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The Journal of Neuroscience, March 15, 1998, 18(6):2301-2308
Action Potentials Are Required for the Lateral Transmission of
Glycinergic Transient Inhibition in the Amphibian Retina
Paul B.
Cook1,
Peter D.
Lukasiewicz2, and
John S.
McReynolds1
1 Department of Physiology, University of Michigan, Ann
Arbor, Michigan 48109, and 2 Department of Ophthalmology
and Visual Science, Washington University, St. Louis, Missouri 63110
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ABSTRACT |
Transient lateral inhibition (TLI), the suppression of responses of
a ganglion cell to light stimuli in the receptive field center by
changes in illumination in the receptive field surround, was studied in
light-adapted mud puppy and tiger salamander retinas using both eyecup
and retinal slice preparations. In the eyecup, TLI was measured in
on-off ganglion cells as the ability of rotating, concentric windmill
patterns of 500-1200 µm inner diameter to suppress the response to a
small spot stimulus in the receptive field center. Both the suppression
of the spot response and the hyperpolarization produced in ganglion
cells by rotation of the windmill were blocked in the presence of 2 µM strychnine or 500 nM tetrodotoxin (TTX),
but not by 150 µM picrotoxin. In the slice preparation in
which GABA-mediated currents were blocked with picrotoxin, IPSCs
elicited by diffuse illumination were blocked by strychnine and
strongly reduced by TTX. The TTX-resistant component was probably
attributable to illumination of the receptive field center. TTX had a
much greater effect in reducing the glycinergic inhibition elicited by
laterally displaced stimulation versus nearby focal electrical
stimulation. Strychnine enhanced light-evoked excitatory currents in
ganglion cells, but this was not mimicked by TTX. The results suggest
that local glycinergic transient inhibition does not require action
potentials and is mediated by synapses onto both ganglion cell
dendrites and bipolar cell terminals. In contrast, the lateral spread
of this inhibition (at least over distances >250 µm) requires action
potentials and is mainly onto ganglion cell dendrites.
Key words:
retina; ganglion cell; receptive field; lateral
inhibition; glycine; action potentials
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INTRODUCTION |
Changes in illumination (the onset,
offset, or movement of a light stimulus) cause inhibition of ganglion
cells over a wide area of the retina. This mechanism, known as
change-sensitive or transient lateral inhibition (TLI), is thought to
be mediated in the inner plexiform layer by wide-field transient
(on-off) amacrine cells (Werblin, 1972
; Werblin and Copenhagen, 1974;
Thibos and Werblin, 1978). The idea that TLI might be mediated by
glycine was first suggested by the finding that in mud puppy ganglion cells glycinergic inhibitory inputs were activated at the onset and
termination of light stimuli but not during steady illumination (Belgum
et al., 1984). A likely source of such input would be on-off amacrine
cells, and in the closely related tiger salamander retina most
wide-field on-off amacrine cells are immunoreactive for glycine (Yang
et al., 1991). The wide-field transient amacrine cells in tiger
salamander retina receive excitatory inputs near their somas and
generate action potentials that propagate away from the soma over long
processes (Cook and Werblin, 1994), suggesting that the lateral spread
of TLI may be via action potentials. It is known that action potentials
increase the spread of signals within some wide-field amacrine cells
(Barnes and Werblin, 1987; Cook and Werblin, 1994; Bloomfield, 1996),
but it is not yet known how action potential-dependent signaling in
amacrine cells affects ganglion cell responses to light. In the present
study, we measured the effects of tetrodotoxin (TTX) and the glycine
antagonist strychnine on light-evoked TLI in ganglion cells in mud
puppy and tiger salamander eyecup preparations and on light-evoked
EPSCs and IPSCs in ganglion cells in the salamander retinal slice
preparation. The results indicate that TLI in ganglion cells involves
glycinergic inhibition onto ganglion cells, that transient glycinergic
inhibition is elicited by both center and surround illumination, and
that action potentials are required for the lateral spread of this
inhibitory signal in the retina over distances >250 µm. There is
also a presynaptic component of transient glycinergic inhibition onto
bipolar cells, but this appears to be mainly from nearby amacrine
cells. This is the first demonstration that action potentials are
required for the lateral transmission of signals affecting the
receptive field organization of retinal ganglion cells.
