Departments of 1Psychology and 2Physiology, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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Burkhardt, Dwight A. and
Patrick K. Fahey.
Contrast Rectification and Distributed Encoding By
ON-OFF Amacrine Cells in the Retina.
J. Neurophysiol. 82: 1676-1688, 1999.
The encoding of luminance
contrast by ON-OFF amacrine cells was investigated by
intracellular recording in the retina of the tiger salamander
(Ambystoma tigrinum). Contrast flashes of positive and
negative polarity were applied at the center of the receptive field
while the entire retina was light adapted to a background field of 20 cd/m2. Many amacrine cells showed remarkably high contrast
gain: Up to 20-35% of the maximum response was evoked by a contrast
step of only 1%. In the larger signal domain, C50, the contrast
required to evoke a response 50% of the maximum, was often remarkably
low: 24 of 25 cells had a C50 value of 10% for at least one contrast polarity. Across cells and contrast polarity, the dynamic ranges varied
from extremely narrow to broad, thereby blanketing the range of
reflectances associated with objects in natural environments. Although
some cells resembled "contrast rectifiers," by showing similar
responses to contrasts of opposite polarity, many did not. Thus for
contrast gain and C50, individual cells could show a strong preference
for either negative or positive contrast. In the time domain, the
preference was strong and unidirectional: for equal contrast steps, the
latency of the response to negative contrast was 20-45 ms shorter than
that for positive contrast. The present results, when compared with
those for bipolar cells, suggest that, on average, amacrine cells add
some amplification, particularly for negative contrast, to the high
contrast gain already established by bipolar cells. In the time domain,
our data reveal a striking transformation from bipolar to amacrine cells in favor of negative contrast. These and further observations have implications for the input and output of amacrine cell circuits. The present finding of substantial differences between cells reveals a
potential substrate for distributed encoding of luminance contrast within the ON-OFF amacrine cell population.
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INTRODUCTION |
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Contrast in luminance provides the primary
dimension for the definition of most objects in natural environments.
Some objects are brighter than their backgrounds (positive contrast),
whereas others are darker (negative contrast), appearing as backlit
objects, shadows, or the myriad of objects that reflect less light than their backgrounds. The retina transforms the resulting images into
complex patterns of neural activity distributed in parallel across
multiple cell types. Many of these cells have the capacity to preserve
the sign of the contrast in the retinal image. These include the
classical types known as the ON cells and the
OFF cells (Dowling 1987; Miller
1994
; Rodieck 1998
; Schiller
1992
; Werblin 1991
). On the other hand, cells of
the ON-OFF type seem indifferent to the contrast sign
because they give similar responses to both decreases and increases of
illumination. ON-OFF cells first were observed in
recordings from the axons of retinal ganglion cells (Hartline
1938
), and in pioneering intracellular recordings three decades
later, Werblin and Dowling (1968)
showed that mechanisms for the generation of ON-OFF responses first were
elaborated earlier in the retina at the level of the ON-OFF
amacrine cells. It now is recognized widely that the amacrine
population of vertebrate retinas is diverse, encompassing a number of
morphological and functional classes (Ammermuller and Kolb
1995
; Bloomfield 1992
; Kolb 1994
;
Kolb et al. 1992
; Miller 1994
;
Morgan 1990
; Rodieck 1998
; Yang et
al. 1991
). Amacrine cells of the ON-OFF class,
responding with transient, graded depolarizing potentials at both the
onset and offset of a light flash, now have been found in a wide range of vertebrates (Ammermuller and Kolb 1995
;
Burkhardt 1975
; Dowling 1987
;
Miller 1994
; Morgan 1990
;
Sakuranaga and Naka 1985b
; Stafford and
Dacey 1995
).
With respect to both retinal design and visual contrast,
ON-OFF amacrine cells are particularly intriguing because
they apparently act as agents for "contrast rectification" of the
retinal image by encoding the contrast magnitude while discounting the
contrast sign. However, little is known in detail about such contrast
rectification or other aspects of the contrast response of
ON-OFF amacrine cells. To date, most studies of
ON-OFF amacrine cells have used light flashes in the dark
or stimuli of unspecified contrast, whereas white-noise studies in the
contrast domain (Naka et al. 1975; Sakai et
al.1995
) do not make direct distinctions between the response
to negative versus positive contrast.
Our goal was to investigate contrast encoding in quantitative detail,
giving special attention to the limits of contrast rectification and
how encoding might be distributed across the ON-OFF
amacrine population. We took the simple approach of applying contrast
steps of variable magnitude and opposite sign so we could compare
responses to negative and positive contrast. The experiments were
carried out in the retina of the tiger salamander (Ambystoma
tigrinum). Because it offers several technical advantages,
including relatively large cells that facilitate intracellular
recording and other analytic procedures (Dong and Werblin
1998; Jacobs and Werblin 1998
; Werblin
1991
; Yang et al. 1991
; Yu and Miller
1996
), it is currently one of the most intensively studied
preparations for the analysis of retinal function. In this report, we
present quantitative data on contrast gain, half-maximal contrasts,
contrast dominance, dynamic range, and contrast/latency relations for
25 ON-OFF amacrine cells along with some related
intracellular recordings from other inner retinal neurons and
ON-OFF ganglion cells.
