Department of Neurobiology, Stanford University, Stanford, California 94305
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ABSTRACT |
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DeVries, Steven H. Correlated firing in rabbit retinal ganglion cells. A ganglion cell's receptive field is defined as that region on the retinal surface in which a light stimulus will produce a response. While neighboring ganglion cells may respond to the same stimulus in a region where their receptive fields overlap, it generally has been assumed that each cell makes an independent decision about whether to fire. Recent recordings from cat and salamander retina using multiple electrodes have challenged this view of independent firing by showing that neighboring ganglion cells have an increased tendency to fire together within ±5 ms. However, there is still uncertainty about which types of ganglion cells fire together, the mechanisms that produce coordinated spikes, and the overall function of coordinated firing. To address these issues, the responses of up to 80 rabbit retinal ganglion cells were recorded simultaneously using a multielectrode array. Of the 11 classes of rabbit ganglion cells previously identified, coordinated firing was observed in five. Plots of the spike train cross-correlation function suggested that coordinated firing occurred through two mechanisms. In the first mechanism, a spike in an interneuron diverged to produce simultaneous spikes in two ganglion cells. This mechanism predominated in four of the five classes including the ON brisk transient cells. In the second mechanism, ganglion cells appeared to activate each other reciprocally. This was the predominant pattern of correlated firing in OFF brisk transient cells. By comparing the receptive field profiles of ON and OFF brisk transient cells, a peripheral extension of the OFF brisk transient cell receptive field was identified that might be produced by lateral spike spread. Thus an individual OFF brisk transient cell can respond both to a light stimulus directed at the center of its receptive field and to stimuli that activate neighboring OFF brisk transient cells through their receptive field centers.
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INTRODUCTION |
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Multielectrode recordings from cat
(Mastronarde 1983a-c
) and salamander (Brivanlou
et al. 1998
; Meister et al. 1995
) retina show
that adjacent ganglion cells have a pronounced tendency to fire
together within ±5 ms. These tightly correlated spikes are not
directly produced by a light stimulus: while a dim stimulus applied to
the receptive fields of adjacent cells will increase activity in both
cells, the increase will have a time course of ~50-100 ms, the
duration of the single photon response at dim intensities
(Brivanlou et al. 1998
; Mastronarde
1983b
). Rather, tight correlations apparently arise through
circuitry within the retina. The existence of specific retinal circuits
that coordinate firing raises the possibility that groups of ganglion
cells could act together to encode a visual stimulus.
To learn about the possible functions of correlated activity,
Mastronarde (1983a,b
) examined the relative timing of
the spikes in neighboring cat X- and Y-ganglion cells in the dark and
in dim, diffuse light. Two types of tightly correlated activity were observed. The first type occurred indiscriminately among pairs of X and
Y cells with the same center sign (i.e., ON or
OFF). The timing of the correlated events suggested that
they were initiated by a transient in a single excitatory interneuron
that made synapses onto both ganglion cells. Consistent with this
"divergent" mechanism, the spike train cross-correlation function
had a single central peak. The second type of rapid correlation was
seen only between Y cells of the same center sign. The
cross-correlation function was bimodal with a trough at 0 ms and peaks
at ±1 ms. A bimodal distribution could result if Y-ganglion cells
activated each other reciprocally with a 1-ms delay. Indeed,
Mastronarde (1983c)
was able to cause one Y cell to fire
at a short latency by retrogradely activating a neighboring Y cell of
the same center sign. It is likely that reciprocal correlations occur
through electrical synapses (Brivanlou et al. 1998
;
Mastronarde 1983c
). For both divergent and reciprocal
activity, correlations were strongest for adjacent cells and much
weaker for distant pairs (Mastronarde 1983a
). Although Mastronarde (1983a)
found correlations among cells in
the four classes studied, mammalian retinas have many additional
ganglion cell types, and it is not known whether correlated firing is a general feature of ganglion cell encoding or whether it is limited to a
few select classes.
Meister et al. (1995) have recently studied the stimulus
properties encoded by tightly correlated spikes produced through a
divergent mechanism. The receptive field profiles of two neighboring ganglion cells were measured and then compared with the "profile" associated with spikes in the two cell's trains that occurred jointly.
Joint-spike receptive fields were located midway between those of
either ganglion cell and were frequently smaller than either of the
original receptive fields. Consequently, it has been suggested that
divergently correlated spikes encode information about small-diameter
stimuli. The stimulus properties encoded by reciprocally correlated
spikes have not been investigated.
