Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, Massachussetts 02114
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ABSTRACT |
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Euler, Thomas and
Richard H. Masland.
Light-Evoked Responses of Bipolar Cells in a Mammalian Retina.
J. Neurophysiol. 83: 1817-1829, 2000.
We recorded light-evoked responses from rod and cone bipolar cells
using patch-clamp techniques in a slice preparation of the rat retina.
Rod bipolar cells responded to light with a sustained depolarization
(ON response) followed at light offset by a slight hyperpolarization. ON and OFF cone bipolar
cells were encountered, both with diverse temporal properties. The
responses of rod bipolar cells were composed primarily of two
components, a nonspecific cation current and a chloride current. The
chloride current was reduced greatly in axotomized cells and could be
suppressed by coapplication of the GABAA antagonist
bicuculline and the GABAC antagonist
(1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid. This suggests
that it largely reflects feedback from GABAergic amacrine cells. The
response latency of intact rod bipolar cells was shorter than that of
the axotomized cells, and the sensitivity curve covered more than twice
the dynamic range. Application of the GABA receptor antagonists
partially mimicked the effects of axotomy. These findings suggest that
functional properties of the axon terminal systemnotably synaptic
feedback from amacrine cells
play an important role in defining the
response properties of mammalian bipolar cells.
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INTRODUCTION |
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Most signal processing by the mammalian retina
takes place within the inner plexiform layer, involving interactions
among bipolar, amacrine, and ganglion cell processes. At the center of
these interactions is the bipolar cell's axon terminal system, a
highly specialized structure capable of releasing glutamate continuously at its ribbon synapses (Heidelberger et al.
1994; Rouze and Schwartz 1998
; von
Gersdorff and Matthews 1994
). A bipolar cell receives graded
photoreceptor signals and translates these into a modulation of its
release of glutamate. This output signal is shaped through several
mechanisms. First, the dendritic glutamate receptors receiving the
photoreceptor input may be modified via intracellular pathways
(reviewed in Hatt 1999
); the second-messenger pathways
that depolarizing (ON) bipolar cells use for signal
transduction offer many such possibilities (Shiells and Falk
1995
; Walters et al. 1998
). Second, bipolar
cells express several types of voltage-gated calcium and potassium
channels. Certain K+ channel subunits
(Klumpp et al. 1995
) and L-type
Ca2+ channels (De la Villa et al.
1998
; Hartveit 1999
; Protti and Llano
1998
; Satoh et al. 1998
) seem to be located
preferentially on the bipolar axon terminal, supporting its role in
shaping the time course of the output. Finally, bipolar cells possess
high densities of ionotropic GABA and glycine receptors on their axon and terminals (reviewed in Wässle et al. 1998
).
There, amacrine cells synapse and deliver reciprocal and nonreciprocal
feedback inhibition (Burkhardt 1972
; Chun et al.
1993
; Dong and Werblin 1998
; Dowling and
Boycott 1966
; Pan and Lipton 1995
; Toyoda
and Fujimoto 1984
).
Bipolar cells come in different morphological types (Cajal
1893; Cohen and Sterling 1990
;
Famiglietti 1981
). It has been suggested that these
types represent parallel signaling pathways through the retina,
carrying different temporal, spatial, and chromatic aspects of the
visual stimulus. In the retinas of cold-blooded animals, especially the
dogfish and tiger salamander, the responses of bipolar cells to light
have been studied extensively, leading to much important information on
the polarity and the time course of their responses, the currents
involved, and their receptive field organization (Ashmore and
Falk 1980
; Kaneko and Shimazaki 1976
;
Lasansky 1992
; Saito and Kujiraoka 1982
;
Saito et al. 1979
, 1981
, 1985
; Werblin
1974
). However, the degree to which information from
cold-blooded animals can be translated to mammals (and vice versa) is
not entirely clear. Although the cell's fundamental biophysical
properties are certainly similar, their wiring is not necessarily the
same. In the mammalian retina, the cells are harder to record from than
in cold-blooded vertebrates, but a systematic anatomic classification
exists: one type of rod bipolar cell (RBC) and seven to nine types of
presumed cone bipolar cells (CBCs) have been described (reviewed in
Euler and Wässle 1995
). In principle, one now can
begin to map the various kinds of physiological responses onto the
morphological types, but in mammals the responses to light have been
little studied.
In this study, we addressed the mechanisms that shape the light-evoked
responses of bipolar cells. We focused on the role of the bipolar
cell's axon terminal system by comparing the functional properties of
intact and axotomized rod bipolar cells. In addition, we studied the
effects of GABA receptor antagonists on the response behavior of these
cells. The results, together with those of others, suggest that events
at the bipolar axon terminalsamacrine cell feedback as well as axonal
membrane conductances
importantly control the dynamic properties of a
bipolar cell's response.
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METHODS |
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Animals
Three- to 7-wk-old albino rats were used. No systematic
age-dependent differences in their responses to light were observed. Although the retinas of hypopigmented rats are susceptible to light-induced damage, the majority of studies on the rat retina have
been done in albinos. In the young animals we used, light-induced retinal damage is probably negligible (Weisse et al.
1990). Because physiological studies show no significant
differences in the sensitivity to light between these animals and
pigmented strains (Green et al. 1991
; Herreros et
al. 1992
), we used albino rats for comparability.
Tissue preparation
The animals were dark adapted for 2 h before the experiment.
To reduce bleaching of the photoreceptors, the preparation was performed under dim red illumination. The rats were anesthetized deeply
with 10 mg/kg xylazine (Bayer AG, Leverkusen, Germany) and 50 mg/kg
ketamine (Fort Dodge Labs, IA). After enucleation, the animals were
killed with an overdose of xylazine/ketamine, in accordance with
institutional guidelines.