Our results show that the ON-OFF amacrine cells are
remarkably sensitive to negative and positive contrast steps but show substantial variation from cell to cell and a considerable degree of
independence with respect to contrast polarity. The present results,
when compared with recent measurements on bipolar cells obtained under
identical conditions (Burkhardt and Fahey 1998), suggest
that, on average, amacrine cells add some amplification, particularly
for negative contrast, to the high-contrast sensitivity already
established by the bipolar cells. In the time domain, we find a
striking transformation from bipolar to amacrine cells in favor of
negative contrast, such that amacrine cells respond with shorter
latencies to negative than to positive contrasts of comparable
magnitude. These and other results have implications for the nature of
the input and output of amacrine cell circuits. The considerable
diversity of amacrine cell types in vertebrate retinas (Dowling
1987
; Miller 1994
; Morgan 1990
;
Rodieck 1998
; Yang et al. 1991
) would
seem to provide a clear basis for distributed encoding of multiple
dimensions of the visual stimulus across the amacrine population as a
whole. The substantial cell-to-cell variations in the contrast response
reported here suggest that distributed encoding may be extended to
ON-OFF amacrine cells for a single stimulus
dimension
achromatic contrast.
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METHODS |
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Preparation and intracellular recording
Intracellular recordings were made from flat-mounted sections
(~2 × 4 mm) of the eyecup of the tiger salamander (A. tigrinum), as described in detail previously (Burkhardt and
Fahey 1998). The retina was maintained at room temperature
(20-23°C) and superfused at ~1 ml/min with a Ringer solution
composed of (in mM) 111 NaCl, 22 NaHCO3, 2.5 KCl,
1.5 MgCl2, 1.5 CaCl2, and 9 dextrose. The pH was regulated at ~7.5 by bubbling the superfusate
with 98% O2-2% CO2.
Intracellular recordings were made with glass micropipettes (0.5 mm ID,
1 mm OD) pulled on a Brown-Flaming puller. They were filled with 2.0 or
0.25 M Kacetate and had resistances of 200-700 M
. Cells were
penetrated by the common procedure of causing the microelectrode
amplifier to oscillate via brief applications of excessive negative
capacitance. Electrodes filled with 0.25 M Kacetate, perhaps due to
their higher resistance, seemed more likely to penetrate cells and thus
generally were preferred despite their somewhat higher noise level.
Light-evoked responses were recorded permanently on video tape and
later digitized (0-5,000 Hz bandwidth) for analysis with the aid of
commercial software (Superscope, GWI Instruments). For the responses
displayed in this paper, low-pass filtering (3 dB down at 250 Hz) was
used to achieve the optimal reduction of noise without producing
detectable distortion of response amplitude and waveform. Low-pass
filtering also was used for all quantitative measurements of response
amplitude in this report. Response amplitude was measured from the
baseline to the peak of the response. All latency measurements were
made on unfiltered records. Latency was taken as the time at which the
rising phase of the response first deviated from the baseline. To make
the latter measurements, two best-fitting straight lines were drawn by
eye to determine the intersection between baseline and the initial
rising phase of the response.
Light stimulation and contrast metrics
Focused light stimuli, arising from a 100-W tungsten-halogen
source, were applied to the retina. An optical system of conventional design was used for standard screening tests to determine the basic
response properties of cells. To investigate responses to contrast
steps, an active-matrix Liquid Crystal Display (Magnabyte m2x, Telex
Communications, Minnneapolis, MN) was inserted at an object plane in
the optical system. The image on the retina was restricted to a field
of 120 × 120 pixels. Each pixel illuminated a 17 × 17 µm
square area on the retina. Custom software made it possible to
stimulate the retina with spots and annuli of variable contrast and
size. Light calibrations were made at the plane of the retina with a
United Detector Technology Model 350 photodiode photometer. Contrast
steps ranging from ±0.02 to ±2.0 log units were generated with the
LCD system on a steady background illumination of 20 cd/m2, a light level ~4-5 log units above
ganglion cell threshold measured in the dark-adapted retina. Thus a
moderately high level of light adaptation was achieved. Further details
of the LCD stimulator are given elsewhere (Burkhardt et al.
1998).
Contrast may be quantified in several ways: as the scaled difference
between spot and background (Shapley and Enroth-Cugell 1984), as the Michelson contrast (e.g., Burkhardt et al.
1984
; Westheimer et al. 1999
), or as the
contrast ratio. Each specification has merits and limitations. We use
each when appropriate but place primary emphasis on the contrast ratio
because it avoids problems of scale compression, allows direct
comparison with our recent work on bipolar cells (Burkhardt and
Fahey 1998
), and there is much evidence that contrast ratios
are fundamental for suprathreshold vision (Goldstein
1998
). Hence in this report, contrast usually is specified as
the logarithm of the contrast ratio: contrast = log10 (F/B), where
B is the steady background intensity and F is the
light intensity prevailing during the flash. In this metric, contrast
of steps that increase or decrease the prevailing light by the same
factor differ only in sign: e.g., 2× increases or 2× decreases from
the background are specified as contrasts of +0.30 and
0.30,
respectively; 10× increases or decreases, amount to contrasts of +1.0
and
1.0, and so forth. In describing our results, we will usually use
the logarithmic specification of contrast and refer to this as contrast
without further qualification. Michelson contrast is a well-known
metric for sinusoidal stimuli that also can be used for contrast steps
(e.g., Burkhardt and Gottesman 1987
; Burkhardt et
al. 1984
; Westheimer et al. 1999
). In the terms
used here, it is defined: Michelson contrast = (F
B)/(F + B), where F and
B are as defined earlier in the paragraph. For contrast
steps, log contrast and Michelson contrast differ insignificantly over
the range of ±0.70, but Michelson contrast compresses all higher
contrasts to the narrow range of 0.7-1.0. Much past work on contrast
gain of retinal and cortical neurons has been reported in units of
percentage Michelson contrast. Hence, when reporting measurements of
contrast gain, we use percentage Michelson contrast and will refer to
this simply as "% contrast." For low to moderate contrasts
(<0.70), percentage Michelson contrast is numerically equivalent to
log contrast × 100.
