
data, and trigger the light stimulus. The light source was a Stanley
red light-emitting diode (LED), which emitted ~10 4 photons/µm2 per second at 620 nm
(Nygaard and Frumkes, 1982). The currents and voltages shown are raw
data, and electrode junctional potentials and access resistances were
not corrected.
Electroretinograms. Electroretinograms (ERGs) were
recorded in the superfused salamander eyecup preparation, as described previously (Dick and Miller, 1985). Field potentials were recorded using a Ringer's filled, low-resistance (~1 M) glass electrode. The location of the recording site was based on the polarity of the ERG
components using conventions established previously (Dick and Miller,
1985). Signals were obtained using an AC amplifier (model 1800; A-M
Systems) and cutoff <0.1 and >50 Hz. Signals were sampled at 100 Hz
with the Digidata 1200 interface in a computer running pClamp software.
Red LEDs were used to stimulate the retina.
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RESULTS |
Sustained and transient On-bipolars cells
Although bipolar cells are considered to respond to light with
sustained graded potentials, most On-bipolar cells show some decay
after the initial peak voltage response. Whole-cell recordings revealed
stark differences in decay among On-bipolar cells. One group of
On-bipolar cells, exemplified in Figure
1A, responded to the
onset of light with a mean ± SD peak depolarization of ~27.3 ± 7.4 mV (n = 6). This initial
depolarization slowly decayed in time and reached a steady state level
of 11 ± 2.3 mV above the dark resting potential (39.2 ± 6.8% of the peak response). A second set of On-bipolar cells,
illustrated in Figure 1B, responded to a step of
light with an initial depolarization of 19.3 ± 4.9 mV
(n = 6) that rapidly decayed to a plateau level only
2.6 ± 0.8 mV above the dark resting membrane potential (14 ± 4.1% of peak response). The time course of the decay of the voltage
responses could be approximated by an exponential function as shown by
the dark lines in Figure 1, A and B.
The first group of bipolar cells had a mean decay time constant ()
of 4019.4 ± 1998.5 msec, whereas the second group had a
considerably shorter mean of 408.6 ± 81.3 msec
(p < 0.005, Student's t test; Fig.
1F). There was also a small difference in the mean
latencies of the two groups of bipolar cells. The mean latency of the
first group of cells was 98.8 ± 6.4 msec, whereas that of the
second set of bipolar cells was 92.7 ± 23.5 msec. However these
latency differences were not statistically significant
(p > 0.05). As might be expected, the time to
peak of the second set of bipolar cells was shorter (233.4 ± 45.5 vs 337.5 ± 56.8 msec; p < 0.05). These data are
summarized in Figure 1E.
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Figure 1.
On-bipolar cells fall into two groups based on the
temporal characteristics of their response to step illumination.
A, B, Current-clamp records of the light-evoked
EPSPS in sustained (A) and transient (B)
On-bipolar cells. C, D, Light-evoked excitatory currents
in the same two bipolar cells, voltage-clamped at 70 mV
(Cl reversal). The solid bar
represents the light stimulus. E, The latencies for the
two types of On-bipolar cells were similar, but the sustained
On-bipolar cells had longer times to peak (337.5 ± 56.8 vs
233.4 ± 45 msec). The mean + SD of the latency and
time to peak of the light-evoked EPSPs are plotted.
(*p < 0.005). Using PClamp software, the voltage
and current responses were fit to single exponential functions (as
shown by the dark curve overlaying the data traces in
A-D). F, The mean values for the
decay of sustained versus transient voltage responses
(VRs) were 4019 and 409 msec, respectively. The mean values for the decay of sustained versus transient current responses
(IRs) were 802 and 320 msec, respectively. Sustained
bipolar cells data are represented by gray bars;
transient bipolar cells are represented by white
bars.
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When On-bipolar cells were clamped at 70 mV, near the calculated
chloride equilibrium potential, the light-evoked synaptic excitatory
currents were found to be more transient than the corresponding voltage
responses (Fig. 1C,D). However, the On-bipolar cells with more prolonged graded potentials also had more prolonged synaptic currents. The time course of the decay of the light-evoked currents could also be approximated by an exponential function (Fig. 1C,D, dark curves). The current responses decayed with a mean of 802 ± 198 msec for the first group of cells and 320 ± 73 msec for the second group of cells (p < 0.05;
see Fig. 3F).