The retina was isolated in a petri dish perfused with Ames medium
(Sigma Aldrich, St. Louis, MO), bubbled with 95%
O2-5% CO2, and cooled to
~20°C. Slices of ~150 µm were cut by hand using a scalpel blade
(Boos et al. 1993). By adjusting the angle of the blade,
vertical or slightly "wedged" slices were produced, increasing the
frequency of intact or axotomized bipolar cells, respectively. The
slices were transferred into a recording chamber and held in place with
a nylon grid. The remaining tissue was maintained intact for
5 h at
room temperature in bubbled Ames medium before being used for more slices.
Patch-clamp recordings
The recording chamber was placed on the fixed stage of an upright microscope (Axioskop, Zeiss, Germany) and perfused with warmed, bubbled Ames solution (35-37°C). A water-immersion objective was used (Acroplan ×63/0.9w Ph3, Zeiss). The microscope was equipped with differential interference contrast (DIC) optics and epifluorescence illumination for visualizing the Lucifer yellow (Sigma Aldrich)-filled cells at the end of the recordings.
Patch electrodes (8-12 M) were pulled from borosilicate glass
(1B150F-4; World Precision Instruments, Sarasota, FL) on a vertical
electrode puller (Narishige, Japan). The recordings were performed with
a PC-501A patch-clamp amplifier (Warner Instruments, Hamden, CT). As
the reference electrode, an Ag/AgCl pellet in contact with the solution
in the recording chamber was used. The recorded signals were low-pass
filtered at 10 kHz or 500 Hz and digitized (4 or 5 times over-sampling)
using AxoScope or Clampex (Axon Instruments, Foster City, CA) in
conjunction with a Digidata 1200A digitizer board (Axon Instruments).
The data analysis was performed with Origin (Microcal Software,
Northampton, MA).
Both whole cell and perforated-patch recordings were performed
(Hamill et al. 1981; Horn and Marty
1988
). For the perforated-patch configuration, nystatin was
used (0.1-0.2 µg/ml intracellular solution). After a successful
perforated-patch recording, the cell membrane was ruptured to fill the
cell with Lucifer yellow. In some whole cell experiments, nystatin was
added at a lower concentration (~0.01 µg/ml) to facilitate
rupturing of the cell membrane under the electrode without breaking the
seal. Perforated-patch recordings tended to be more stable than whole
cell recordings. Apart from this, no differences relevant to our
results were observed between the two recording modes.
Seal resistances >1 to 2 G were commonly obtained. Series
resistances (RS) were estimated from
the peak amplitudes of the capacitative currents or automatically
determined by Clampex7. Because capacitative currents are capped by the
limited sampling frequency of the system,
RS was overestimated. For the cells
that were analyzed in more detail, RS
ranged from 35 to 85 M
(whole cell; for perforated-patch mode:
65-125 M
). Recordings of cells with higher
RS were only used to determine the
polarity of their light responses (ON or OFF).
The built-in RS compensation of the amplifier was not activated routinely because it often caused oscillations that damaged the seal. Corrections for voltage errors related to RS were not made.
The experiments were performed in a dark room. The slices were
visualized using dim red illumination. Immediately after sealing onto
the targeted cell and while establishing the whole cell configuration, the illumination was turned off. Before the first light stimulus was
applied, the slice was kept in darkness for 5 min to allow the
photoreceptors to recover.
Light stimulus
Below the recording chamber, a conventional yellow light-emitting diode (LED; emission maximum at 585 nm) diffusely illuminated the microscope field. The LED was controlled either directly by an analogue output channel of the Digidata or by an external stimulator (S88, Grass Instrument, West Warwick, RI), which then also triggered the recording.
The stimulus duration was varied between 1 ms and 2 s. The brightness of the LED was calibrated using a photodiode (S1133-12; Hamamatsu Photonics, Japan) and a photometer (LS-100; Minolta, Japan). The stimulus intensity could be varied from 0.02 to 100 cd/m2, covering the range from high scotopic into the photopic regime. Although the recording chamber was shielded, a very dim background illumination (not visible to the dark-adapted eye of the experimenter) may have been present during the recordings, precluding the observation of single photon responses.
The recorded bipolar cells generally were located near the upper
surface of the slice, so that the stimulating lightentering the slice
perpendicular to the outer segments of the photoreceptors
had to pass
through tissue before it reached the photoreceptors connected to the
examined cell. On its way, the light was attenuated, mainly by the
intervening outer segments, in an amount depending on the thickness of
the slice. This attenuation was probably similar in all experiments,
though difficult to estimate. Therefore we give all intensities as
luminance at the bottom of the recording chamber (in
cd/m2).
Analysis of the responses of RBCs
Bipolar cells are thought to be electrotonically compact
(Mennerick et al. 1997; Protti and Llano
1998
), thus the potential recorded at the cell's soma must be
very similar to the potential at the axon terminals. The output of
bipolar cells
the release of glutamate from their terminals
is
modulated by the membrane potential of the cell (Tachibana et
al. 1993
; von Gersdorff and Matthews 1994
),
therefore the voltage response is presumably an adequate representation
of the cell's output signal. For this reason, we analyzed and compared
properties of the voltage responses.
Several parameters were used to characterize the light responses of
RBCs (illustrated in Fig. 3A). First, the maximum amplitudes of the two phases of the typical voltage responsethe depolarizing phase at light onset and the succeeding hyperpolarization at light offset
were measured (Vdep,
Vhyp; in mV). For the sensitivity curves, the response amplitudes of both phases were plotted over the
logarithm of the stimulus luminance (L; Figs. 3C
and 7) or the normalized luminance
(L/L50; Fig. 4). When comparing the
curves of several cells in a diagram, the response amplitudes were
normalized. The values for Vdep were fitted
using the Hill function: V/Vmax = Lh/(Lh + L50h) (see also
intensity-response function: Normann and Werblin 1974
), with the maximal amplitude (Vmax), the
luminance (L; in cd/m2), the slope of
the curve (h; the Hill coefficient), and the luminance evoking a half-maximal response (L50).