Protocol
After a cell was penetrated, the following protocol typically
was used to identify cell types and obtain contrast/response measurements: 1) the center of the receptive field was found
by flashing a 100 × 2,000-µm slit at various positions on the
retina. 2) The LCD stimulator was used to present
low-contrast stimuli in steps of variable diameter (from 100 to 2,000 µm) at the center of the receptive field to determine the optimum
diameter, i.e., the stimulus diameter giving the largest response.
3) A centered spot of the optimum diameter and an annulus
(typically 600 µm ID and 2000 µm OD) were flashed at several
contrast levels to screen for center/surround antagonism (see following
text). 4) The relation between contrast and response was
investigated by presenting contrast flashes of the optimal stimulus
diameter for 500 ms at the center of the receptive field. Flashes were
presented every 10 s at each of 14 contrast levels covering a
range from about 2.0 to +2.0. Because recordings from amacrine and
ganglion cells were relatively noisy (see following text), the series
was repeated, at least once when possible, and average responses were computed. The retina was always light-adapted to a steady background field of 20 cd/m2 covering the entire retina.
Preliminary work showed that the interstimulus interval of 10 s
was sufficiently long to eliminate effects of previous flashes, thus
keeping the retina in a steady state of light adaptation. 5)
When recording time permitted, interference filters (~10 nm
half-band) were used to present flashes of variable intensity at 630 and 530 nm on the background field of 20 cd/m2.
The resulting measurements were used to determine the cell's 630/530
nm sensitivity ratio and thereby classify the cell for spectral type.
Identification of intracellular recordings
The origin of intracellular recordings was determined from
functional criteria based on past work in amphibian retinas
(Burkhardt and Fahey 1998; Frumkes et al.
1981
; Hare and Owen 1990
; Hare et al.
1986
; Vallerga 1981
; Werblin
1977
; Werblin and Dowling 1968
). Cells
penetrated within 0-30 µm of the retinal surface that exhibited
graded depolarizing responses to contrast steps with superimposed
bursts of ~ 5-20 impulses, were identified as ganglion cells.
The present analysis is restricted to ON-OFF ganglion cells
because very few ON cells were detected, and recordings from OFF cells, although more common, were rarely
sufficiently stable to permit quantitative studies. Recordings assigned
to ON-OFF amacrine cells were identified according to the
following functional criteria: 1) recordings were obtained
after the electrode was advanced through an inactive region some 30-40
µm distal to the level of the ganglion cells. 2) The
response waveform consisted of a transient, graded depolarization at
the onset and at offset of light flashes. Only graded responses were
seen in most recordings but a few cells showed a brief, stereotypical
burst of one to three impulses on the rising phase of the graded
depolarization (Miller and Dacheux 1976
; Werblin
and Dowling 1968
). The number of spikes changed little, if at
all, as a function of stimulus contrast, and no quantitative
observations on these spikes were made. Thus all analyses in this paper
are based on the graded depolarization. 3) Flashed annuli
(600 µm ID/2000 µm OD), unlike the case for bipolar cells
(Burkhardt and Fahey 1998
; Hare et al.
1986
), never inverted the response polarity. 4)
Cells typically gave their largest response to stimuli of 250-500
µm. Larger spots attenuated the response but never reversed the
polarity of the response.
On the criteria outlined in the preceding paragraph, ~60% of
recordings obtained in the inner region of the inner plexiform were
classified as depolarizing ON-OFF amacrines. Of the
remaining 40%, some clearly resembled responses of ON-OFF
hyperpolarizing or sustained, depolarizing amacrine cell types, as will
be discussed in more detail in RESULTS. Other recordings in
the inner retina were not readily classified as a simple or known
functional type and will not be considered further. When the electrode
was advanced more distally in the inner nuclear layer, recordings were
observed that clearly satisfied the functional criteria for bipolar
cells (Burkhardt and Fahey 1998; Hare et al.
1986
). Resting membrane potentials of on/off amacrines were in
the 25- to 40-mV range but were not studied in detail. All statistical
probabilities cited in the text are based on Student's
t-test unless noted otherwise.
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RESULTS |
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ON-OFF amacrine cells
RECEPTIVE FIELD ORGANIZATION: A SUPPRESSIVE SURROUND.
The receptive field of the majority (84%) of ON-OFF
amacrine cells showed evidence for surround antagonism. When the
diameter of a centered flash of fixed contrast was varied in steps from 100 to 2,000 µm, the response typically increased to reach a maximum response at some optimal diameter and then decreased for larger fields.
As a rule, the optimal diameter was either 240 or 500 µm, and the
response to a 2,000-µm field was attenuated by ~35 ± 5%
(mean ± SE, n = 25) relative to the response
evoked by the optimum diameter. Because the responses to large fields
as well as to annuli were attenuated but never reversed in polarity,
the surround mechanism, as in turtle ON-OFF amacrines
(Marchiafava and Torre 1978), is suppressive and not
overtly opponent in nature. By contrast, the surround mechanism of
bipolar cells is polarity reversing, i.e., overtly opponent (see
METHODS).
RESPONSES TO CONTRAST STEPS IN THE CENTRAL RECEPTIVE FIELD.