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MATERIALS AND METHODS |
Eyecup preparations were made from mud puppies (Necturus
maculosus) and larval tiger salamanders (Ambystoma
tigrinum). Mud puppies were obtained from Kons Scientific
(Germantown, WI), and tiger salamanders were from Charles Sullivan
(Nashville, TN). The care and use of animals was in accordance with the
University of Michigan and the Society for Neuroscience policies on the
use of animals in research. Details of the preparation, electrical recording, and light stimulation are described elsewhere (Cook and
McReynolds, 1998). Intracellular voltage recordings were made from
on-off ganglion cells using micropipettes, filled with 2 M
potassium acetate (resistance 300-500 M
), and conventional electronics. Light stimuli were a 400 µm diameter spot in the receptive field center (determined before the experiment) and concentric annuli [inner diameter (i.d.) 1200 or 500 µm, outer diameter (o.d.) 2600 µm]. All light stimuli were 560 nm, the
intensity of which was controlled with calibrated neutral density
filters and expressed as log quanta/cm2/sec. Retinas
were superfused with amphibian Ringer's solution (in
mM/l): NaCl 110, KCl 2.5, CaCl2 1.8, MgCl2 1.2, glucose 11, HEPES buffer 5, adjusted to pH 7.8 with NaOH. Drugs were added by switching to another Ringer's solution
containing either 2 µM strychnine, 150 µM
picrotoxin, or 500 nM TTX.
One measure of TLI in the retina is the degree to which a
response of the ganglion cell to illumination of the receptive field center is suppressed by rotation of a concentric windmill pattern (Werblin, 1972; Werblin and Copenhagen, 1974; Thibos and Werblin, 1978). In the present experiments, this measurement of TLI was made by
comparing the response to a 400-µm-diameter spot in the presence of a
concentric windmill pattern (i.d. 500 or 1200 µm, o.d. 2600 µm)
that was either stationary or rotating at 0.25 rps. Because TLI is the
additional suppression produced by rotation of the windmill pattern,
the percentage suppression attributed to TLI is
100·(Rstat 
Rrot)/Rstat,
where Rstat = spot response in the presence of
the stationary windmill, and Rrot = spot
response in the presence of the rotating windmill. Voltage responses
were digitized at 1 KHz. Four to eight responses in each condition were
averaged after digital filtering using a Gaussian with 3 dB rolloff
at 10 Hz, and the area of the EPSP above the baseline (dark) potential
was calculated between 250 and 1250 msec after the onset of the spot
stimulus. The spot response measured in this way correlated well with
the number of action potentials produced by the spot stimulus.
Retinal slice preparations (200 and 400 µm thick) were made from
larval tiger salamander eyes as described in detail by Lukasiewicz et
al. (1994). Whole-cell voltage-clamp recordings were made using patch
electrodes containing (in mM/l): cesium gluconate 99, tetraethylammonium chloride 8, NaCl 3.4, MgCl2 0.4, CaCl2 0.4, EGTA 11, HEPES buffer 10, adjusted to pH 7.7 with NaOH. The bath solution contained (in mM/l): NaCl 112, KCl 2, CaCl2 2, MgCl2 1, glucose 5, HEPES buffer 5, adjusted to pH 7.8 with NaOH. Light stimuli were diffuse flashes of white light in which the intensity at the retinal surface was equivalent to 3.6 × 108
quanta · cm2
1 · sec1
at 560 nm. The slice was superfused and drugs were applied through a
large diameter pipette connected to a gravity-driven superfusion system
that permitted rapid switching between control and test solutions.
Ganglion cells were identified by the location of their somas in the
ganglion cell layer and large (>1000 pA) inward currents elicited by
depolarizing voltage steps. Some cells were also identified visually by
inclusion of Lucifer yellow in the recording pipette.
Current recordings from slice preparations were digitized at 2 kHz.
GABAA- and GABAC-mediated synaptic currents
were blocked by the presence of 150 µM picrotoxin in the
superfusate. In some experiments the retina was electrically stimulated
by application of 1 msec positive current pulses (0.5-2 µA) using a
constant current stimulator (Grass S48 with stimulus isolation unit
PSIU6) through a Ringer's solution-filled pipette, the tip of which
was located in the outer plexiform layer (OPL) ~300 µm lateral to the recording site. The return path for the current was a
silver-silver chloride electrode, separate from the recording ground
electrode, connected to the bath through an agar bridge filled with 1 M KCl. The OPL site was used because it produced larger and
more consistent responses than when the electrode was located in the
inner retina. Focal application (puffs) of glycine was made by pressure
(5 msec at 5 psi) ejection (Picospritzer) from a pipette containing 0.5 mM glycine. Strychnine and picrotoxin were obtained from
Sigma (St. Louis, MO), and TTX was from Research Biochemicals (Natick, MA).
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RESULTS |
Eyecup experiments
As previously shown in mud puppy, both sustained and transient
lateral inhibition in tiger salamander ganglion cells are strongly affected by the state of adaptation. Light adaptation enables TLI (it
is disabled in dark-adapted retinas) and reduces the amount of lateral
inhibition produced by distant stationary stimuli (Cook and McReynolds,
1998). Therefore, to study the effects of pharmacological agents on
TLI, most of the eyecup experiments reported here were performed under
conditions in which TLI was relatively strong and suppression by
stationary windmills was relatively weak, i.e., using light-adapted
retinas and windmill stimuli of large (1200 µm) inner diameter.