Although most experiments were performed using a red light stimulus
applied on a dark background, in a few cells (n = 6)
green adapting light was used. This background light was used to
eliminate rod contributions to the bipolar cell response and also to
determine whether retinal adaptation contributed to the
transient-sustained dichotomy that we observed. When the light
response of bipolar cells was compared under a dark background and
again under a green light background, the temporal characteristics were
only slightly changed. Thus, sustained and transient responses were not
caused by selective cone and rod input, as reported in fish (Saito and Kujiraoka, 1982). These results are consistent with another recent study (Roska et al., 2000) and indicate that transient responses are
not simply the result of light adaptation.
Another distinguishing feature of these two types of On-bipolar cell
was the hyperpolarizing Off light response. It was observed that cells
with long decay constants also had slower, less pronounced Off
responses than cells with short decay constants (Fig. 1, compare A, B). In the former group, this
afterhyperpolarization had a mean maximum amplitude of 14.33 ± 4.13 mV (52% of peak On response), peaked at ~1 sec, and decayed to
the dark potential ~5 sec after the termination of the stimulus. In
contrast, in the latter group, a transient Off hyperpolarization of
~17.42 ± 5.5 mV occurred at light offset (90.4% of peak On
response). This afterhyperpolarization consisted of two components: a
transient fast component that peaked 224.6 ± 10.24 msec after the
offset of the stimulus and a second more sustained component that
decayed to the dark potential within 5-7 sec. In voltage-clamp
recordings, the Off responses were much less pronounced. Part of the
reason might be that the current is small in voltage clamp.
Additionally, voltage-gated conductances may play an important role in
shaping this response.
Analysis of the responses of all On-bipolar cells studied indicated
that they fell into two categories. When the peak amplitudes of the On
response were normalized and superimposed, the traces fell into two
nonoverlapping groups as shown in Figure
2A. Similar analysis of
the Off hyperpolarization also revealed two groups (Fig.
2B). As another means of measuring the decay of
bipolar cells responses, the ratio of the amplitude of the response 1 sec after the onset of the light to the peak response was computed. This ratio gives a measure of the rate of decay, as well as the relative magnitude of the sustained component. This is graphed as the
tonic-to-phasic ratio (T/P) in Figure 2D. In cells
with long decay constants (gray bars), the mean T/P
was 0.811 ± 0.05 for the On voltage responses and 0.85 ± 0.08 for the Off responses. In contrast, cells with short decay
constants (white bars) had average T/P values of 0.36 ± 0.05 and 0.28 ± 0.09 for the On and Off responses,
respectively. When the On and Off T/P values were plotted against each
other, the cells fell into two distinct groups (Fig. 2C).
Bipolar cell responses with phasic On components also had transient Off
responses. Conversely, cells with more tonic On responses had sustained
Off responses.
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Figure 2.
Normalizing On-bipolar light responses revealed
two nonoverlapping sets of responses. A, The responses
at light onset of 12 On-bipolar cells were normalized to their peak
voltages, and the responses were superimposed. B,
Similarly, the responses at light offset were normalized and
superimposed. C, The ratio of the voltage amplitudes at
the points marked by the dotted lines in
A and B relative to the peak
amplitudes was plotted for each On-bipolar cell.
D, The mean tonic-to-phasic ratios at light onset and
offset are graphed for the sustained (dark bars) and
transient (light bars) cells for both the On and Off
responses.
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Visualization of the morphology of a few On-bipolar cells, using
Lucifer yellow, suggested distinct differences in the axonal arborization of transient (n = 7) and sustained
(n = 2) bipolar cells. In transient On-bipolar cells,
the axonal terminals ramified in the middle of the inner plexiform
layer, whereas in sustained On-bipolar cells the arborization was more
proximal. On the basis of these anatomical distinctions and the
discrete decay kinetics of the light responses at both onset and offset
of the light stimulus, On-bipolar cells were parsed into two groups:
sustained and transient.
From these observations it is reasonable to conclude that transient and
sustained responses of ganglion cells are the result of segregated
inputs from the two types of bipolar cell. We found an antagonist of
the On-bipolar metabotropic receptor that allowed us to confirm this
supposition. Furthermore, if bipolar cells are inherently either
transient or sustained, then this raises a question about the mechanism
that initiates this distinction. It might be attributable to the
temporal properties of photoreceptor transmitter release or to response
kinetics of bipolar cell synaptic transduction. The metabotropic
receptor antagonist revealed that On-bipolar response kinetics depends
on the latter and not on temporal properties of photoreceptor release.
Before addressing these two points, the properties of the metabotropic
glutamate receptor need to be described.