To analyze the response of the central receptive field mechanism,
centered spots of optimal diameter were used in all the following
experiments. The retina was light adapted to a steady background field
of 20 cd/m2 that covered the entire retina.
Preliminary observations indicate that this background is sufficiently
intense to bring ON-OFF amacrine cells close to their
maximum achievable contrast sensitivity. At this level of light
adaptation, it has been shown previously that input from rods is
negligible and the 610 nm cones provide the overwhelming input to
bipolar cells (Burkhardt and Fahey 1998), so it would be
expected that the responses of ON-OFF amacrine cells also
would be cone-driven. This was confirmed by measurements of the 630/530
sensitivity ratio in a subset of cells (see METHODS). They
yielded a mean value of +0.23 ± 0.03 log units (n = 11), in very close agreement with the value of 0.22 expected for
exclusive input from 610-nm cones (Burkhardt and Fahey
1998
).
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CONTRAST/RESPONSE FUNCTIONS: SYMMETRY AND CONTRAST DOMINANCE. Quantitative contrast/response curves were analyzed by plotting peak response to contrast onset versus contrast magnitude. The maximum response varied from cell to cell [mean = 11.6 ± 6.6 (SD) mV]. The factors responsible for the variation are unknown, although differences in electrode seal are one likely possibility. However, a regression analysis failed to reveal any significant correlations between the magnitude of the maximum response and other aspects of the contrast response, so the present report concentrates exclusively on the analysis of normalized contrast/response measurements. The features of the response that will be analyzed are illustrated in the hypothetical contrast/response curve of Fig. 2. These features are defined in the legend and will be discussed in detail later.
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CONTRAST GAIN.
The plots in Fig. 3 are compressed to show the entire contrast/response
curve, but it is still obvious that most cells are exceedingly
sensitive to small contrast steps. This characteristic was analyzed
quantitatively by calculating the contrast gain: for each contrast
polarity, the lowest point of the contrast/response curve was used to
estimate the gain in units of percentage normalized amplitude/percentage contrast. Two values were obtained:
CGp and CGn, the contrast gain for positive
and negative contrasts, respectively. (Because contrast gain has most
often been expressed in percentage Michelson contrast in past work, we
use this contrast metric here. For low contrasts, percentage Michelson
contrast is equivalent to log contrast × 100, as noted in
METHODS). The average contrast gains were high and slightly
greater for negative than positive steps:
CGn = 17.5 ± 1.8%,
CGp = 13.4 ± 1.9%,
P = 0.015. The measurements for each cell in our sample
are plotted in Fig. 4. The + near the
origin shows the contrast gain, of ~0.9%, previously measured for
cones (Burkhardt and Fahey 1998). Thus 84% of the
amacrine cells in Fig. 4 have contrast gains that are 10-30 times
higher than that of cones. Some of the data points fall very near the 45° locus, providing clear examples of cells with nearly equivalent (balanced) negative and positive contrast gains, whereas other cells
have very different contrast gains for negative versus positive steps.
Although the present measurements are not adequate for a rigorous
analysis of the issue of linearity, it seems quite likely that the
balanced cells in Fig. 4 may generate quasi-linear responses for small
contrasts (
0.02), whereas those cells showing significant departures
from contrast equivalence, of which there are many in Fig. 4, must
already be responding in a nonlinear fashion. Overall, Fig. 4 suggests
a fair degree of independence for negative versus positive contrast
because a regression analysis showed that
CGp could only account for ~43% of
the variance of CGn.
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C50.
For each cell, the positive and negative contrasts required to evoke a
half-maximal response, C50p and C50n, were determined by interpolation
from the contrast/response curve. The results, plotted in logarithmic
coordinates in Fig. 5, show cases of
clear symmetry as well as cases of marked disparity between negative and positive contrast. Many of the values are remarkably low. Thus 12 of 25 cells have values of 0.10 for both contrast polarities and 24 of the 25 cells have a C50 values of
0.10 for at least one polarity.
The means for C50n and C50p, were 0.12 and 0.22, respectively. However,
the standard deviations were large and the difference in the means was
not significant (P = 0.37).
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DYNAMIC RANGE.
The dynamic range of the contrast/response curve was assessed for
both contrast polarities by measuring C10 and C90, the contrasts required to evoke responses of 10 and 90% of the cell's maximum response, respectively (see Fig. 2). Figure
6 shows C10-C90 measurements () for
all cells in our sample. For cells with highly asymmetric contrast/response curves (e.g., Fig. 3, A and H),
a C90 value could not be determined for the less effective contrast
polarity. These instances are shown by
. The cells in Fig. 6 are
sorted in ascending order with respect to the magnitude of C90n.
Several main points are evident: a number of cells show remarkably
narrow ranges, particularly for negative contrast; other cells show
larger dynamic ranges, particularly for positive contrast, although
C90p is indeterminate for nearly half of the cells; if C90 is
recalculated relative to the maximum response of the less effective
contrast polarity, then these recalculated C90 values are typically
small, as shown by
in Fig. 6. This is expected from the finding
that, in many cells, the lesser limb of the contrast response curve rises quite rapidly (see Fig. 3). Also evident is that the ordering of
the dynamic range for positive contrast is erratic with respect to that
for negative contrast, suggesting that dynamic range varies somewhat
independently with the contrast polarity in any given cell, i.e., cells
that have small dynamic ranges for negative contrast do not necessarily
have small dynamic ranges for positive contrast.
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EFFECT OF CONTRAST POLARITY ON RESPONSE LATENCY.