Strychnine blocks transient lateral inhibition in
ganglion cells
Figure 1 shows responses of an
on-off ganglion cell from tiger salamander retina to a 400 µm
diameter spot centered in the receptive field under various conditions.
Each row of traces shows averaged responses to the spot alone
(left), and to the same spot in the presence of a 1200 µm
i.d. windmill stimulus that was either stationary (middle)
or rotating (right), as indicated by the stimulus markers at
the bottom of the figure. In these traces the shaded areas indicate the
area of EPSP above the baseline (resting potential in darkness) that
was measured for quantifying the effects of the windmill patterns (see
Methods and Materials). In control Ringer's solution (top
row), there was a transient hyperpolarization at the onset of the
stationary windmill that became sustained when the windmill was
rotating. The spot response was affected only slightly by the
stationary windmill but was shifted to a more negative potential level
when the windmill was rotating. Although not shown here because the
action potentials were filtered out, both the number of spikes
generated by the spot stimulus and the maximum spike frequency were
significantly reduced while the windmill was rotating. The changes in
spike number and maximum spike frequency correlated well with the
changes in EPSP area above baseline (see Methods and Materials). The
middle row of traces in Figure 1 shows responses to the same stimuli 5 min after the addition of 2 µM strychnine to the
superfusate. Strychnine blocked the transient hyperpolarization at the
onset of the stationary windmill, the sustained hyperpolarization
produced by the rotating windmill, and the suppression of the spot
response by rotation of the windmill. Strychnine also altered the
kinetics of the spot response (see below). The bottom row of traces
shows the responses to these same stimuli 20 min after return to
control Ringer's solution. Recovery from strychnine was slow, but
complete recovery was usually observed if the recording could be
maintained for >30 min. Similar results of 2 µM
strychnine were seen in all on-off ganglion cells tested with 1200 µm i.d. windmill stimuli (n = 6), as summarized (see
Fig. 3). Strychnine caused a significant reduction in TLI (paired
Student's t test; p = 0.004). The small amount of suppression produced by stationary 1200 µm i.d. windmills in these light-adapted retinas was not significantly reduced by strychnine (p = 0.30).
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Figure 1.
Strychnine blocks TLI in ganglion cells. Responses
are from an on-off ganglion cell in tiger salamander eyecup. Each
trace is the average of four to eight responses that had been filtered to eliminate action potentials (see Methods and Materials). Each of the
three groups of traces shows the average response to a 400-µm-diameter spot alone (left) and in the presence
of a windmill pattern (1200 µm i.d., 2600 µm o.d.) that was either
stationary (middle) or rotating (right).
The timing of the spot and windmill stimuli are indicated by the
horizontal bars at the bottom. Spot and
windmill intensity were both 8.4 log quanta. Responses in the
middle row of traces were obtained after 5 min in 2 µM strychnine, and those in the bottom row
of traces were obtained 20 min after return to control Ringer's
solution. Resting potential in darkness was 50 mV in control
Ringer's solution and 53 mV in the presence of strychnine.
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Although not shown here, strychnine also blocked TLI elicited by
windmills of smaller inner diameter (500 µm). With the smaller inner
diameter windmill patterns, there was also significant suppression of
the spot response when the windmill was stationary, but this sustained
lateral inhibition (Cook and McReynolds, 1998) was not reduced by
strychnine.
The same experiments were repeated using the
GABAA/GABAC antagonist picrotoxin (150 µM). In contrast to strychnine, picrotoxin did not reduce
TLI (see Fig. 3). Picrotoxin also did not reduce the transient
hyperpolarization at the onset of a stationary windmill stimulus or the
sustained hyperpolarization produced by rotating the windmill,
indicating that neither GABAA nor GABAC
receptors are necessary for the activation of the glycinergic
inhibition associated with TLI.
Tetrodotoxin blocks the lateral spread of transient lateral
inhibition in the retina
To test whether the lateral spread of TLI in the retina requires
action potentials, we measured the effects of steady versus rotating
1200 µm i.d. windmills before and after blocking action potential
generation with 500 nM TTX. Figure
2 shows that in the presence of TTX
neither the stationary nor rotating windmill stimuli produced any
detectable hyperpolarization. The stationary windmill caused a slight
suppression of the spot response, but there was no further suppression
of the spot response when the windmill was rotating. Thus, TTX had the
same effects as strychnine on all of the manifestations of TLI elicited
by the 1200 µm i.d. windmill stimulus: it blocked the transient
hyperpolarization at the onset of the stationary windmill, the
sustained hyperpolarization produced when the windmill was rotating,
and the suppression of the spot response by the rotating windmill.
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Figure 2.