The rabbit retina is well suited to in vitro recordings with a
multielectrode array because it is thin and avascular. In addition, the
responses of rabbit ganglion cells have been characterized extensively
both in single electrode (Amthor et al. 1989a,b
;
Caldwell and Daw 1978
; Levick 1967
) and
paired or multielectrode recordings (Arnett and Spraker
1981
; DeVries and Baylor 1995
, 1997
). The purpose of this work is to identify the classes of rabbit retinal ganglion cells that fire tightly correlated spikes. Once these classes
are identified, the mechanisms that produce the correlated activity
will be characterized. Finally, the receptive field properties of
ON and OFF brisk transient cells will be
compared with each other to show how reciprocal correlations extend the
receptive field span of an individual ganglion cell.
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METHODS |
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The methods for recording ganglion cell activity with a
multielectrode array and the use of a flickering stimulus to measure receptive field profiles have been described previously (DeVries and Baylor 1995, 1997
; Meister et al. 1994
,
1995
). In brief, pigmented New Zealand white rabbits were
maintained in darkness overnight. All subsequent manipulations were
done under dim red light or using an infrared to visible light image
converter to maintain the retina in a dark-adapted state. An eye was
enucleated, and the retina was separated from the pigment epithelium
and mounted ganglion cell side down on a glass substrate bearing 61 closely spaced extracellular electrodes. Most recordings were obtained from the central retina, slightly below the visual streak. While mounted on the multielectrode array, voltage spikes occurring at each
electrode were recorded continuously, and their amplitudes, widths, and
times of occurrence stored in a computer. At the end of a recording
session, the spikes were sorted and assigned to individual neurons
(Meister et al. 1994
). The responses of ganglion cells
and spike-generating displaced amacrine cells could not be
distinguished. It was assumed that a majority of the recorded cells
were ganglion cells because displaced amacrine cells comprise <20% of
the cells in the ganglion cell layer (Vaney 1980
).
During recordings, the retina was superfused continuously with
bicarbonate-buffered Ames medium at 35°C. Pharmacological agents were
added to the superfusate before solution entered the recording chamber.
All reagents were obtained from Sigma Chemical (St. Louis) unless otherwise mentioned.
Ganglion cell receptive field profiles were determined by analyzing
responses to random flicker stimulation (DeVries and Baylor 1997). The retina was stimulated with a Macintosh
high-resolution RGB color monitor the screen of which was imaged onto
the photoreceptor layer. The stimulus was a randomly varying
checkerboard with squares (100-160 µm on a side) that were made
green or black according to a pseudorandom sequence. The mean stimulus
intensity, calculated as described in DeVries and Baylor
(1995)
, was 0.5-5.0 Rh* · rod
1 · s
1. At this intensity, rods were the predominantly active
photoreceptor (DeVries and Baylor 1995
). The
checkerboard pattern was updated continuously at 13.3 or 16.7 Hz
because a stimulus refresh interval of 60-75 ms is well matched to the
response properties of the transduction cascade in dark-adapted rabbit
rods (Nakatani et al. 1991
). The sequence of patterns
imaged on the retina then was cross-correlated with a ganglion cell's
spike train to obtain the cell's mean effective stimulus. This is the
stimulus, a function of space and time, that on average preceded the
occurrence of a spike. It has units of light intensity. The spatial
profile of a ganglion cell's receptive field was obtained from the
mean effective stimulus at the temporal maximum. Plots of the spatial dependence of the mean effective stimulus usually were normalized such
that +1 and
1 corresponded to the high and low intensities in the
checkerboard, the average normalized intensity being zero.
Receptive field centers were fitted with equations that specified
either a single generalized Gaussian surface or the sum of two
generalized Gaussians. The single Gaussian fit provided estimates of
Gaussian amplitude and width along the major and minor axes
(x or
y), the position of the receptive
field center, and the angle relative to the checkerboard stimulus
(DeVries and Baylor 1997
). The two Gaussian fit provided
estimates of the amplitudes of both Gaussians, the widths along the
major and minor axes of each Gaussian, and the position of the
receptive field center and angle relative to the checkerboard stimulus,
which were constrained to the be same for both Gaussians. The root mean
square (RMS) error was used to compare how well the same profile was
fitted by the two surfaces.
In some experiments, the receptive field spatial profile was measured
by using contrast reversal spatial sinewave gratings as described in
DeVries and Baylor (1997; see also Hochstein and Shapley 1976
). In brief, the gratings had a contrast,
Imax
Imin/Imax + Imin, of 1 where Imin and
Imax are the peak and trough intensities. The
mean intensity was 1.5 Rh* · rod
1 · s
1.