In discussing Fig. 1, it was noted that the response latency to
contrast onset was shorter for negative than for positive contrast
steps. Quantitative evidence for this generalization is summarized in
Fig. 7. For the cell of Fig.
7A, it can be seen that the minimum (asymptotic) latency is
~50 ms for negative contrast and 65 ms for positive contrast. Over
all 25 cells, the minimum latency averaged 60.9 ± 3.6 and
79.3 ± 4.3 (SE) ms for negative and positive contrast,
respectively, a highly significant difference (P = <0.001). Figure 7B shows results for another cell with the measurements now plotted against the absolute value of the contrast. This illustrates the general finding that the difference in latency holds for all comparisons between responses to steps of equal absolute
contrasts. This effect was quantified by measuring the latency
difference, Lp Ln, for all negative/positive contrast pairs, where Lp and
Ln are the latencies evoked by the
onset of positive and negative contrast steps of equal absolute
magnitude. Thus a positive value indicates that the latency to negative
contrast is shorter than that to positive contrast. Figure
7C summarizes data for all cells in our sample. The
difference decreases in quasiexponential manner from ~45 ms at very
low contrast to ~20 ms at maximum contrast. The differences found in
Fig. 7, A-C, are impressive when it is realized that the
absolute magnitude of the light step (
L) is considerably
smaller for negative than for positive contrast steps. To highlight
this point, all the data for the cell of Fig. 7B are
replotted in terms of
L in Fig. 7D. This
clearly illustrates that for light steps of equal absolute magnitude
(
L), the latency of the on-off amacrine cell is
invariably shorter for the negative step.
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Other inner retinal neurons
In addition to the preceding recordings from depolarizing
ON-OFF amacrines, several other response types were seen in
the inner retina (see METHODS). As displayed in Fig.
8, one type showed hyperpolarizing
transients at stimulus onset and offset regardless of the spatial
configuration or contrast polarity and thus appeared similar to
ON-OFF hyperpolarizing amacrine cells described previously by others (Ammermuller and Kolb 1995; Werblin
1970
). Such recordings were typically very noisy, but eight
were studied quantitatively. Their contrast/response curves were within
the range of variation described in the preceding text for depolarizing
ON-OFF amacrines and included several cells with
exceedingly steep curves. Mean contrast gain for the eight cells were:
CGp = 17.3 ± 2.9%,
CGn = 20.0 ± 3.1%. Like their
depolarizing counterparts, they consistently showed shorter latencies
to negative than to positive contrast steps. Despite the compressed
time scale, this effect can be seen in Fig. 8. The - - - in the +0.30
trace corresponds to the latency of the onset of the
0.30 trace and
falls ~20 ms before the onset of the response to the +0.30 contrast
step. Figure 8, inset, shows a full set of contrast/latency
measurements. Over all contrasts and cells in our sample, the mean
difference in latency for positive and negative contrast was 22 ms, a
robust difference (P < 0.002).
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Several recordings were obtained in the inner retina that resembled
those described for sustained, depolarizing amacrine cells (Ammermuller and Kolb 1995; Chan and Naka
1976
; Dixon and Copenhagen 1992
; Frumkes
et al. 1981
; Maguire et al. 1989
;
Vallerga 1981
). In response to positive steps, these
cells showed a peak depolarization followed by a sustained phase. An
example is shown in Fig. 9. Usually,
oscillations of ~5-20 Hz were superimposed on the depolarization, as
found for sustained amacrine cells (type Na) in catfish retina (Sakai and Naka 1992
; Sakuranaga and Naka
1985a
). Negative contrast steps evoked an
ON-OFF response. Seven of these cells were studied quantitatively. Without exception, they were strongly positive-contrast dominant, showing steep contrast/response curves for positive contrast
and considerably weaker responses for negative contrast. An example is
shown by Fig. 9, inset. Mean results for the seven cells
sampled were: CGp = 19 ± 5.3%;
CGn = 3 ± 1.5%; C50p = 0.06 ± 0.02; C50n =
1.64 ± 0.28. When responses to
negative contrast could be adequately measured, they usually showed
shorter latencies than those for positive contrasts for most contrasts
of
1.0 (mean difference = 15.6 ms, P <0.01,
n = 27), whereas in some cells, the difference was
reduced or reversed at very high contrasts.
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ON-OFF ganglion cells
Figure 10 shows intracellular
responses of an ON-OFF ganglion cell to positive and
negative contrast steps (light and dark traces, respectively). At both
contrast onset and offset, the response consists of a burst of nerve
impulses riding on a graded, excitatory postsynaptic potential (EPSP).
For both EPSP and spikes, the cell generates vigorous responses to very
small contrast steps and shows clear latency differences in favor of
negative contrast at onset and positive contrast at offset,
respectively, as previously found for amacrine cells (Figs. 1 and 7).
These properties were characteristic of the 15 ON-OFF
ganglion cells studied. As shown in Fig. 10, the latency of the first
spike of the impulse discharge was triggered on the rising phase of the
EPSP. Thus depending on the cell and contrast magnitude, the first
spike occurred ~5-15 ms after the onset of the EPSP and showed the
same dependence on contrast as the EPSP, i.e., for ON
responses, the first spike latency was typically shorter for negative
than for positive contrast of comparable magnitude, whereas the reverse
was typically found at contrast offset. As with amacrine cells, a small
hyperpolarizing deflection (Fig. 10, ) is apparent at the offset of
negative contrast and may be responsible for the increased latency of
the subsequent EPSP and spike discharge. Although the waveform of the
EPSPs of Fig. 10 differ from those of Fig. 1, on the whole, no
consistent differences were apparent in the range of the EPSP waveforms
observed across our amacrine and ganglion cell samples. The EPSPs of
ganglion cells in our sample remained stable for some 15-45 min after
impalement, whereas the spike discharge often deteriorated or was lost
with time. Hence, quantitative measurements of the contrast response were confined to the EPSP.