TTX blocks TLI in ganglion cells. Responses are
from the same cell as in Figure 1. Except for the use of TTX rather
than strychnine, all conditions are the same as in Figure 1. The
responses in the middle row of traces were obtained
after 2 min in 500 nM TTX, and those in the bottom
row of traces were obtained 10 min after return to control
Ringer's solution. Resting potential was 51 mV in both control
Ringer's solution and TTX.
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Figure 3 summarizes the effect of 500 nM TTX on the six cells tested with 1200 µm i.d. windmill
patterns. TTX caused a significant reduction in TLI
(p = 0.003). TTX also blocked TLI elicited by 500 µm i.d. windmill stimuli (not shown), indicating that the lateral
spread of TLI over distances as short as 250 µm requires action
potentials.
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Figure 3.
Summary of effects of strychnine, picrotoxin, and
tetrodotoxin on transient lateral inhibition in ganglion cells. TLI was elicited by 1200 µm i.d. windmill stimuli, intensity 8.4 log quanta. Ordinate indicates the magnitude of TLI expressed as percentage suppression of the spot response by rotation of the windmill pattern, calculated as described in Materials and Methods. Each pair of bars shows TLI in control Ringer's solution
(open) and in the presence of the indicated drug
(hatched): 2 µM strychnine
(STR) (n = 6 cells), 150 µM picrotoxin (PTX)
(n = 3 cells), and 500 nM tetrodotoxin
(TTX) (n = 6 cells). In three
of the cells both strychnine and TTX were tested. Error bars indicate 1 SEM. Strychnine and TTX each caused a significant decrease in TLI
(p = 0.004 and 0.003, respectively; paired
Student's t test), but picrotoxin did not
(p = 0.45). For comparison, the data shown
above include only those cells in which the spot and windmill
intensities were 8.4 log quanta, but similar results were obtained in
other cells from both tiger salamander and mud puppy using windmill
stimuli of other intensities.
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Strychnine, but not tetrodotoxin, blocks transient inhibition
produced by illumination of the receptive field center
In addition to blocking TLI elicited by rotating windmill
patterns, strychnine also blocked inhibition elicited by illumination of the receptive field center. Figure 4
shows responses to a centered 400-µm-diameter spot in the absence of
a windmill pattern. The thick trace shows the response in control
Ringer's solution. In the presence of 2 µM strychnine,
the response to the spot reached its peak earlier and began to decay
sooner than in the absence of strychnine. The increase in rate of rise
of the spot response probably results from blocking glycinergic
transient inhibition elicited by the onset of the spot stimulus (Belgum
et al., 1984). The effect of strychnine on the decay of the spot
response is more difficult to explain, but it may be attributable to
removing glycinergic inhibition of a GABAergic input (Roska and
Werblin, 1997; Zhang et al., 1997), either to the ganglion cell or onto the bipolar terminals responsible for the EPSP. TTX (500 nM) did not mimic this effect of strychnine, suggesting
that glycine release from the nearby amacrine cells (those stimulated
by the small spot) does not require action potentials.
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Figure 4.
Effect of strychnine and TTX on the waveform of
the response elicited by center illumination. The traces show
superimposed responses of a tiger salamander on-off ganglion cell to a
400-µm-diameter spot (8.4 log quanta) in the receptive field center
in control Ringer's solution, in the presence of 2 µM
strychnine (STR), and in the presence of 500 nM TTX. Each trace is the average of eight responses. The
retina was washed with control Ringer's solution for 20 min between
the two drug applications. Resting potential was 55 mV in all
traces.
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Strychnine and tetrodotoxin block the conductance increase
associated with transient lateral inhibition
The above results indicate that TLI is mediated by glycine but
does not distinguish between a direct action (inhibition of ganglion
cells) and an indirect action (inhibition of bipolar cell terminals).
To test whether TLI involves direct inhibition of ganglion cells we
measured the changes in ganglion cell input conductance produced by
stationary and rotating windmill patterns. In the cells used in these
experiments, the effects of the 1200 µm i.d. windmill patterns on the
spot response were similar to those described above. The stationary
windmill caused only a transient hyperpolarization and did not
significantly suppress the spot response, whereas rotation of the
windmill caused a sustained hyperpolarization and significant
suppression of the spot response. Figure
5 shows the voltage deflections produced
in a ganglion cell by a 0.1 nA current pulse (250 msec duration) in
the dark (left), and in the presence of a 1200 µm i.d.
windmill that was either stationary (middle) or rotating
(right). Each trace is the average of 20 responses to the
current pulse. The hyperpolarizing voltage deflection produced by the
current pulse in darkness was balanced using the bridge circuit of the
amplifier, so that an upward voltage deflection indicates an increase
in conductance relative to the dark level. The stationary windmill did
not cause a sustained hyperpolarization or a change in membrane
conductance, whereas rotation of the windmill caused an increase in
membrane conductance and a sustained hyperpolarization (note downward
shift of right trace). The increase in conductance indicates
that TLI involves a direct inhibition of ganglion cells. In the three
ganglion cells tested, both strychnine and TTX blocked the
hyperpolarization and conductance increase associated with TLI.