From this fit, the luminance evoking a 90%-response
(L90) also was determined. The width
of the dynamic range (w; in log units) was estimated using
following equation: w = 2·(log
L90
log
L50).
Four timing parameters were measured (in ms) from the start of the stimulus (t = 0): tVdep is the latency of the maximal amplitude of the depolarization Vdep; tMaxSlope is the time when the rising depolarizing response reached its maximal slope. The latter was determined by differentiating the rising phase and fitting a Gaussian function to find its peak. At t0, the voltage response crossed the baseline, marking the start of the hyperpolarizing response phase, and at tVhyp the hyperpolarizing phase reached its maximal amplitude, Vhyp.
The Student's t-test (significance level 0.05) was
used to determine whether or not a parameter was significantly
different between two groups of cells.
Solutions
The following intracellular solution was used (in mM):
10 NaCl, 1 CaCl2, 1 MgCl2,
1.1 EGTA, 4 KCl, 126 K+-gluconate, 10 HEPES, 1 Mg2+ATP(H2O)2,
and 1 Na+3GTP(H2O)2
(pH 7.2). All chemicals were purchased from Sigma Aldrich. The solution
also contained 0.025% Lucifer yellow (potassium salt) to visualize the
recorded cells after the experiment. The chloride reversal potential
(in Ames) was 48.8 mV; the (mean) reversal potential for nonspecific
cation currents was +0.2 mV (assuming similar permeability for
Na+ and K+). This
difference in reversal potential enabled us to distinguish excitatory
cation currents from inhibitory chloride currents. For the combination
intracellular solution-Ames, a liquid junction potential of 18 mV was
measured, close to the estimated value of 15.7 mV, and was used to
correct all measured potentials. Antagonists were dissolved in Ames and
applied directly with the bath. The solution in the recording chamber
was completely exchanged within 3 min.
The GABAA antagonist bicuculline (50 to 100 µM) and the GABAC antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA; 25-50 µM) were purchased from Sigma RBI (Natick, MA).
Identification of the bipolar cells
Because Lucifer yellow was included in the intracellular
solution, we were able to classify the recorded cells according to their morphology (Euler and Wässle 1995;
Hartveit 1997
). RBCs can be identified easily from the
stereotypical branching pattern of their axon terminals, by the size
and the location of their somata, and especially by the characteristic
appearance of their dendrites; their narrow and more tightly clustered
dendritic tree enabled us easily to distinguish axotomized RBCs from
other axotomized bipolar cells (Fig. 1).
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In most mammals, only the RBC, one of the 8-10 types of bipolar cells,
is known to exclusively contact rods (Cajal 1893). It is
assumed that the remaining types of bipolar cells contact only cones,
and they are hence referred to as cone bipolar cells. However, in many
mammals, including rats, detailed knowledge of the wiring between
photoreceptors and bipolar cell types is still lacking and recent
evidence suggests that some "cone bipolar cells" in the mouse
retina may connect also to rods (Brandstätter et al.
1998a
; Soucy et al. 1998
). Nonetheless, for
simplicity's sake, we refer to these "nonrod bipolar cells" as
cone bipolar cells (CBCs).
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RESULTS |
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Thirty-two RBCs and 35 CBCs were studied. Twelve of the RBCs lost their axon terminals during the preparation. These axotomized RBCs seemed to be as healthy as their intact counterparts: they could be studied for similar periods of time and showed no abnormal rundown of their light responses. Twenty-nine RBCs, including all axotomized cells, were ON cells. The remaining three RBCs hyperpolarized at light onset. Twelve of the CBCs were OFF cells; 23 CBCs were ON cells.
Resting potentials were measured in darkness, immediately after the
recording had stabilized. The resting potential of intact RBCs was
45.4 ± 10.6 mV (n = 15), that of axotomized RBCs was
39.0 ± 7.4 mV (n = 11). The resting potentials were
38.5 ± 13.6 mV (n = 12) and
35.9 ± 9.8 mV (n = 9) for ON and OFF CBCs,
respectively. Axotomized RBCs being slightly more depolarized than
normal (although this difference was not statistically significant),
could indicate that they leaked ions and thus were damaged. For two
reasons, we think this was not the case. The cell's axons were cut off during the dissection, generally
1 h before the recordings. By then,
damaged cells most likely have died off. The axotomized RBCs displayed
stable responses to light during the whole experiment, indicating that
the intracellular milieu in these cells was widely undisturbed. The
likely explanation for the drop in resting potential is instead the
lack of inhibitory axonal input from amacrine cells or the loss of
axonal conductances, as studied further in the following text.
Rod bipolar cellsLight-induced voltage responses
A typical voltage response of an intact RBC is shown in Fig. 2. At light onset, the cell displayed a strong depolarization (ON response), followed by a smaller hyperpolarizing phase after light offset.
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With increasing light intensity, the amplitude of the depolarization
(Vdep) increased (Fig. 2A,
bottom 4 traces). At higher intensities,
Vdep saturated, but the width of the
depolarizing phase continued to increase (Fig. 2A, upper 6 traces). Note that the hyperpolarization also increased with
increasing stimulus intensity. The maximal
Vdep ranged from 3.9 to 26 mV (15.1 ± 7.1 mV, n = 24) and no differences were observed
between intact and axotomized cells. The maximal amplitude of the
hyperpolarizing phase (Vhyp) ranged from
1.0 to
11.5 mV (
4.5 ± 3.1 mV, n = 9) in
intact RBCs and from
0.6 to
5.0 mV (
2.2 ± 1.2 mV,
n = 11) in axotomized cells. This difference, though
small, was statistically significant suggesting that at least part of
the hyperpolarization is caused by functional properties of the axon.
Stimuli of varying duration showed the responses of RBCs to have a large sustained component (Fig. 2C). To quantify this, the percentage of the amplitude remaining at the offset of saturating stimuli was determined: for 0.5 s stimuli this percentage ranged from 47 to 93% (75 ± 15, n = 8); for 2 s stimuli, it ranged from 62 to 89% (75 ± 12, n = 5). Note also that the longer the duration of the stimulus, the longer the time required for the cell to repolarize (Fig. 2C).