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Figure 11, A-C, shows
representative contrast/response curves for measurements of normalized
EPSP amplitude [maximum amplitude varied from cell to cell: 10.9 ± 2.6 (SE) mV]. Cells showed some clear differences in contrast
dominance across the population sampled. A few showed considerably
larger responses to positive contrast (A), some were
relatively symmetrical or balanced (B), whereas others
showed maximum responses to negative contrast (C). The mean
contrast gains for the EPSPs were high:
CGp = 15.5 ± 2.0% and
CGn = 20.9 ± 2.7%,
and the difference in the gains was statistically significant
(P < 0.01). Figure 11D shows a
representative contrast/latency plot for the EPSP evoked by contrast
onset. Measurements of latency differences
(Lp Ln, as defined earlier in the text for
amacrine cells) over all contrast levels for a total of 10 cells
showed, on average, that the negative contrast latency was shorter by
25.1 ± 1.2 (SE) ms than that for the equivalent positive contrast. This difference was highly significant (P = 0.001), providing strong support for the conclusion that for contrast steps of comparable magnitude, the response to negative contrast arises
earlier than that to positive contrast. The minimum asymptotic latencies of the EPSPs to contrast onset were:
Lmin,
Cp = 76.3± 5.0 ms,
Cn = 58.0 ± 4.3 ms, a highly
significant difference (P < 0.001). However, 1 cell of
the 15 in our sample showed the reverse result, i.e., positive contrast
latency shorter than negative contrast latency. This result also was
found previously for a very small minority of ON-OFF cells
in extracellular recordings (Burkhardt et al. 1998
).
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DISCUSSION |
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Although the light-evoked responses of ON-OFF amacrine
cells have been described in various species since the first report of
Werblin and Dowling (1968), this report supplies new
information by providing a quantitative analysis of contrast processing
in the light-adapted retina. Most of the cells in our sample showed evidence for a strong suppressive surround, so it useful to emphasize that all the following discussion applies to stimuli designed to
optimally stimulate the center of the receptive field.
Dynamic range and the reflectance of objects in natural environments
In natural environments, virtually all objects are seen by
reflected light. Reflectance (percent of light reflected) typically covers a range from ~5% for black objects to ~85% for white
objects, with midgray at ~22% (Goldstein 1998). If it
is assumed that the background corresponds to the midgray reflectance,
then white and black objects (85 and 5% reflectances), respectively,
will correspond to contrasts of about ±0.60. These limits are shown in
Fig. 6 (- - -). The distribution of dynamic ranges in Fig. 6 is thus
sufficient to encode this range but also includes some cells with
exceedingly small ranges, particularly for negative contrast. The
latter cells are well designed to detect very dim gray objects but are
ill-equipped to represent the full range of contrasts in natural
environments. Recent measurements (Vu et al. 1997
)
suggest that the contrast of most objects in natural scenes falls
within a range of about ±0.25 log contrast, thus covering about half
of the ±0.60 range shown in Fig. 6. With respect to the extreme case,
i.e., a cell adapted to an object of either extreme reflectance (i.e.,
85 or 5%) and subsequently exposed to the other, the resulting
contrast step would be 1.2 log units, which is within the dynamic range
of a subset of cells in Fig. 6.
Contrast sensitivity
The contrast sensitivity of many ON-OFF amacrine cells
was remarkably high. Contrast gain, an index of the small signal
response, often ranged as high as 20-35%. Thus 20-35% of the
cells' total response could be evoked by a contrast of only 1%.
Furthermore a contrast of <1% was sufficient evoke a response of
10% of the maximum (C10) in more than half of the cells in our
sample. The rather remarkable sensitivity of these cells can be
appreciated from the observation that a simple contrast step of ~1%
is just detectable (i.e., the threshold contrast) when applied to the fovea of the human retina under favorable conditions (Burkhardt et al.1987
; Westheimer et al. 1999
). The mean
contrast gains, ~13 and 17% of the maximum response, for
CGp and
CGn, respectively, correspond to
signals in the 1.6- to 2.0-mV range, signals that would be large enough
to often be detectable even in the face of the physiological and
electrode noise in our recordings. [The corresponding signals in cones
(Burkhardt and Fahey 1998
) are in the range of 0.13 and
0.07 mV for positive and negative steps, respectively.] Because the
slope of the contrast/response curve typically remained quite steep for
relatively large responses (Fig. 3), C50, the contrast required to
evoke a response 50% of the maximum, was remarkably low in many cells.
Thus 24 of 25 cells had a C50 value of
0.10 (i.e.,
10% contrast)
for one contrast polarity and for 12 of 25 cells, C50 was
0.10 for
both contrast polarities.
The observed differences between cells in contrast sensitivity and dynamic range seemed unrelated to differences in maximum response, latencies, resting membrane potential, age of the preparation, or other factors that might be indicative of compromised cells or abnormal retinal function. Thus it seems likely that the observed differences in the contrast response are largely due to normal physiological differences within the amacrine cell population.