CPPG: an antagonist at the AP4 receptor
In the dark, glutamate released by photoreceptors activates mGluRs
on the dendritic terminals of On-bipolar cells. These receptors are
negatively coupled to a cationic conductance; thus glutamate hyperpolarizes On-bipolar cells. At the onset of light, photoreceptors hyperpolarize and stop releasing glutamate, and consequently On-bipolar cells depolarize. Figure 3A
shows a current-clamp recording of the light-evoked EPSP in an
On-bipolar cell obtained in dark-adapted retinal slice preparation. The
dark potential of this cell was approximately 40 mV, and a 5 sec
light stimulus evoked an initial peak followed by a smaller, sustained
depolarization. At the offset of the light stimulus, the membrane
potential returned to the dark resting level. Application of 200 µM CPPG depolarized this On-bipolar cell to
approximately 25 mV, a few millivolts above the sustained level of
the light response. Although CPPG depolarized the On-bipolar cell and
suppressed its response to the onset of light, a large hyperpolarizing
transient at light offset was often observed. This offset response was
not blocked by picrotoxin and strychnine and had a reversal near 0 mV
(data not shown). The offset probably represents a peak of transmitter
release from the photoreceptor as it depolarizes at light offset (Roska
et al., 1998). Hence, 200 µM CPPG inhibits the
action of steady-state levels of glutamate released from the
photoreceptors in the dark but is less effective in antagonizing higher
levels of glutamate released initially at light offset. Because the
effects of 200 µM CPPG were prominent and
reversible, this concentration was used in this study. Moreover, at
this concentration CPPG had negligible effects on the potential and
light response of rod photoreceptors (n = 4; Fig.
3C) and Off-bipolar cells (n = 4; Fig.
3B) or the Off response of ganglion cells (see below),
indicating that it specifically affected receptors on On-bipolar
cells.
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Figure 3.
Effects of CPPG on the light-evoked EPSPs in the
outer retina. A, CPPG suppressed the light response of
an On-bipolar cell. CPPG has no significant effect on the light-evoked
potentials of an Off-bipolar cell (B) or a rod
photoreceptor (C). Whole-cell recordings of
neurons in control Ringer's solution (left), then in
the presence of CPPG (middle), and after the mGluR
antagonist has been washed out (right) are shown. The
solid bar represents a 5 sec light stimulus.
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AP4 has often been used as a pharmacological tool to separate On and
Off pathways in the retina (Slaughter and Miller, 1981). In the brain
and spinal cord, group III mGluRs (mGluR4, 6, 7, and 8) are
distinguished by their selective affinity for this drug. In the retina,
AP4 mimics the effect of glutamate on the On-bipolar cells, suggesting
that the On response is mediated via group III mGluRs. Confirming the
pharmacological findings, molecular characterization of these receptors
in mouse identified them as mGluR6 (Nakajima et al., 1993). To define
the properties of CPPG, we tested its ability to antagonize the effects
of AP4 on On-bipolar cells (Fig.
4A). Application of 2 µM AP4 hyperpolarized the membrane potential of
the On bipolar cell, indicating that steady-state dark levels of
endogenous glutamate did not saturate the mGluRs. Two micromolar AP4
almost completely suppressed the light-evoked response. Further
application of 200 µM CPPG in the presence of 2 µM AP4 antagonized and reversed the effects of
AP4. CPPG produced a depolarization of the bipolar cell and a
suppression of the light response. Note that CPPG depolarized this cell
slightly beyond the voltage of the sustained light response. The
effects of these drugs were reversible. Figure 4B,
left, shows the I-V relationship for the same
On-bipolar cell. The cell was held at 70 mV and then stepped for a
duration of 50 msec, from 120 to 20 mV in increments of 25 mV.
These neurons exhibited both inward and outward rectification as noted
previously (Lasater, 1988; Tian and Slaughter, 1994). The protocol was
then repeated in the presence of 2 µM AP4 (Fig.
4B, middle). Consistent with current-clamp recordings, application of AP4 induced a small outward current. The
ligand-gated current was small at each command potential and outweighed
by the much larger, voltage-gated currents. Therefore, control currents
were subtracted from the currents observed in the presence of AP4,
thereby isolating the ligand-gated currents. The differences in current
at various voltages, with and without AP4, are plotted in Figure
4C (squares). The difference current revealed a
net outward current that reversed near 10 mV, confirming that AP4
closed a cationic channel (Shiells et al., 1981). When the same
protocol was repeated in the presence of 200 µM
CPPG and 2 µM AP4, an inward current was
observed (Fig. 4B, right). The difference
in currents in the presence and absence of CPPG and AP4 was a net
inward current that also reversed near 10 mV (Fig. 4C,
circles). The results indicate that CPPG antagonized the action of
endogenous glutamate as well as exogenously applied AP4. CPPG
depolarized On-bipolar cells by opening a channel with a reversal
potential near 10 mV, which probably represents a nonspecific cation
conductance.