Experiments in the slice preparation (see below) indicated that there
is also a presynaptic component of transient glycinergic
inhibition.
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Figure 5.
TLI is associated with an increase in ganglion
cell conductance. Each trace shows voltage deflections produced in a
tiger salamander ganglion cell by a 0.1 nA current pulse (250 msec duration) in the dark (left) and in the presence of a
1200 µm i.d. windmill that was stationary (middle) or
rotating (right). Each trace is the average of 20 responses to the current pulse. The voltage deflection produced by the
current pulse in darkness was balanced using the bridge circuit of the
amplifier, so that a positive voltage deflection indicates an increase
in conductance. The downward shift of the right trace
indicates the amount of sustained hyperpolarization (7 mV) produced
by the rotating windmill. Resting potential was 58 mV.
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Experiments in the retinal slice preparation
Experiments in tiger salamander retinal slice preparation were
undertaken to determine whether glycinergic inhibitory currents in
ganglion cells also consisted of a lateral component that required action potentials and a local component that did not. We also wanted to
determine whether there was a presynaptic component of TLI mediated by
glycinergic inhibition of bipolar cells, and if so, whether this
presynaptic inhibition also had TTX-sensitive and TTX-resistant
components. The presynaptic component was studied by recording the
excitatory inputs to ganglion cells when inhibitory inputs to the
recorded cell were eliminated by voltage-clamping the ganglion cells at
the reversal potential for chloride ions. The results reported here
were obtained only from ganglion cells that were reasonably well
space-clamped, as evidenced by reversal of the excitatory and
inhibitory currents near their expected reversal potentials (0 and 60
mV, respectively) and absence of the spontaneous, voltage-gated inward
currents usually seen in poorly space-clamped spiking cells.
Tetrodotoxin blocks the lateral spread of transient
glycinergic inhibition
The whole-cell recordings in Figure
6 show light-evoked, glycine-mediated
IPSCs in an on-off ganglion cell. GABAA- and
GABAC-mediated responses were eliminated by 150 µM picrotoxin in the bathing solution, and excitatory
currents were eliminated by voltage-clamping the cells at 0 mV, which
is near the reversal potential for the excitatory input. Under these
conditions, diffuse illumination elicited an outward current that was
completely blocked by 2 µM strychnine (Fig.
6A), indicating that this current was entirely glycinergic. The light-evoked glycinergic current was a transient response elicited by the onset of illumination, because even with long
(3-5 sec duration) light stimuli the IPSC was transient and decayed to
zero in <1 sec. In the same cell, 500 nM TTX strongly reduced this light-evoked current (Fig. 6B). Because
the diffuse light stimulus also illuminated the receptive field center,
the small TTX-resistant component of the inhibitory current was
probably attributable to glycine released by amacrine cells near the
receptive field center. This is consistent with the finding in eyecup
preparations that illumination of the ganglion cell receptive field
center activated glycinergic transient inhibition that was not blocked by TTX (Fig. 4).
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Figure 6.
Effects of strychnine and TTX on light-evoked
inhibitory currents in ganglion cells. Responses are from an on-off
ganglion cell in a tiger salamander slice preparation. The control
Ringer's solution contained 150 µM picrotoxin to block
GABA-mediated responses. The cell was voltage-clamped at 0 mV to
eliminate glutamate-mediated excitatory currents. Horizontal
bars below responses indicate duration of diffuse light
stimulus. A, Currents recorded in control solution
(control), in the presence of 2 µM
strychnine (STR), and 20 min after return to control
solution (wash). B, Currents recorded in
control solution (control), in the presence of
500 nM TTX, and 10 min after return to control solution
(wash). All responses were from the same cell. Holding
current was 20 pA in A and 34 pA in B.
Calibration bars are for both A and
B.
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Although separate stimulation of the receptive field center and
surround with light stimuli was not possible in the slice preparation,
we tested the hypothesis that TTX blocked only the glycinergic
inhibition from laterally distant sources by comparing the effects of
TTX on inhibition produced by local and laterally displaced electrical
stimulation of the retina. Figure 7 shows the effects of TTX on glycinergic IPSCs elicited by focal electrical stimulation of the retina directly above the recording site
(A), and ~300 µm lateral to the recording site
(B). The cells were voltage- clamped at 0 mV to
eliminate excitatory currents, and the bath contained 150 µM picrotoxin to eliminate GABA-mediated responses. Under
these conditions the IPSCs elicited by electrical stimulation were
purely glycinergic because they were completely blocked in the presence
of 2 µM strychnine. TTX reduced the glycinergic IPSC
elicited by laterally displaced electrical stimuli (Fig. 7B)
to a much greater extent than it reduced that elicited by electrical
stimuli near the recording site (Fig. 7A). Although the
local and laterally displaced electrical stimulations were made in
different cells, the differential effect of TTX on inhibition from
local versus laterally displaced electrical stimulation was quite
clear. For electrical stimulation directly over the recording site, TTX
reduced the IPSC area by 18.8 ± 11.3% (n = 4 cells), and for laterally displaced electrical stimulation TTX reduced the IPSC area by 64.3 ± 12.0% (n = 6 cells).