Contrast rectification and the effects of contrast polarity
The degree to which negative and positive contrast generate similar responses depends on the cell and response measure in question. For very small signals, it might be expected that the response would approximate linearity and thus the contrast gain would be independent of contrast polarity. Although some cells in Fig. 4 do show similar contrast gains, many clearly do not and of the latter, more show higher gains for negative contrast. Over all cells, a regression analysis showed that only 43% of the variance in contrast gain was dependent on contrast polarity. For the half-maximal contrast (C50), some cells fall near the 45° locus for equivalence in Fig. 5 but more than half do not. These departures are bidirectional and so large (note that the scale in Fig. 6 is logarithmic) that, statistically, they swamp out the symmetrical cases. Consequently, the overall correlation between C50n and C50p is exceedingly low (R2 = 0.02). The results for contrast gain and C50 indicate that the departure from contrast equivalence increases as one goes from the small to the larger signal domain. This generalization also applies for the dynamic range results of Fig. 6. Thus the C10p and C10n values are relatively similar, whereas the C90 values show some striking differences as a function of contrast polarity. For contrast dominance based on response maxima, many cells were relatively balanced, but across the population ratios as large as 2 to 1 in both directions were found (Fig. 3). We also have described clear cases (see Fig. 1 and text) in which the response waveform is strongly dependent on the contrast polarity. In sum, our findings show that some ON-OFF amacrine cells respond in a relatively similar fashion independent of the contrast sign, but many show variable degrees of bias in favor of positive or negative contrast. Thus "contrast rectification" rarely holds exactly and the consequent variation in contrast dominance provides a distributed parameter across the ON-OFF amacrine population.
Relations between the contrast response of amacrine and bipolar cells
Although the details are not established, it is widely believed
that the response of the ON-OFF amacrine cells is shaped by interaction between synaptic inputs from the hyperpolarizing (Bh) and
depolarizing (Bd) bipolar cells. Our results highlight some new aspects
of this issue because the contrast responses of bipolar cells recently
have been studied under identical conditions (Burkhardt and
Fahey 1998). Figure 12
summarizes some of the measurements made here for amacrine cells
(row Am) along with a new analysis of our previous work on
hyperpolarizing (row Bh) and depolarizing (row
Bd) bipolar cells. In addition, mean results for cones
(Burkhardt and Fahey 1998
) are shown by - - - in the
Bh figures.
|
Contrast gain values for amacrine cells are often as much as 10-20
times higher than that for cones (Fig. 12A), so there is very clear evidence for contrast enhancement occurring between the
level of cones and amacrine cells. However, Fig. 12 suggests that much
of this amplification of the amacrine contrast response could be
accounted for by the striking increase in contrast gain that occurs
across the cone bipolar synapse. Thus many Bh and Bd cells show
gains as high as that of amacrine cells, so high-gain amacrine cells
might simply be the consequence of selective connections with high-gain
bipolar cells. However, there is evidence that amacrine cells on
average may show slightly higher contrast gain for negative contrast:
The Am n distribution (
) in Fig. 12A differs markedly from that for Bh n (P = 0.005) and
the difference between Am n and Bd n (
)
approaches statistical significance (P = 0.017).
Amacrine cells show much lower C50 values than do cones (Fig.
12B), indicating a substantial transformation in
suprathreshold contrast processing from cones to amacrine cells.
Moreover, Fig. 12B suggests that the C50 distributions for
amacrine and bipolar cells differ. This question was evaluated with a
nonparametric test (Wilcoxon signed rank) because C50 was of
indeterminate value (i.e., >2.0) for a good number of Bh and Bd cells,
thus precluding parametric statistical tests. The signed-rank test
showed that Bh n and Am n differed greatly
(P < 0.001). When all measurements were combined
across both bipolar cells types and contrast polarities, it also was
found that the composite bipolar distribution differed from the
composite amacrine population (P < 0.001). The origins of these differences seem evident on inspection of Fig. 12B
because the amacrine cells show a relatively greater proportion of very low C50 values and fewer cases of high, indeterminate C50 values. Thus
the C50 results suggest that, on average, the suprathreshold contrast
response of amacrine cells is somewhat enhanced relative to that of
bipolar cells. Possible mechanisms for this enhancement might involve
contributions of voltage-sensitive conductances of amacrine cells
(Barnes and Werblin 1986; Cook and Werblin
1994
; Werblin 1977
) and/or signal shaping
by the amacrine-bipolar feedback synapse (Lukasiewicz and
Werblin 1994
; Yu and Miller 1996
; Zhang et al. 1997
).
A comparison of the contrast dominance distributions for response maxima (Fig. 12C), show that both cones and Bh cells are positive contrast dominant. Thus the means for cones (0.65) and Bh cells (0.60) were both much higher (P < 0.01) than would be expected for a perfectly balanced distribution with a mean of 0.50. The amacrine cells were, on average, slightly negative dominant (mean = 0.46) and clearly differed from cones and Bh cells (P < 0.001). Bd cells fell between the two extremes, with a mean of 0.53 that approached but did not quite reach statistical significance when evaluated against either Bh cells (P = 0.19) or amacrine cells (P = 0.12). In sum, Fig. 12 suggests that contrast dominance tends to shift in the direction of negative contrast as signals are transmitted from cones to bipolars to amacrine cells.
Although the latency of Bh and Bd cells may differ slightly (see next
paragraph), both cell types tend to show a fundamentally similar
relation between contrast and latency; this may be summarized: for
contrast steps of equal magnitude but opposite polarity, positive contrast evokes a shorter latency than negative contrast
(Burkhardt and Fahey 1998). Thus the latency
differences, Lp
Ln (as defined in
RESULTS), tend to be negative in sign in Fig. 12D,
top and middle. The means for both Bh and Bd cells are
negative in value and differ statistically from the null (0 difference
case): P = 0.014 for Bd cells and P < 0.001 for Bh cells. Just the opposite results were found here for
amacrine cells (Fig. 7, B and C). Thus the differences in Fig. 12D are predominantly positive in sign.