The spatial intensity profile of the grating was periodically reversed
by a temporal sinusoid at 0.5 Hz. Monitor intensities were corrected
for the nonlinear relation between command voltage and phosphor
emission. The retina was stimulated for 120-s intervals at each spatial
frequency. Time histograms of each ganglion cell's spike train then
were analyzed by integrating the peak in their Fourier transforms at
the fundamental frequency. The peak area then was plotted as a function
of the spatial frequency of the stimulus. The effective diameters of
the center and surround of the receptive field were obtained by fitting
plots of peak area versus spatial frequency with a sum of two Gaussian
curves.
When the same receptive field profiles were measured with sinusoidal
gratings and checkerboard patterns, the surrounds obtained from
gratings appeared to be more prominent. However, for the larger
receptive fields measured here, a checkerboard stimulus (containing 240 squares) that provided useful information about the receptive field
center profile typically did not extend far into the receptive field
surround. For ganglion cell types with small receptive fields
encountered during the same experiments, the checkerboard stimulus
usually encompassed most of the surround. In these cases, the center
and surround profiles calculated from both checkerboard and sinusoidal
stimuli were found to be similar after the making the appropriate
conversion (see Eq. 5, DeVries and Baylor
1995; unpublished results).
Neurons were classified into functional groups by two criteria
(DeVries and Baylor 1997). The first criterion was the
shape of the autocorrelation function of the spike train. In the rabbit retina, the autocorrelation functions of ganglion cells in the same
class have nearly identical shapes, which are distinct from those of
other classes. The second criterion was the time course of the mean
effective stimulus, which was obtained by spatially averaging the
stimulus intensity over the center of a cell's receptive field.
Ganglion cells in the same class had similarly shaped mean effective
stimulus time courses.
The tendency for two neighboring ganglion cells to fire together was
studied by plotting their cross-correlation function. A spike train
cross-correlation function plots the mean firing rate of one cell as a
function of time relative to the occurrence of a spike in its neighbor.
Figure 2 shows typical cross-correlation functions for pairs of
ON and OFF center ganglion cells. The function has up to three regions. First, at times greater than about ±100 ms,
the function is flat, indicating that spikes are not correlated over
long intervals. Within ±100 ms, there is a broad increase in the
tendency of the two cells to fire together. Finally, a sharp peak in
the center of the cross-correlation shows that the cells have a
pronounced tendency to fire within ±5 ms of each other. The fraction
of a cell's spikes that are "rapidly" correlated was calculated
as follows: the crude number of rapidly correlated spikes was
determined by counting the number of spikes in cell 1's
train that occurred within ±5 ms of a spike in cell 2's
train. The crude number was corrected for the pairings that would occur due to both chance and to the broad correlations in the same interval. This correction was based on the observation that the peak height of
the broad correlation is similar to the height within the intervals of
15 to
10 ms and +10 to +15 ms. The number of spikes in cell 1 occurring within these intervals of a spike in cell 2 then was determined and subtracted from the crude value, and the result divided by the total number of spikes in the train to give the fraction
of rapidly correlated spikes. The average fraction of correlated spikes
for both cells usually is cited (Mastronarde 1983a
).
The strength of correlated firing decreases with distance between
receptive fields (Mastronarde 1983a). The spacing ratio, s, used to express the distance between receptive fields is
defined as
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RESULTS |
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Rapid correlations occur in a subset of ganglion cell classes
Eleven functional classes of rabbit ganglion cells were identified
previously during recordings with a multielectrode array (DeVries and Baylor 1997). The occurrence of tightly
correlated firing among the cells in these classes is now
characterized. In Fig. 1, the percentage
of rapidly correlated spikes in pairs of ganglion cells is plotted as a
function of the distance between receptive field centers. To compare
results from different retinas, the distance between receptive fields
was normalized so that Gaussian receptive field profiles which touched
at their 1
borders had a spacing of 1. Rapid correlations were
categorized arbitrarily as either strong (>8% of the spikes in
adjacent cells were correlated rapidly), moderate (2-8%), or weak
(<2%) according to the strength of the correlations at 0.5 spacing
units. Figure 1A plots the results for pair-wise
combinations of ON-center ganglion cells including the
ON
cells, a type of ganglion cell with distinctive physiological properties that are described in the next
section. Pairs of ON brisk transient cells had the
strongest correlations with a shared activity of 13% at a spacing of
0.5. The spikes in ON brisk transient cells also were
correlated strongly with those in neighboring ON
and
ON brisk sustained cells. Moderate correlations were
observed in ON brisk sustained cell pairs, ON
cell pairs, and in pairs consisting of an ON brisk
sustained and an ON
cell (Table
1). Weak correlations were observed when pairs contained ON sluggish, ON-OFF direction
selective (DS), or ON DS cells. For both moderately and
strongly correlated pairs, the strength of coupling declined with
distance so that an exponential curve fit to the data had a "length
constant,"
, of 0.42-0.67 spacing units (Table 1). A
<1
indicates that rapid correlations were prominent between adjacent cells
where the receptive fields overlapped significantly, but much weaker
for pairs where there was little receptive field overlap.