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Figure 7.
Effect of strychnine and TTX on glycinergic
inhibitory currents elicited by electrical stimulation. IPSCs were
recorded from ganglion cells held at 0 mV to eliminate excitatory
currents, and the control Ringer's solution contained 150 µM picrotoxin to block GABA-mediated responses. Responses
were elicited by 1 msec, 1 µA current pulses applied through a
pipette located in the OPL directly over the recording site
(A) and ~300 µm lateral to the recording site
(B). Superimposed traces show current recorded under control conditions (control), in the
presence of 2 µM strychnine (STR), and in
the presence of 500 nM TTX. A and
B were from different cells. Holding current was 15 pA
in A and 12 pA in B. The
dot below traces indicates time of electrical
stimulation.
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Another observation supporting the idea that TTX blocked primarily the
lateral spread of glycinergic inhibition is that TTX caused much less
reduction in the IPSCs evoked by diffuse illumination in thin (200 µm) slices than in thick (400 µm) slices, presumably because in the
thinner slices a greater fraction of the inhibitory input was from
nearby regions and did not require action potentials.
To rule out the possibility that the reduction in glycine-mediated
currents by TTX was attributable to some action on the ability of the
ganglion cell to respond to glycine, the effect of TTX on inhibitory
currents elicited by locally applied puffs of glycine was tested. The
current traces in Figure 8 were obtained when the cell was voltage-clamped at 0 mV to eliminate excitatory inputs and in the presence of 150 µM picrotoxin to block
GABA-mediated responses. The puff-evoked IPSCs were blocked by 2 µM strychnine but not by 500 nM TTX. This
indicates that the effect of TTX on the light-evoked inhibitory current
was attributable to an action on the glycinergic input pathway rather
than to some effect of TTX on the ability of the ganglion cell to
respond to glycine.
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Figure 8.
Effect of strychnine and TTX on responses to
direct application of glycine. The superimposed traces show IPSCs
recorded from the same ganglion cell in control solution
(control), in the presence of 2 µM
strychnine (STR), and in the presence of 500 nM TTX. The cell was voltage-clamped at 0 mV to eliminate
excitatory currents, and the control solution contained 150 µM picrotoxin to block GABA-mediated responses. Responses
were elicited by a 5 msec puff of 0.5 mM glycine from a
pipette located in the inner plexiform layer just above the ganglion
cell. The dot below traces indicates time of glycine
puff. Holding current was 35 pA.
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Effect of strychnine and tetrodotoxin on light-evoked excitatory
inputs to ganglion cells
Because there is evidence for glycine-mediated synaptic input onto
bipolar cell axon terminals in tiger salamander (Maple and Wu, 1998),
it is of interest to know whether these inputs also contribute to TLI.
We looked for evidence of such a presynaptic or disfacilitatory
component of TLI by examining the effects of strychnine and TTX on the
light-evoked excitatory currents in ganglion cells, because
the excitatory currents presumably reflected transmitter
release from bipolar cells. Light-evoked EPSCs were isolated
by voltage-clamping ganglion cells at the reversal potential for
chloride ions (65 mV) to eliminate the inhibitory currents. For
consistency with the other experiments in the slice, the bathing solution also contained 150 µM picrotoxin to block
GABA-mediated responses. Figure 9 shows
the effects of strychnine and TTX on EPSCs isolated in this manner.
Strychnine did not cause a change in the holding current in darkness,
but it caused a significant increase in the light-evoked EPSC. This
result was seen in four of the five cells tested; it had no effect on
the EPSC in one cell. The increase in EPSC amplitude in the presence of
strychnine was likely caused by the blocking of light-evoked
glycinergic input to bipolar cells. In this cell, 500 nM
TTX had little effect on the EPSC. The effect of TTX on the EPSC was
variable, but in the five cells tested it was always much smaller than
that of strychnine. This suggests that in bipolar cells a large
fraction of the glycinergic input does not require action potentials
and therefore may come from nearby amacrine cells.
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Figure 9.
Effect of strychnine and TTX on excitatory input
to ganglion cells. The superimposed traces show the
inward currents elicited by the same diffuse light flash (indicated by
horizontal line above responses) in control solution
(control) and in the presence of 2 µM strychnine (STR) and 500 nM
TTX. Strychnine was applied after recovery following washout of TTX.