In the nonsaturating intensity range, the latency of the depolarizing response decreased with increasing luminance as Fig. 2B clearly shows (traces from A, marked by arrows). For saturating stimuli of increasing duration, the latency of the depolarizing phase remained constant (Fig. 2D; arrows in C).
To characterize the light-induced voltage responses of RBCs in more detail, we separately analyzed three aspects: the response latency, the relationship between the response phases, and the sensitivity of the cells. As an example, Fig. 3 shows the results from an intact RBC.
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Rod bipolar cellsLatency
Latencies were measured from the onset of the light stimulus to the time points marked by the symbols next to the voltage trace in Fig. 3A. The changes in the response latencies over the intensity range examined are revealed in Fig. 3B. First, we first determined the latency of the maximum depolarization (tVdep; upward triangles in Fig. 3B). For both intact and axotomized cells, tVdep was relatively constant; in a few cells, a slight decrease in tVdep was observed for increasing intensities. However, this trend was small. Therefore we calculated the mean tVdep which was 176 ± 23 ms (n = 10) in intact cells and 216 ± 29 ms (n = 10) in axotomized cells. This difference was statistically significant and suggests that intact cells have shorter latencies.
To overcome the difficulties of pinpointing the exact latency of the maximum response, we also determined the latency of the maximal slope (tMaxSlope; circles in Fig. 3B). In both intact and axotomized cells, tMaxSlope decreased significantly with increasing intensity until it reached a minimum for the highest intensities. However, intact cells had consistently shorter latencies. For intact cells, tMaxSlope was 132 ± 25 ms (n = 10) at low light levels and 73 ± 9 ms (n = 10) at saturating illumination, while the corresponding values for axotomized RBCs were 160 ± 26 ms (n = 11) and 106 ± 21 ms (n = 11). In both cases, the difference in tMaxSlope was statistically significant. Thus the depolarizing phase of RBC responses speeded up with increasing stimulus intensity. Furthermore, intact RBCs responded faster, on average, than axotomized cells, suggesting that the axon terminal system is involved in shaping the time course of the depolarizing phase.
Increasing the intensity of the stimulus also affected the latency of the hyperpolarizing portion of the RBC response. Both the latency of the hyperpolarization (tVhyp, downward triangles in Fig. 3B) and the time when the trace crossed the baseline (t0, squares in Fig. 3B) increased with increasing stimulus intensity. The fit appears linear in the double-logarithmic plot, indicating that it can be fitted with a power function of the form f(x) = alog(x/b). This change in latency reflects the lengthening of the depolarizing phase.
Rod bipolar cellsRelationship between the response
phases
We next wanted to explore the relationship, if any, between the size of the depolarizing and hyperpolarizing phases of the light response. Finding a proportional relationship between the two response phases could indicate that the depolarization directly causes the hyperpolarization, for example by a very simple form of reciprocal feedback.
The ratio of the amplitudes of the depolarizing and hyperpolarizing phase was studied to see whether it remained constant over the examined range of intensities. For each cell, we took the ratio of Vhyp/Vdep at different intensities and calculated the mean. The ratio of Vhyp/Vdep was quite different between axotomized and intact RBCs. We found a mean ratio of 0.29 ± 0.13 (n = 8) for intact cells, and a significantly smaller average ratio of 0.17 ± 0.06 (n = 9) for axotomized cells. The standard deviation (SD) of Vhyp/Vdep was used as an estimate of the constancy of the ratio. The averaged SD was 0.11 (n = 8) for intact and 0.08 (n = 9) for axotomized cells. The variability of this ratio suggests that the relationship, if any, between the two response phases in both intact and axotomized RBCs is not straightforward. This means that the hyperpolarizing phase probably results from a more complex interaction than reciprocal feedback alone.
Rod bipolar cellsSensitivity
Figure 3C shows the sensitivity curve for an RBC. The
peak amplitudes, Vdep (upward triangles) and
Vhyp (downward triangles), of the response are
plotted over the stimulus intensity. The data points for
Vdep can be well fitted with the Hill
function (see METHODS), describing the light intensity
range within which the cell responds with graded potentialsthe
dynamic range of the cell. The data points for
Vhyp were simply fitted by linear regression.
We noted that the intact RBCs behaved differently than axotomized RBCs in terms of dynamic range and curve profile. To explore this quantitatively, we compared the normalized sensitivity curves of intact and axotomized RBCs. The curves for four cells of each group are shown: Fig. 4A contains the data for the intact RBCs; Fig. 4B shows the same plot but for the axotomized RBCs. The response amplitude of each cell was plotted as a function of the stimulus luminance on normalized axis. The amplitude was normalized to the maximal amplitude (V/Vmax) and the luminance was normalized to the L50 value, the luminance needed to evoke a half-maximal response. Solid lines represent the fits for the data points. For comparison, the fit from the other group of cells is added to the plots (dashed lines).
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The comparison of the fits reveals a surprising difference: The curve for the intact RBCs is shallower and extends over a wider range of intensities than the curve for their axotomized counterparts. This is expressed in the slope of the curve (h; the Hill coefficient), which was 1.07 ± 0.19 (n = 7) for intact RBCs and 2.39 ± 0.84 (n = 9) for axotomized cells. The dynamic range of intact cells (1.84 ± 0.32 log units; n = 7) was more than twice as wide as that of axotomized RBCs (0.87 ± 0.23 log units; n = 9). The L50 value ranged from 0.15 to 0.58 cd/m2 (average = 0.34; n = 7) for intact RBCs and from 0.26 to 9.75 cd/m2 (average = 2.01; n = 9) for axotomized RBCs. The relatively high variability of the L50 value in both cell groups may reflect variations in number and sensitivity of rods connected to the recorded cell, but probably also depends on the synaptic integrity of the slice and the different attenuation of the stimulus by the tissue (see METHODS). The results suggest that the axon terminal system is involved in widening the dynamic range and lowering the threshold of RBC voltage responses.