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Figure 4.
CPPG antagonized and reversed the effects of AP4
on On-bipolar cells. A, Light-evoked EPSPs in an
On-bipolar cell in control, in the presence of 2 µM AP4,
in the presence of 2 µM AP4 + 200 µM CPPG,
and after the drugs had been washed out (left to
right). The solid bar represents a 4 sec
light stimulus. B, The same cell was voltage-clamped
from 120 to 20 mV in 25 mV steps for 50 msec. Each trace was
separated by 1 sec during which the cell was held at 70 mV. Membrane
currents in this cell are shown in control Ringer's solution
(left), in response to AP4 (middle), and
in the presence of AP4 + CPPG (right). Current-voltage
relationships shown in C were calculated by
subtracting the control current-voltage relationship from those
obtained in the presence of the drugs (squares, AP4
data; circles, CPPG + AP4 data). Lines
are a linear fit to the data points using PClamp software.
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Another method of measuring the effectiveness and specificity of CPPG
is to examine the effects of this drug on the ERG in the intact eyecup
preparation. The ERG is a field potential that consists of waveforms
generated by neurons in the outer retina. The "b" wave of the ERG
is thought to represent the field potential generated by the
depolarization of On-bipolar cells (Stockton and Slaughter, 1989).
Blocking the light-evoked depolarization of On-bipolar cells by
applying AP4 completely eliminates the b wave, whereas the other
components of the ERG remain intact (Gurevich and Slaughter, 1993).
Figure 5A compares the effects of CPPG with those of AP4 on the ERG. In these recordings the b wave
was completely abolished by 50 µM AP4 and
significantly reduced by CPPG (500 µM). These
results are consistent with the finding that both of these agents
prevent the light-evoked depolarization of On-bipolar cells (Figs. 3,
4). The amplitude of the b wave, measured from the positive peak of the
a wave to the negative peak of the b wave, was completely suppressed by
AP4 (n = 4). Saturating doses (2 mM) of CPPG maximally suppressed the b wave by
~80 ± 3% (n = 4). Comparing various doses of
CPPG indicated that the IC50 was ~185
µM (Fig. 5B). Drug concentrations in
the retinal slice and the eyecup are not always comparable, because diffusion barriers in the eyecup often necessitate higher drug doses.
Although CPPG suppressed the b wave, it left the a and d waves intact,
indicating that its action was specific for the On-bipolar cells. Taken
together, these results demonstrate that CPPG is a selective antagonist
at the AP4-sensitive, group III mGluRs in the retina.
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Figure 5.
AP4 and CPPG suppress the b wave of the ERG.
A, The ERG recorded in response to full-field light
stimulation in control Ringer's solution (left) and in
the presence of CPPG (right) is shown in the top
panel. An ERG recorded in another eyecup in control
(left) and in the presence of AP4 (right)
is shown in the bottom panel. The solid
bar represents a 2 sec light stimulus. B,
Dose-response curve for CPPG. Each point represents the
mean ± SEM percentage of the maximal suppression of the b wave by
CPPG (from 4 preparations). The data were fit to the logistic equation
using Origin software.
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Synaptic block depolarized sustained but not transient
On-bipolar cells
CPPG blocked the action of endogenous transmitter and depolarized
the On-bipolar cell to a level slightly above the sustained phase of
the light response. This phenomenon was observed in all On-bipolar
cells tested. However, CPPG had different effects on sustained and
transient bipolar cells because the amplitudes of their steady-state
responses were different. This is illustrated in Figure
6. In the sustained On-bipolar cell, the
peak light response amplitude was 25 mV, and the sustained response was
10 mV. CPPG depolarized the resting membrane potential by 12 mV
(dotted line). In the transient bipolar cell, CPPG
suppressed most of the peak light response but only minimally
depolarized the membrane potential. Hence, CPPG reproduced the action
of maintained light in both types of bipolar cell.
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Figure 6.
Differential effect of CPPG on sustained and
transient On-bipolar cells. Responses of a sustained On-bipolar cell
(A) and transient On-bipolar cell
(B) in control, CPPG, and after the drug had been
washed out (left to right) are
shown.
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CPPG evoked distinctly different responses in the two groups of bipolar
cells, yet in both groups it was blocking the postsynaptic receptors at
On-bipolar dendrites. This indicates that the steady-state responses
that distinguish sustained and transient bipolar cells are not
attributable to the release kinetics at photoreceptor terminals.