Bath contained 150 µM picrotoxin. Cells were
voltage-clamped at the reversal potential for chloride ions (65 mV)
to eliminate glycine-mediated responses. Holding current was 12
pA.
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DISCUSSION |
The results of these experiments show that in mud puppy and
salamander retinas TLI and the associated hyperpolarization and conductance increases in ganglion cells are mediated by glycinergic amacrine cells and do not require GABA, and that the lateral spread of
this transient glycinergic inhibition in the retina requires action
potentials. In some ganglion cells, the effect of rotating the windmill
pattern was primarily a sustained hyperpolarization that shifted the
spot response to a more hyperpolarized level with relatively little
change in its amplitude, but in other ganglion cells windmill rotation
also caused both a hyperpolarization and a significant reduction in
spot response amplitude. The relative contributions of these two
effects varied considerably among ganglion cells in both mud puppy and
tiger salamander. The functional significance of these differences is
not known and will require further study. However, both effects were
always blocked by strychnine and TTX, indicating that both were the
result of laterally transmitted glycinergic inhibition.
The fact that transient glycinergic inhibition was elicited by
illumination of the receptive field center, as well as by surround (windmill) stimuli, indicates that transient glycinergic inhibition has
both local and lateral components. Because the receptive field of the
glycinergic inhibitory input is much wider than that of the excitatory
input to ganglion cells, Lukasiewicz and Werblin (1990), illumination
of the receptive field center with a 400-µm-diameter spot elicits
both excitation and transient glycinergic inhibition, whereas distant
light stimuli such as the 1200 µm i.d. windmill patterns elicit only
TLI. The fact that TLI elicited by windmill stimuli of 500-1200 µm
i.d. was blocked in the presence of TTX indicates that the lateral
spread of the TLI signal in the inner retina requires action
potentials. However, because TTX did not block the transient
glycinergic inhibition elicited by illumination of the receptive field
center and did not block all of the glycinergic IPSCs elicited by
diffuse light stimuli in the slice recordings, transient glycinergic
inhibition near the site of illumination probably does not require
action potentials. The greater effectiveness of TTX in blocking
glycinergic IPSCs elicited by laterally displaced versus local
electrical stimulation supports this idea.
The finding that light-evoked excitatory inputs to ganglion cells were
enhanced in the presence of strychnine indicates that light also
activates glycinergic inhibition of bipolar cell output. Because this
effect of strychnine was not mimicked by TTX, it is likely that most of
the glycinergic input to bipolar cell terminals comes from nearby
amacrine cells. The present results thus establish a functional role
for the recently described glycinergic inputs to bipolar cell terminals
in tiger salamander retina (Maple and Wu, 1998).
The simplest explanation of these results is that transient glycinergic
inhibition elicited by both local and distant illumination is mediated
by the wide-field transient amacrine cells described by Cook and
Werblin (1994). These cells receive excitatory input near the soma and
generate action potentials that propagate away from this area over long
processes. Figure 10 shows the basic
elements of the proposed circuitry for the glycinergic wide-field
transient amacrine cell. These cells receive excitatory input from
bipolar terminals near the soma and make output synapses onto bipolar and ganglion cells at sites both near the input region and on the long
processes. Depolarization from the excitatory inputs near the soma
spreads passively to nearby transmitter release sites, but action
potentials are required for transmission of this signal to the more
distant release sites. In the diagram of Figure 10, the thick processes
of the wide-field amacrine cell indicate the region over which
transmitter release can by elicited by passive spread of the input
signals (local output region), and the thin processes indicate the more
distant regions over which information is conducted only via action
potentials. Because most of the light-evoked, transient glycinergic
input to bipolar terminals appears to be resistant to TTX, the action
potential-dependent portion of the glycinergic input to bipolar
terminals is shown as a dashed line. Because the wide-field transient
amacrine cells typically generate only a single action potential at the
onset or termination of a light stimulus, the "sustained"
inhibition produced in ganglion cells by rotation of a distant,
concentric windmill pattern is likely attributable to the temporal
summation of IPSPs produced by action potentials generated in many
different wide-field transient amacrine cells that are successively
activated as the windmill vane edges pass over their input regions.
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Figure 10.
Proposed pathway for TLI in mud puppy and
salamander retina. Filled and open
terminals indicate excitatory and inhibitory synapses,
respectively. Glycinergic wide-field transient amacrine cells
(GLY WFTA) receive excitatory input from bipolar cells
(BC) over a restricted region near their somas and send
action potentials laterally (arrows) over long processes
(thin lines). Output synapses are onto bipolar terminals
and ganglion cell dendrites. It is not known whether GLY-WFTA cells
also make feedback synapses onto the bipolar terminals from which they
receive excitatory input. Depolarization produced by excitatory inputs
can spread passively (over region indicated by thick
processes) to affect glycine release at nearby sites, but
action potentials are required to elicit glycine release at distant
sites (region indicated by thin processes). Alternatively, some or all of the short-range TTX-resistant transient glycinergic inputs to bipolar and ganglion cells may come from a
separate population of glycinergic narrow-field transient amacrine cells (not shown). In addition to their role in mediating TLI, glycinergic amacrine cells may also make synapses onto GABAergic amacrine cells (not shown).