For both groups of RBCs, Vhyp increased with increasing light intensity. This increase was smaller in axotomized cells, further supporting the hypothesis that the hyperpolarization partially reflects an axonal contribution.
To illustrate the significance of the difference between the two cell groups, the separate fits for the 8 cells are plotted in Fig. 4C (axotomized: thick lines; intact: thin lines). The error bars designate the width of a 95% confidence interval around the fits shown in panels A and B.
Rod bipolar cellsLight-induced current responses
To characterize the currents involved in the light responses of
RBCs, we recorded in voltage clamp at different holding potentials (VH). We used a low intracellular chloride
concentration, setting the Cl reversal
potential (ECl
) to about
50 mV,
to distinguish inhibitory chloride currents and excitatory cation currents. By clamping the cells to VH =
3
mV (ECation) and to VH =
48 mV
(ECl
), respectively, we could
isolate these two components of the light response.
A current family recorded from an intact RBC is shown in Fig.
5A. For negative holding
potentials, the stimulus evoked a strong initial inward current,
followed by a weak outward current rebound. For positive holding
potentials, the polarity of the currents was reversed. At both the
reversal potentials of cations (ECation; marked a) and of chloride
(ECl; marked b), currents were observed. The outward current at
ECation was probably a chloride current,
while the inward current at
ECl
was very likely a
nonspecific cation current. The two different currents are combined in
the responses: when the cell was held at an intermediate VH (e.g.,
18 mV), one can see the
two components, the outward and the inward current, contributing to the
response.
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Figure 5C1 shows the initiation of the responses at higher
magnification. The inward currents developed smoothly. The outward currents often began with large, apparently discrete events and had a
noisy characteristic. Nonetheless, both inward and outward currents
started with approximately the same latency. Similar events were also
seen in darkness (Fig. 5C2). Since these events reversed
around 50 mV (ECl
) both in
the light response and in the dark they probably reflect postsynaptic
inhibitory input, carried by chloride, and thus are IPSCs.
A similar current family for an axotomized RBC is shown in Fig.
5B. In contrast to the intact RBC, no outward current was measured when the cell was held at VH = 3 mV (ECation; marked a). However, at
ECl
(marked b) the response was very similar to that of the intact RBC. This effect of axotomy is
also clearly seen when comparing the response of the two cells at
VH =
18 mV (Fig. 5, A and
B). The response of the axotomized RBC is a "simple"
inward current whereas the intact RBC shows a combination of inward and
outward currents. These results suggests that the chloride component is
absent or largely reduced in axotomized RBCs. Note also that the
rebound at light offset is smaller but still present in the axotomized cell.
We attempted to separate the different current components by
determining the net charge flowing through the membrane (area under the
current trace) within the time limits marked by the shaded boxes
underlying the traces (Fig. 5, A and B). This
charge was normalized and plotted over the holding potential
(charge-voltage relation; Fig. 5, D-F). For intact RBCs,
the reversal potentials of the first (black squares) and the second
(black circles) phases were between
ECl and
ECation, suggesting a mixture of chloride
and cation currents and indicating that the separation of the
components could not be achieved. However, since the first phase
reversed at a more negative potential than the second phase (
30 mV
compared with
20 mV), chloride seemed to be more prominent in the
initial phase of the response.
In axotomized RBCs, the first phase of the response was nearly absent
(open squares). The second phase was pronounced and reversed around 0 mV. This "shift" in reversal potential further supports the idea
that the chloride current is largely reduced in axotomized cells. The
reversal potential of the second phase (circles) is an indicator of the
composition of the currents: it was 15.7 ± 10.4 (n = 6) in intact RBCs and 2.3 ± 4.7 (n = 7) in axotomized cells (statistically significant).
The current rebound at light offset partially remained in axotomized
cells and it probably caused the smaller hyperpolarization observed in
those cells. Its charge-voltage relationship (triangles in Fig.
5F) is nonlinear, making it difficult to pinpoint the reversal potential between 20 and 0 mV. However, the negative slope
suggests a reduction in current flow. This effect could be due to the
kinetic properties of the APB receptors, the subsequent second
messenger cascade or intrinsic membrane properties.
Rod bipolar cellsGABA receptors shape the light-induced responses
The fact that GABAergic amacrine cells provide significant
synaptic input to RBC terminals (Fletcher et al. 1998;
Kim et al. 1998
; Sterling and Lampson
1986
; Strettoi et al. 1990
) suggests that the
observed chloride current reflected inhibitory input mediated by
ionotropic GABA receptors (GABARs). We tested this hypothesis by
co-applying the GABAAR antagonist bicuculline
(100 µM; Feigenspan et al. 1993
) and the GABACR
antagonist TPMPA (50 µM; Ragozzino et al. 1996
) to intact and
axotomized RBCs.
Figure 6 shows the charge-voltage
relations for two RBCs. The total area under the
responsescorresponding to the first two shaded boxes in Fig. 5,
A and B
was plotted. Under control conditions, the curve for the intact RBC (Fig. 6A, open circles)
reversed between ECl
and
ECation, reflecting a mixture of cation and
chloride currents (
33.8 ± 17.0 mV; n = 3). The co-application of the GABAR antagonists decreased the charge and shifted the reversal potential close to
ECation (shaded circles), indicating that
the chloride component was strongly reduced (+3.7 ± 10.4 mV;
n = 3). The shift was reversible (not shown). In
axotomized RBCs (Fig. 6B), neither a shift in the reversal
potential nor a decrease in charge was observed (3 of 3 cells). These
results support the conclusion that the light-induced chloride current reflects inhibitory input from GABAergic amacrine cells to the axon
terminals of RBCs.