However, these results do not exclude the possibility that synaptic
feedback determines the steady-state voltage. Because CPPG caused a
depolarization of some On-bipolar cells, it was conceivable that this
activated neural circuits and provided inhibitory feedback to
selectively truncate the responses of transient On-bipolar cells. To
test this possibility, we examined the effects of blocking all synaptic
transmission on the membrane potential of On-bipolar cells. Consistent
with previous findings (Dacheux et al., 1979; Slaughter and Miller,
1981) Cd2+ depolarized and eliminated the
light responses of On-bipolar cells (Fig.
7). Cd2+,
like CPPG, significantly depolarized sustained On-bipolar cells but
produced only a small depolarization in transient bipolar cells (Fig.
7A,B). In every instance, CPPG and
Cd2+ depolarized bipolar cells to a
voltage just slightly more positive than the plateau voltage produced
by maintained light stimulation. Figure 7C plots the
depolarizations produced by CPPG and Cd2+
against the sustained phase of the light responses of On-bipolar cells,
showing that these blockers mimic the sustained phase of the light
response. The Cd2+ results confirm the
CPPG findings and furthermore indicate that the steady-state voltages
in the two sets of bipolar cells are intrinsic to the bipolar cell and
not attributable to synaptic feedback.
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Figure 7.
Similar effects produced by blocking glutamate
transmission presynaptically and postsynaptically. A,
Plot of light response of a transient On bipolar cell under control
conditions, during application of 50 µM cadmium, after
recovery from cadmium, during application of 200 µM CPPG,
and after recovery from CPPG. The dark bars under the
voltage traces indicate the timing of a 5 sec light stimulus.
B, Same protocol as in A but while
recording from a sustained On-bipolar cell. C, Plot of
the depolarization produced by CPPG and cadmium versus the sustained
depolarization during the light response of On-bipolar cells.
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Sustained and transient ganglion cell light responses
With knowledge of these distinct effects of CPPG on the membrane
potential of sustained and transient On-bipolar cells, it would be
anticipated that CPPG would hold sustained On-bipolar cells in a
depolarized state that would favor transmitter release but would hold
transient On-bipolar cells near their dark resting state, which would
prevent transmitter release. We therefore examined the ramifications of
these effects of CPPG on the postsynaptic ganglion cells.
At the onset of a light, some ganglion cells responded with a transient
depolarization that returned to the dark potential in the presence of
the light stimulus (Fig.
8A). Other ganglion cells responded with a transient depolarization that was followed by a
sustained depolarization that persisted for the duration of the light
stimulus (Fig. 8B). The synaptic currents reflected a
similar distinction in the relative amplitude of the peak and the
plateau currents (Fig. 8C,D). This dichotomy seemed to
reflect the properties of the two groups of On-bipolar cells. When
transient ganglion cells were stained with Lucifer yellow
(n = 6), their dendritic processes were found to
terminate near the middle of the inner plexiform layer. The dendrites
of sustained ganglion cells (n = 2) ramify at the
border of the inner plexiform layer. This anatomical separation has
been described in rabbit retina, where transient ganglion cells
terminate in the middle of the inner plexiform layer, whereas sustained
ganglion cells ramify at the edges of this plexiform layer (Roska and
Werblin, 2000). This distribution of transient and sustained ganglion
cell processes correlates with the axonal terminations of transient and
sustained bipolar cells, respectively.
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Figure 8.
Light responses of transient and sustained
ganglion cells. A, C, Voltage and current
recordings from a transiently responding ganglion cell. Note the
responses return to resting levels while the light is still on.
B, D, Voltage and current responses in a
ganglion cell that responds in a more sustained manner
(Vhold, 70 mV).
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Figure 9A shows a transient
ganglion cell that responded to a 6 sec light stimulus with ~35 mV
EPSPs at light onset and offset. When 200 µM
CPPG was applied, the On EPSP was suppressed significantly, but the
resting membrane potential and the Off EPSP were only slightly
affected. The simplest explanation for this outcome is that CPPG acted
directly and selectively to suppress transient On-bipolar input to this
ganglion cell. After removal of CPPG the light responses recovered. A
similar observation was made when the ganglion cell was voltage-clamped
at 70 mV. Under control conditions there were large On and Off EPSCs,
which decayed back to baseline during the light stimulus. CPPG
suppressed the On EPSP but did not significantly alter the holding
current, and the Off EPSC remained. Figure 9, C and
D, shows continuous traces of the membrane potential and the
membrane current while a different transient ganglion cell was recorded
in the dark. When CPPG was applied, it increased the spontaneous
activity in this ganglion cell but had only a small effect on the
baseline voltage or current. Similar results were observed in 12 other
transient ganglion cells. This correlates well with the effect of CPPG
on transient bipolar cells, in which the light responses were
suppressed attendant with a small depolarization of the resting
potential.
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Figure 9.