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In the above circuit the wide-field transient amacrine cell releases
glycine both locally and at distant sites. It is also possible that the
local (TTX-resistant) transient glycinergic input to bipolar and/or
ganglion cells may come from a separate population of narrow-field
glycinergic transient amacrine cells that have not yet been described.
At present we cannot distinguish between these possibilities. If local
and laterally conducted transient glycinergic inhibition are mediated
by the same cell type, as suggested above, then it is appropriate to
consider transient glycinergic inhibition as a wide-field mechanism,
including both local and lateral components, the latter of which is
referred to as TLI.
The circuit in Figure 10 also does not show any glycinergic feedback
from the wide-field transient amacrine cells onto the bipolar terminals
from which they receive excitatory input. It is not known whether such
feedback synapses exist, but this certainly is a possibility because a
given bipolar terminal may synapse onto both ganglion and amacrine
cells.
Although picrotoxin did not block TLI in ganglion cells, in some cases
it caused an increase in the transient glycinergic IPSP at the onset of
a windmill stimulus and the glycinergic sustained hyperpolarization
elicited by rotation of the windmill. This observation suggests that
GABAergic neurons may modulate the pathway mediating TLI, and such
modulation may be one function of the recently reported reciprocal
inhibitory interactions between GABAergic and glycinergic amacrine
cells (Roska and Werblin, 1997; Zhang et al., 1997). The effects of
GABA antagonists on glycinergic inhibitory inputs to ganglion cells are
quite complicated, however, and much additional work is necessary
before the role of GABA in modulating TLI can be understood.
TLI evoked by rotating windmill patterns is not a peculiarity of
amphibians; it has also been described in cat retina (Enroth-Cugell and
Jakiela, 1980). A type of wide-field transient amacrine cell that
appears to receive excitatory input over a narrow range near the soma
and send information laterally over long axonal processes via action
potentials has been described recently in rabbit retina (Taylor, 1996).
Although the transmitter used by these cells is not known, their
properties are similar to those of the wide-field transient amacrine
cells in salamander, and such cells would be well suited for mediating
long-range TLI. It is not known whether TLI in mammalian retinas is
mediated by glycine or whether its lateral spread requires action
potentials.
Because TLI is concerned with changes in illumination, it is tempting
to speculate that it may have something to do with directional selectivity. In mammalian retinas, directional selectivity is believed
to involve mainly GABA (Caldwell et al., 1978; Kittila and Massey,
1997), but it has been suggested that glycine may play a role in
directional selectivity of ganglion cells in amphibian retinas (Pan and
Slaughter, 1991).
The role of amacrine cell action potentials in retinal signaling is not
completely understood. In rabbit retina, TTX reduced the spread of
signals within the dendritic arbors of large-field amacrine cells but
not in small-field amacrine cells or in any ganglion cells (Bloomfield,
1996); although TTX did not affect ganglion cell receptive field center
size, no other receptive field properties were examined in that study.
In tiger salamander retina, TTX blocked the inhibition of amacrine
cells by laterally displaced transretinal current stimulation (Barnes
and Werblin, 1987), but its effect on ganglion cell receptive field
organization was not studied. The present results are the first
demonstration that action potentials are required for the lateral
transmission of light-evoked signals affecting ganglion cell receptive
field organization. One might ask why action potentials are necessary, because lateral transmission of signals over similar distances via
horizontal cells in the outer retina does not require action potentials. One reason may be that the increased speed of conduction afforded by action potentials is more important for TLI, because TLI
signals changes in illumination rather than steady illumination. Another possibility is that the processes of the amacrine cells mediating TLI must be small in diameter because of space restrictions in the inner plexiform layer, and the resulting short length constants do not allow passive spread of signals for any significant distance in
the inner plexiform layer. If this is the case, then it is possible
that other types of lateral signaling over any appreciable distance in
the inner retina may also require action potentials. We are currently
studying this possibility.
| |
FOOTNOTES |
Received Nov. 20, 1997; revised Jan. 5, 1998; accepted Jan. 7, 1998.
This work was supported by National Institutes of Health Research
Grants EY01653 and EY08922, Core Grants EY07003 and EY02687, and a
grant from Research to Prevent Blindness to P.D.L.
Correspondence should be addressed to Dr. Paul B. Cook, Department of
Physiology, The University of Michigan Medical School, Ann Arbor, MI
48109-0622.
| |
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Top
Abstract
Introduction
Compound via MeSH