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To evaluate the role of the GABAergic inhibition in shaping the cell's responses, we examined the effects of the antagonists on the sensitivity curves. Figure 7 shows the curves of two RBCs, first under control conditions (open triangles, dotted lines), then during the co-application of the antagonists (shaded triangles, solid lines). Over the extended time course of the pharmacological experiments the response amplitudes slightly decreased in some cells (Fig. 7A), however, this had no effect on the slope of the sensitivity curve. In intact cells, the slope of the sensitivity curve increased to 2.74 ± 0.78 (n = 3) which is 221% of the value before the drug application (Fig. 7A), corresponding to a decrease in curve width to 47%. Thus the antagonists partially mimicked the effects of axotomy on the sensitivity curve. Two (of 3 tested) axotomized RBCs experienced a small decrease in slope to 63% and 68% of the control value (curve width: 159% and 147%). One axotomized RBC experienced an increase in slope to 171% (curve width: 58%). This suggests that the predominant (but not sole) role of GABAergic inhibition in RBCs seems to be at the axon, where it widens the dynamic range of the cell.
|
The antagonists did not significantly reduce the hyperpolarizing phase
of intact RBCs (Fig. 7A, downward triangles). In axotomized cells, small and inconsistent effectsboth increases and decreases
on the hyperpolarizing phase were observed. In both intact and axotomized RBCs, the response latencies slightly decreased during the drug application, but this tendency was not significant.
Cone bipolar cells
While RBC responses were consistent across cells, CBCs displayed a
wide variety of responses (Fig. 8). We
recorded more ON than OFF CBCs, probably
because we targeted cells in the outer third of the inner nuclear
layer, where the somata of RBCs and CBCs stratifying in sublamina b are
located (Euler and Wässle 1995). All
ON responding bipolar cells
CBCs and RBCs
had their axon
terminals in the inner part of the inner plexiform layer, while all
OFF responding CBCs stratified in its outer portion; bipolar cells obey the ON/OFF subdivision of
the inner plexiform layer (Euler et al. 1996
;
Famiglietti and Kolb 1976
; Hartveit 1997
;
Kaneko 1979
; Nelson et al. 1978
;
Werblin and Dowling 1969
). Although we found no
exception to this rule, we cannot exclude that exceptions exist given
that we recorded from only 35 CBCs.
|
Figure 8, A and B, shows light-induced voltage responses of two representative ON CBCs. Some cells displayed sustained responses: they depolarized for the duration of the stimulus (Fig. 8A). Other ON CBCs responded with a brief depolarization at the onset of the stimulus, independent of the stimulus duration (transient response; Fig. 8B). Interestingly, axotomized CBCs tended to be more sustained than intact cells (data not shown), although this was not examined quantitatively.
Figure 8, C and D, shows two examples of light-induced responses for OFF CBCs. They also differed in the time course of their responses, showing more sustained (Fig. 8C) or more transient responses (Fig. 8D), although these characteristics were not as pronounced as in ON CBCs. We generally used light-on stimuli to reduce the effect of photoreceptor bleaching; light-off stimuli might reveal transient and sustained OFF CBCs more clearly.
Most of the CBCs displayed biphasic voltage responses. For the
brightest LED stimulus, ON CBCs displayed an initial
depolarization at light onset ranging from 1.8 to 10.8 mV, and a
hyperpolarization at light offset between 0.9 and
8.4 mV. For
OFF CBCs, the initial hyperpolarization ranged from
16.0
to
1.6 mV, while the depolarization at light offset was 0.6 to 2.1 mV. However, since the intensity of the light stimulus was limited to
the low photopic range (see METHODS), the brightest of our
stimuli did not evoke the maximal responses. For several cells, we
confirmed this by briefly switching on the fluorescent illumination of
the microscope. This produced larger light responses than any of our
LED stimuli.
Responses to light of CBCs were smaller and noisier that those of RBCs (compare traces in Fig. 2 with Fig. 8). In many CBC recordings, low frequency noise, probably a summation of discrete events, was observed. Larger single excitatory (EPSPs) and inhibitory events (IPSPs) can be clearly resolved, but were not further analyzed. The fact that the CBC responses were small can probably be explained by the relatively low density of cones in the rat retina, as well as by technical limitations: The probability of having enough intact cones connected to the recorded bipolar cell is low in a slice, especially for bipolar cells close to the surface of the slice.
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DISCUSSION |
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By comparing the light-evoked responses of intact RBCs with those
of axotomized or pharmacologically treated ones, we found evidence that
the bipolar axon terminalespecially the GABAergic inhibition it
receives
plays an important role in determining the cell's dynamic
response properties. The responses of various types of cone bipolar
cells, whose most distinguishing characteristics are the shape and the
stratification level of their axon terminals, differed not only in
polarity but also in time course. Taken together, these findings
support the view that these cell types represent parallel signaling
pathways to the inner retina and that the axon terminals may be an
important source of their functional specificity.
Responses of rod bipolar cells to light
The great majority of RBCs responded with a robust, sustained
depolarization at light onset (ON response), followed by a
smaller hyperpolarization at light-off. Only a few previous studies
have described light responses of RBCs in other mammals. The
depolarizing responses from rabbit RBCs in whole-mounts (Dacheux
and Raviola 1986) resembled those we recorded in slices from
the rat. Despite some sagging back after an initial peak, the cells
stayed depolarized during the whole stimulus presentation. A
hyperpolarizing phase was not noted for rabbit RBCs, but a small dip at
light offset is visible in some of the published traces. As for rabbit
RBCs, we observed a widening of the depolarizing phase and longer
repolarization periods for postsaturating stimulus intensities. This
could reflect nonlinearities in the rod responses (Dacheux and
Raviola 1982
; Kraft and Schnapf 1998
;
Schneeweis and Schnapf 1995
).
Using voltage clamp recordings and separate reversal potentials for
cation and chloride currents, we identified two components in the
response of RBCs: a nonspecific cation current and a chloride current.