Effect of CPPG on a transient ganglion cell.
A, B, The light-evoked EPSP and EPSCs
(Vhold, 70 mV) of a transient ganglion cell in
control solution, in the presence of 200 µM CPPG, and
after the drug had been washed out. The three bars
between A and B indicate the timing of
the light stimuli. C, Effect of CPPG
(bar) on the membrane potential of a transiently
responding ganglion cell. D, Effect of CPPG on the
membrane current of the same cell (Vhold, 70
mV).
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Application of CPPG had a different effect on sustained On-ganglion
cells as illustrated in Figure 10.
Figure 10A shows the voltage response of a ganglion
cell to a 6 sec light stimulus. There was an initial peak
depolarization of 38 mV followed by a sustained depolarization of 23 mV. When 200 µM CPPG was applied, the cell
depolarized by 30 mV, and all light responses were abolished. The
membrane potential and light responses of the cell returned when CPPG
was removed. Similar observations were made when the cell was
voltage-clamped at 70 mV. Under control conditions there was a large
peak current a light onset, followed by a smaller sustained current
that persisted for the duration of the light response. When CPPG was
applied, it produced a standing inward current and suppressed the
On-response but left the Off response. The effect of CPPG on the
resting potential of sustained cells is illustrated in Figure 10,
C and D. When the cell potential was monitored in
the dark, CPPG caused a depolarization of 30 mV, and this reversed as
the antagonist was washed out. CPPG produced a concomitant inward
current of ~30 pA. Similar results were found in 10 other sustained
ganglion cells. These observations correlate with the effect of CPPG on
sustained bipolar cells.
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Figure 10.
Effect of CPPG effect on a sustained ganglion
cell. A, B, The light-evoked EPSPs and
EPSCs of a sustained ganglion in control solution, in the presence of
200 µM CPPG, and after the drug has been washed out. The
three bars between A and B
indicate the timing of the light stimuli. C, Effect
of CPPG (bar) on the membrane potential of a sustained
responding ganglion cell. D, Effect of CPPG on the
membrane current of the same cell (Vhold, 70
mV).
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In some ganglion cells, such as that illustrated in Figure 10, CPPG had
an effect on the Off response. This is not unexpected, because CPPG
depolarization of sustained bipolar cells will result in excitation of
sustained amacrine cells. These inhibitory amacrine cells can affect
the Off pathway. In support of this explanation, preliminary
experiments indicate that glycine and GABA antagonists (strychnine,
picrotoxin, and CGP35348) eliminated most of the effect of CPPG
on the Off-response in ganglion cells.
| |
DISCUSSION |
Pharmacological properties of AP4-sensitive mGluRs in
the retina
The synaptic receptor at the On-bipolar cell dendrite was the
first mGluR to be described (Shiells et al., 1981; Slaughter and
Miller, 1981; Nawy and Jahr, 1990). Yet it has proven difficult to find
a selective antagonist for this receptor. Although retinal AP4-sensitive receptors are pharmacologically similar to other group
III receptors in their sensitivity to various agonists (Slaughter and
Miller, 1985; Thoreson and Miller, 1993; Tian and Slaughter, 1994), the
mGluR antagonists
(S)-2-amino-2-methyl-phosphonobutanoic acid and
(RS)--methyl-4-carboxyphenylglycine had little effect on
these receptors (Thoreson et al., 1995, 1997; Dixon and Copenhagen, 1997). Other phenylglycine derivatives, such as
S-4-carboxy-3-hydroxyphenylglycine, S-3-carboxy-4-hydroxyphenylglycine, and
S-4-carboxyphenylglycine, which are known to antagonize
group III receptors, were found to act as agonists at the AP4-sensitive
receptor in the retina (Thoreson et al., 1995).
(RS)-4-Chloro-3,5-dihydroxphenylglycine and
(RS)-3,4,5-trihydroxphenylglycine were shown to have some antagonistic activity at retinal mGluRs, but these drugs had low potency and little specificity (Thoreson et al., 1997). The inability to find a specific antagonist to AP4-sensitive receptors has hampered the functional study of this receptor in the retina. CPPG is
severalfold more potent than the previously described mGluR antagonists
and is found to antagonize the inhibitory effects of AP4 on synaptic transmission in the spinal cord (Jane et al., 1996), cerebral cortex
(Toms et al., 1996), rat taste cells (Lin and Kinnamon, 1999) and the
neurons of the auditory system (von Gersdorff et al., 1997). In
this study we identify CPPG as the first specific antagonist at the
retinal mGluR on the On-bipolar cell.