When measured over all contrasts, the mean latency difference of the distribution for amacrine cells (Fig. 12D) was 28.5 ± 1.6 ms (n = 182), a value significantly more positive
(P < 0.001) than that expected for the null, zero
difference case. Moreover, the amacrine distribution differs very
significantly from those of Bh and Bd cells (P < 0.001 for both cases). Thus the comparisons in Fig. 12D strongly
support the conclusion that, in the latency domain, there is a striking
transformation occurring between the bipolar and ON-OFF
amacrine cells. This might be a general feature of inner retinal
neurons, regardless of response polarity and type because it also was
observed for the hyperpolarizing and sustained neurons of Figs. 8 and
9.
Under identical conditions as used here, the latency of Bh cells was
found to be ~20 ms shorter than that of Bd cells (Burkhardt and Fahey 1998) in qualitative agreement with past work in
mudpuppy and turtle (Frumkes and Miller 1978
; Kim
and Miller 1993
; Marchiafava and Torre 1978
;
Nelson 1973
). Thus the faster response of amacrine cells
to negative contrast steps might be explained if it is assumed that Bh
and Bd cells provide the dominant input, respectively, for negative and
positive contrast. This seems consistent with well-known
pharmacological evidence that Bd and Bh cells mediate, respectively,
the ON and OFF responses of amacrine cells
(Miller 1994
; Slaughter and Miller 1981
).
However, we can provide no further evidence for this hypothesis, and
alternative possibilities, including differences in rise and peak times
in bipolar cells and/or voltage-sensitive conductances in amacrine
cells, might be proposed. Indeed, several observations suggest that the
amacrine response arises from operations more complex than simple
polarity inversion and algebraic summation of input from Bh and Bd
cells (Marchiafava and Torre 1978
; Miller 1979
; Sakuranaga and Naka 1985b
).
Contrast responses of ON-OFF amacrine and ON-OFF ganglion cells
Our finding that the contrast responses of the EPSPs of amacrine
and ganglion cells are quite similar suggests that the bipolar cells
may drive both ON-OFF ganglion and amacrine cells by very similar synaptic mechanisms. With respect to the view that
ON-OFF amacrine provide a strong inhibitory input to
ganglion cells (Frumkes et al. 1981; Morgan
1990
; Werblin 1977
; Wunk and Werblin
1979
; Yu and Miller 1996
; Zhang et al.
1997
), our results suggest that the inhibitory input may be,
apart from the difference in polarity, quantitatively similar to the
ganglion cell's excitatory input.
Evidence that the latency to negative contrast is shorter than that for
positive contrast previously was seen in the extracellular impulse
discharge of ON-OFF ganglion cells (Burkhardt et al.
1998). The present results clearly suggest this finding is a
direct consequence of latency differences in the ganglion cell EPSP
(Fig. 10) and show that the difference first appears earlier, at the
level of the ON-OFF amacrine cells (Figs. 1 and 7). At the
offset of contrast steps, the extracellular recordings showed that the
latency difference usually was reversed, i.e., the latency to positive
contrast was shorter than that for negative contrast (Burkhardt
et al. 1998
). This somewhat unexpected result is confirmed and
perhaps explained by the present results (Figs. 1 and 10). Both
ganglion cells and amacrine cells exhibit an initial hyperpolarization
at the offset of negative contrast that may delay the appearance of the
subsequent depolarizing response.
Distributed encoding in amacrine cells
It now is recognized widely that the amacrine population of
vertebrate retinas is diverse, encompassing a number of morphological and functional classes (Ammermuller and Kolb 1995;
Kolb et al. 1992
; Morgan 1990
;
Yang et al. 1991
), so it seems evident that there is
distributed encoding of visual information across the amacrine cell
population as a whole. However, the present paper focuses on only one
general class of amacrine cell, the transient, depolarizing
ON-OFF type. Our results (Figs. 3-6 and 12) show that ON-OFF cells vary substantially in dynamic range, contrast
dominance, half-maximal contrast (C50), and contrast gain. Whether the
distributions are continuous or composed of identifiable functional
subtypes (Yang et al. 1991
) remains to be established.
In either case, our findings suggest that differences across the
depolarizing ON-OFF amacrine cells could provide a
substrate for distributed encoding. Thus differential activation of
cells within the population could enhance discrimination between the
varied permutations of contrast polarity and magnitude found across
objects in the environment. Cells with preferential responses to
negative contrast might be critical for detecting shadows produced by
predators overhead. Cells with high contrast gain and narrow dynamic
ranges seem all the more important when it is realized that
high-contrast objects in the environment end up as low-contrast images
on the retina whenever the eye is out of focus or light is scattered by
fog, rain, or snow or by turbidity underwater.
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ACKNOWLEDGMENTS |
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We thank M. Sikora and M. Bergman for technical support.
This research was supported by National Eye Institute Grant EY-00406 to D. A. Burkhardt and Training Grant EY-07133 to P. K. Fahey.
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FOOTNOTES |
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Address for reprint requests: D. A. Burkhardt, n218 Elliott Hall, University of Minnesota, 75 E. River Rd., Minneapolis, MN 55455.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 November 1998; accepted in final form 28 May 1999.
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REFERENCES |
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