Both currents were induced by light. Similar components have been
reported in depolarizing bipolar cells in the salamander retina
(Lasansky 1992). The cation current caused the
depolarization at light-on and is most likely attributed to the
APB-receptor/cGMP-gated channel mechanism (Nawy and Jahr
1991
; Shiells and Falk 1990
). Chloride currents
in bipolar cells have been shown to originate from ligand-gated
inhibitory receptors (postsynaptic currents, IPSCs) or
Ca2+-activated chloride channels (Hartveit
1999
; Okada et al. 1995
). Since the recorded RBC
was voltage-clamped during the light stimulus, Ca2+-activated chloride currents may have been
continuously activated but were not modulated by the cell's light
response. Hence, the measured light-evoked chloride current instead
reflected receptor-mediated inhibitory input, probably from amacrine
cells at the RBC axon terminals. Two lines of evidence support this
assumption. Under control conditions, the integrated current response
of intact RBCs reversed at a value between the reversal potentials for
chloride and cations, indicating a mixture of the two currents. During the co-application of GABAA/C receptor
antagonists, the overall current response decreased and its reversal
potential shifted toward that for cation currents, consistent with the
suppression of a chloride current. Thus the chloride current is
mediated by ionotropic GABA receptors, presumably located on the
bipolar cell itself. Furthermore, axotomized RBCs lacked most if not
all of this chloride current, which is consistent with the fact that RBCs receive their major inhibitory input at their axon terminals (Hartveit 1999
; Karschin and Wässle
1990
; Suzuki et al. 1990
).
Our findings fit well into a picture in which GABAergic amacrine cells
provide the bulk of inhibitory synaptic input to RBCs (Fletcher
et al. 1998; Kim et al. 1998
; Sterling
and Lampson 1986
; Strettoi et al. 1990
).
Anatomical studies have established that in the inner plexiform layer,
RBCs make dyad synapses onto both glycinergic AII and GABAergic A17
amacrine cells (Famiglietti and Kolb 1975
; Kolb
and Nelson 1983
; Nelson and Kolb 1985
;
Raviola and Dacheux 1987
; Sandell et al.
1989
; Sterling et al. 1988
). In the rat, about
40% of the input synapses onto RBC axon terminals are reciprocal
feedback synapses (Chun et al. 1993
), presumably from
A17-like amacrine cells (Perry and Walker 1980
). This
reciprocal feedback has been shown to be reflected by chloride-carried
IPSCs
similar to those we observed (Fig. 5C)
and can be
elicited in RBCs in intact slices using voltage step protocols
(Hartveit 1999
; Protti and Llano 1998
).
The chloride current we observed was activated quickly almost
simultaneously with the cation current
suggesting the involvement of a
simple circuit, such as reciprocal feedback. Since, however, the
light-evoked current should not have induced any transmitter release
from the voltage-clamped cell, it is more likely that the chloride
current resulted from amacrine cell input induced by neighboring RBCs.
In current-clamp recordings, direct reciprocal feedback from the A17
amacrine may play a role, because the recorded cell is then able to
modulate its release glutamate release.
Alternative pathways are conceivable and cannot be excluded. For
example, rod signals may pass through gap junctions to cones and reach
the inner plexiform layer via CBCs (DeVries and Baylor 1995; Nelson 1977
; Smith et al.
1986
). In this case, an ON CBC would receive rod
signals, relay them to amacrine cells presynaptic to RBC terminals and
cause a light-induced chloride current similar to the one we observed.
Also, a recent set of experiments indicated the existence of
rod/cone-contacting OFF bipolar cells in mice (Soucy
et al. 1998
).
Since receptor antagonists did not significantly reduce the
hyperpolarizing phase and, hence, GABAergic inhibitory feedback seems
not to be involved, the mechanism of this after-hyperpolarization remains unknown. The current reboundobserved in both intact and axotomized cells
could cause the hyperpolarizing phase and may reflect
intrinsic conductance changes, for example to quickly terminate the
light-evoked depolarization.
Hyperpolarizing rod bipolar cells
We found three RBCs that responded by hyperpolarizing at light
onset (not shown). For several reasons we believe that these RBCs
obtained reduced input from photoreceptors while still receiving inhibitory input at their axon terminals. First, current families indicated that the hyperpolarizing response of these RBCs was composed
predominately of chloride currents. Thus these cells are not
OFF cells in the sense that ionotropic glutamate receptors are probably not involved. Second, AII amacrine cells are
ON cells (Bloomfield et al. 1997;
Kolb and Nelson 1983
; Nelson et al. 1976
) and depolarize when glutamate is applied (Boos et al.
1993
). Since they receive most of their bipolar cell input from
RBCs (Famiglietti and Kolb 1975
), RBCs should be
ON cells as well. Third, the dendritic input of RBCs can be
compromised
by damage to the fragile dendritic tips or the rods distal
to the recorded cell
even when the dendritic tree appears visually to
be intact (Euler et al. 1996
; Hartveit 1996
). Hence, while axotomized RBCs clearly had lost the
majority of chloride currents, the hyperpolarizing cells behaved as
though they had their dendrites cutoff and thus lost their cation
currents (the chloride current would originate from amacrine cells
driven by neighboring bipolar cells).
Responses of cone bipolar cells to light
The responses of CBCs varied in polarity and time course. Four major types of responses were observed: transient ON, sustained ON, transient OFF, and sustained OFF. Given the difficulties of patching specific CBCs, we have not recorded enough examples of each morphological type to firmly establish the physiological behavior of each type. Nonetheless, it is clear that different types of bipolar cells in mammals, as in cold-blooded vertebrates, carry different signals.