In the retina, light reduces photoreceptor transmitter release and
depolarizes On-bipolar cells. Similar to the action of light, CPPG
caused an increase in membrane conductance and a depolarization of
sustained On-bipolar cells. The membrane potentials of rod photoreceptors and Off-bipolar cells were not blocked by CPPG. Although
we did not record from cones, the responses of Off-bipolar cells and
the d wave of the ERG in the light-adapted retina imply that cone
responses were intact. Thus, CPPG specifically blocks the action of
glutamate on mGluRs in On-bipolar cells but does not affect its release
from photoreceptors or its action on the ionotropic receptors in the
Off-bipolar cells. Consistent with CPPG being a group III antagonist in
other parts of the CNS (Jane et al., 1996), it reversed the
hyperpolarizing effects of the group III mGluR agonist AP4. More
evidence to support the role of CPPG as an mGluR antagonist that
affects On-bipolar cells comes from recordings of light-evoked
intraretinal field potentials (ERGs) in the intact eyecup. In the ERG,
the b wave represents the light-evoked electrical activity of the
On-bipolar cells (Stockton and Slaughter, 1989), whereas the d wave
reflects activity of Off-bipolar cells (Xu and Karwoski, 1995). The
suppression of the b wave and not the d wave by CPPG supports the
hypothesis that it specifically disrupts the light responses of
On-bipolar cells. Last, most ganglion cells receive excitatory inputs
from On- and Off-bipolar cells, and these inputs give rise to On and Off responses, respectively. CPPG blocked the On response in ganglion cells, but the Off response remained relatively intact. Taken together
these results demonstrate that CPPG specifically blocks the action of
glutamate at On-bipolar mGluRs.
Temporal characteristics of the light responses of
On-bipolar cells
On the basis of a number of criteria, On-bipolar cell responses
fall into two groups: transient and sustained. First, light response
decay time constants parse into two distinct domains. Second, the
tonic-to-phasic ratios in current clamp or voltage clamp place bipolar
cells in two groups. Additionally, in transient bipolar cells, the
undershoot of the voltage response at light offset has a fast component
that is easily distinguishable from the slow undershoot observed in
sustained cells. Figures 1 and 2 tabulate these differences,
manifesting that there are two types of On-bipolar cells. Last, cell
staining indicates that transient and sustained On-bipolar cells are
morphologically distinct, with the axon processes of the former
ramifying near the middle of the inner plexiform layer and the latter
terminating more proximally in the inner plexiform layer.
Wu et al. (2000) have recently produced a catalog of 12 bipolar cell
subclasses based on correlates between light responses and morphology.
The transient cells in our experiments appeared similar to their cell
types 7 and 8, whereas sustained cells resembled cell types 9 and 10. They did not report kinetic differences between these subclasses,
although several laboratories have recently reported transient and
sustained bipolar cells (Euler and Masland, 2000; Roska et al.,
2000).
Besides different response kinetics, these two types of On-bipolar cell
also respond differently to presynaptic or postsynaptic blockade of the
action of glutamate. Blocking the release of glutamate with
Cd2+ or antagonizing the postsynaptic
mGluRs using CPPG caused On-bipolar cells to depolarize slightly above
the plateau phase of their light response. Sustained bipolar cells
depolarized to ~39% of their peak response, whereas transient
bipolar cells depolarize to 14% of their peak light response. It is
interesting to note that although the peak depolarization of the
On-bipolar cell corresponds to minimal glutamate released by
photoreceptors, yet completely blocking glutamate release or
antagonizing the mGluR did not depolarize these cells to this peak
potential. In fact, blocking the action of glutamate mimicked the
sustained phase of the light response, not the early transient
component. One interpretation of these data is that the transient
component of the cationic conductance desensitizes after prolonged
activation. Because of the relatively slow application of
Cd2+ or CPPG, the activation and
desensitization processes occur in tandem during drug application,
resulting in a depolarization to the plateau level. Moreover,
Cd2+ blocks all light responses and all
inhibitory neural feedback that is known to modulate these cells
(Lukasiewicz and Werblin, 1994; Zhang and Slaughter, 1995; Dong and
Werblin, 1998). Hence, the mechanisms underlying the modulation of the
transient component appear to reside, at least in part, within the
On-bipolar cell.
The signaling pathways in photoreceptors and On-bipolar cells may be
analogous. In photoreceptor adaptation, calcium inhibits guanylate
cyclase and thus modulates the responses of these cells (Matthews et
al., 1988; Nakatani and Yau, 1988). If this is used as a model for
bipolar cell response kinetics, then elevation of internal calcium
during depolarization may serve as a negative feedback that reduces the
On-bipolar cell light response. Consistent with this model, increasing
internal calcium reduces the light responses of On-bipolar cells and
occludes their light adaptation (Shiells and Falk, 1999). Hence,
calcium influx through the cGMP-gated channel may initiate the decay of
the On-bipolar cell light response. This internal feedback may be more
pronounced in one type of On-bipolar cell, thereby accounting for
transient responses.
Another potential contributor to the temporal responses in bipolar
cells is the kinetics of glutamate in the synaptic cleft. But previous
studies, using AMPA/KA receptor-mediated responses in horizontal cells
as indicators of glutamate concentration in the synaptic cleft,
suggested that glutamate levels decrease in a sustained manner in
response to bright light (Gaal et al., 1998; Roska et al., 1998).
Consistent with these findings, relatively sustained disfacilatory
currents are observed in Off-bipolar cells (Maple et al., 1998; Roska
et al., 2000). Because On- and Off-bipolar cells make similar contacts
with photoreceptors (Lasansky, 1972), this would suggest that transient
responses in On-bipolar cells are not shaped by the kinetics of
photoreceptor transmitter release.
Voltage-sensitive channels may also contribute to the intrinsic
differences between sustained and transient cells (Lasater, 1988; Mao
et al., 1998; Euler and Masland, 2000). But we observed that the
transient-sustained dichotomy remained under voltage clamp, negating
an essential role for voltage-gated channels. Nevertheless, a much
larger separation between sustained and transient On-bipolar responses
was observed in current versus voltage clamp. Thus, voltage-gated
channels may augment the transient-sustained dichotomy.
In addition to factors intrinsic to On-bipolar cells, it is important
to acknowledge that extrinsic factors also contribute to the formation
of transient bipolar cells responses. Probably the largest extrinsic
factor is negative feedback from amacrine cells (Dong and Werblin,
1998), acting through GABAC receptors (Lukasiewicz et al., 1994).
Origin of the transient and sustained light responses in
ganglion cells
The finding that CPPG significantly depolarized sustained but not
transient On-bipolar cells allowed for the evaluation of synaptic input
from these bipolar cells to ganglion cells. The prediction would be
that ganglion cells that received input from sustained On-bipolar cells
would depolarize in the presence of CPPG, whereas ganglion cells
receiving input from transient bipolar cells would not. Our experiments
disclosed that CPPP caused a depolarization in only sustained ganglion
cells. This supported the conclusion that transient and sustained
bipolar cells provide, respectively, the predominant input to transient
and sustained ganglion cells. The similarity in dendritic ramifications
in bipolar and ganglion cells with the same temporal properties
provides an anatomical substrate for this model. Hence the distinct
temporal characteristics of the bipolar cells are faithfully
conveyed and represented in phasic and tonic responses of ganglion
cells (Fig. 11).
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Figure 11.
Model. Two separate channels, originating in
bipolar cells, generate transient and sustained responses. The light
responses of bipolar cells and ganglion cells are shown; the
dark traces show voltage responses under control
conditions, and the light traces show the responses in
the presence of CPPG.
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In addition to the nature of the synaptic input from bipolar cells,
other factors contribute to the kinetics of light responses in ganglion
cells. For example, AMPA receptor desensitization at ganglion cell
dendritic terminals affects the time course of the light response
(Lukasiewicz et al., 1995). Feed-forward inhibition is also known to
contribute to the temporal properties of ganglion cells (Miller et al.,
1981; Barnes and Werblin, 1987; Cook and McReynolds, 1998). In
addition, voltage-gated channels may also play an important role in
amplifying small input currents (Diamond and Copenhagen, 1995; Tabata
and Ishida 1996). However, none of these factors is shown to be
specific for transmission to sustained or transient ganglion cells.
Hence the sustained-transient distinction seems to arise from
different inputs to the ganglion cells, which are then shaped by these
different factors.
Transient and sustained responses represent the extraction of discrete
aspects of the visual world. The former convey information about motion
and edge detection, whereas the latter relay signals associated with
color and shape (Jacobs and Werblin, 1998; Boycott and Wassle, 1999).
Therefore, the intricacies involved in the formation of these responses
are not surprising. The present experiments indicate that this
distinction first arises from the intrinsic properties of bipolar cells.
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FOOTNOTES |
Received Feb. 14, 2000; revised July 5, 2000; accepted July 5, 2000.
This work was supported by National Eye Institute Grant EY05725. We
thank Dr. Werblin's laboratory for assistance with the slice technique.
Correspondence should be addressed to Gautam Awatramani, Department of
Physiology and Biophysics, 124 Sherman Hall, 3435 Main Street, Buffalo,
NY 14214. E-mail: gba{at}acsu.buffalo.edu.
| |
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