In the salamander retina, inhibitory feedback at the axon terminals of
bipolar cells has been shown to make amacrine and ganglion cells
respond more transiently, presumably by shaping the bipolar cell's
output (Dong and Werblin 1998). Furthermore, it has been suggested that the temporal properties of bipolar cells results, at
least partially, from the differential expression of inhibitory receptors (Lukasiewicz 1996
; Lukasiewicz and
Shields 1998
; Pan and Lipton 1995
). Supporting
this hypothesis, recent studies (Euler and Wässle
1998
; Lukasiewicz and Wong 1997
; Shields
et al. 1998
) have found differences in the contribution of
GABAA and GABAC receptors
to the GABA responses of different types of bipolar cells. Because of
their different kinetic properties, GABA receptors can tune the output
of bipolar cells: GABAA relays fast, transient inhibition, while GABAC provides delayed,
sustained inhibition (Pan and Lipton 1995
). Furthermore,
given the variety of GABA receptor subunits that have been localized to
mammalian bipolar cells (Enz et al. 1996
;
Greferath et al. 1995
; Grünert and Hughes 1993
), each cell type could well express a unique combination of GABA receptors with specific subunit compositions, as recently demonstrated for RBCs (Fletcher et al. 1998
;
Koulen et al. 1998
). Thus the axon terminals, and their
associated amacrine cells, are probably important in generating the
diversity of dynamic temporal properties of CBCs.
The role of the axon terminal system
The comparison of intact and axotomized RBCs enabled us to evaluate the role of the axon terminals in shaping the light response of RBCs. We quantitatively examined the response parameters listed in Table 1. Three major differences were found: intact RBCs 1) had generally a significantly shorter response latency (tVdep, tMaxSlope), 2) displayed a larger hyperpolarizing phase (Vhyp), and 3) had wider sensitivity curves. In other words, functional properties of the axon terminals seem to speed up the responses of RBCs and extend their dynamic range. We believe that this is achieved through two mechanisms: inhibitory feedback from amacrine cells and axonally concentrated voltage-gated channels.
|
It has been proposed, that feedback from amacrine cells could extend
the dynamic range in which a bipolar cell responds with graduated
signals (Tachibana and Kaneko 1988). Here, we
demonstrated that by suppressing the
GABAA/C-mediated inhibitory input of intact RBCs,
their shallow sensitivity curve could be transformed into a much
steeper one. The transformed curve covered a smaller dynamic range and
matched closely the sensitivity curves of axotomized RBCs. Thus
inhibitory feedback from GABAergic amacrine cells at the bipolar axon
terminal is an important factor in modifying the bipolar cell's
dynamic range
Inhibitory feedback does not explain the longer latencies and smaller
after-hyperpolarization observed in axotomized RBCs. During drug
application, intact cells did not become significantly slower or
displayed a reduced after-hyperpolarization. Both effects may be linked
with the loss of voltage- and/or ion-gated conductances predominantly
located in the axon. Voltage-gated channels play an important role in
shaping the responses of bipolar cells (Mao et al. 1998;
Mennerick et al. 1997
). In addition to controlling transmitter release, calcium channels are thought to amplify the cell's responses and decrease their latency (Zenisek and
Matthews 1998
). Potassium channels may accelerate the response
by attenuating the gain (Mao et al. 1998
). In rat
bipolar cells, several types of both calcium and potassium channels
have been described (Karschin and Wässle 1990
).
For contributing to the differences between intact and axotomized RBCs
L-type Ca2+ channels are good candidates. These
channels are located in the axon terminals (Hartveit
1999
; Pan and Lipton 1995
; Protti and Llano 1998
) and rapidly activate at membrane potentials between
50 and
40 mV, close to the resting potential of RBCs. Hence, Ca2+ currents could be elicited by small
depolarizations, amplifying the responses and decreasing the latency in
dim light. This would result in "pushing" up the left side of the
sensitivity curve. And indeed, we have found that axotomized RBCs,
which lack L-type Ca2+ channels (Hartveit
1999
; Kaneko et al. 1989
; Protti and
Llano 1998
), have longer latencies and need higher stimulus
intensities to respond.
The sensitivity curve for the intact RBCs resembles the non
co-operative Michaelis-Menten function (slope h =1), and is
similar to the sensitivity curve found in rods and cones
(Normann and Werblin 1974; Schneeweis and Schnapf
1995
). The sensitivity function of axotomized RBCs, however, is
best fitted using the Hill equation with h >1 (positive
co-operativity). In general, increasing the slope of the curve improves
the cell's ability to threshold its input and respond in a more binary
fashion (Rao et al. 1994
; Smith et al.
1986
; Sterling 1998
). One could speculate that
interactions at the axon may modulate the thresholding transfer
function of the input stage (equivalent to the axotomized RBC) to
switch the cell between a `binary' detection mode at scotopic
conditions (steep curve) and an `analogue' mode at mesopic conditions
(shallow curve). The shallow sensitivity curves we found in intact RBCs support this speculation, since we recorded in the high scotopic to
mesopic range.
Support for an important role of the axon in modulating the bipolar
cell's response comes also from recent evidence that shows bipolar
cells to be quite homogenous in their direct response to photoreceptor
input. All ON bipolar cells use the same type of
metabotropic glutamate receptor, mGluR6, to relay photoreceptors signals (Brandstätter et al. 1998b; Nomura
et al. 1994
; Vardi and Morigiwa 1997
),
suggesting that all ON bipolar cells have similar dendritic
responses. Recently, DeVries and Schwartz (1999)
recorded the response
of OFF cone bipolar cells to direct depolarization of
single cones. Under their conditions, with feedback onto the bipolar
cells eliminated pharmacologically, all cells responded similarly
even though the OFF bipolar cells studied clearly
represented several morphological types. Thus the glutamate receptors
directly involved in relaying the photoreceptor signals seem not to
contribute much to the diversity of bipolar cell responses.
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ACKNOWLEDGMENTS |
---|
We thank S. P. Brown for helpful comments and discussion.
This work was supported by a postdoctoral grant from the Deutsche Forschungsgemeinschaft (DFG), Germany.
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FOOTNOTES |
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Address for reprint requests: R. H. Masland, Wellman 429, Massachusetts General Hospital, Fruit Street, Boston, MA 02114.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 May 1999; accepted in final form 1 November 1999.
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REFERENCES |
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