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A plot of correlated activity versus spacing is shown in Fig. 1B for pairs of OFF-center cells. The largest correlations were observed in pairs of OFF brisk transient cells, where >20% of the spikes in nearest neighbors were correlated tightly. Spikes in OFF brisk transient cells were correlated moderately with those in OFF brisk sustained cells (2.4% at 0.5 spacing units), although the strength of the correlations varied significantly from retina to retina. Thus rapid correlations were not evident between OFF brisk transient and OFF brisk sustained cells in most retinas (48 of 50). In two retinas, however, the percentage of rapidly correlated spikes was found to exceed 4%. The plot in Fig. 2B includes results from these two retinas. The source of this variability is not known. OFF delayed, OFF sluggish, and local edge detector (LED) cells showed no evidence of rapid correlations. In addition, rapid correlations were not seen between OFF brisk transient and ON-OFF DS cells. When rapid correlations were observed, they were strong between neighboring OFF cells but much weaker for cells with receptive fields that did not overlap.
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Three experimental approaches suggested that the rapid correlations
were not produced by photoreceptor activity during stimulation with the
dim checkerboard. The first approach compared correlated activity in
the dark and during exposure to the checkerboard stimulus. A dim
checkerboard stimulus was used to probe correlated activity because
most rabbit ganglion cells were silent in the dark or in dim, diffuse
light. For those cells that did maintain a modest firing rate in the
dark, primarily brisk transient and ON brisk sustained
cells, rapid correlations could be observed, and their strength and
extent were similar to that measured during checkerboard stimulation
(Table 1). Although the narrow central peak changed little in the dark
and during stimulation (Fig. 2, A and B), the height of the broad hump was increased markedly in the presence of
the stimulus. Results from cat (Mastronarde 1983b)
and salamander (Brivanlou et al. 1998
) suggest that the
broad correlations are produced by light that activates photoreceptors
the signals of which drive both recorded ganglion cells. Indeed, the
temporal width of the broad correlation is similar to the duration of
the dim flash response in dark-adapted rabbit rods (Nakatani et
al. 1991
).
The second approach used 2-amino-4-phosphonobutyric acid (APB) to block
transmission at the rod synapse. As a consequence of its action at
receptors on rod bipolar cells, APB eliminates activity in
ON center ganglion cells while strongly suppressing light
responses and increasing spontaneous activity in OFF center ganglion cells (DeVries and Baylor 1995; Muller
et al. 1988
). In APB, the rapid correlations between
OFF brisk transient cells were strengthened and the
correlations in OFF brisk transient-OFF brisk
sustained pairs were more evident (Fig. 2C; Table 1). Cells that lacked rapidly correlated spikes in control solution also lacked
these spikes in APB-containing solution. Thus blocking communication at
the rod synapse did not stop the rapid correlations.
Further evidence that the rapid correlations were not directly caused by the light stimulus was obtained by recording from adjacent OFF brisk transient cells during two presentations of the same 20-min stimulus sequence (Fig. 2D). Within a trial, the cross-correlation function had the expected broad region and a narrow central peak. However, when the spike trains were cross-correlated across trials, the narrow peak disappeared while the broad hump remained. The simplest interpretation is that the broad correlations are produced by the light stimulus, while the rapid correlations originate in synaptic activity within the retina.
A narrow central peak was not observed when the spikes trains of
ON center and OFF center ganglion cells were
cross-correlated, as was also the case in cat retina
(Mastronarde 1983a). Although tight negative or
anticorrelations were seen commonly between cat ON and
OFF center ganglion cells (Mastronarde
1983
), tight negative correlations were not prominent in the
rabbit. The lack of distinctive tight negative correlations in the
rabbit retina probably results from the relatively low firing rate of
rabbit ganglion cells. In summary, tightly correlated activity was
significant in 5 of the 11 identified classes of rabbit ganglion cells.
Correlations were strongest for cells the receptive fields of which
overlapped but much weaker for second or third order neighbors.
ON eta cells
Correlated activity was observed in four classes of ganglion cells
in cat retina: ON and OFF X and ON
and OFF Y. As shown above, correlated activity is found in
similar cell types, the brisk cells (Caldwell and Daw
1978), in the rabbit. However, in rabbit, there appears to be
an additional type of ON-center cell, the ON
cell, the spikes of which were correlated strongly with those of
neighboring ON brisk transient and brisk sustained cells. The criteria for distinguishing between ON
and other
rabbit ON center ganglion cells are described in the next
paragraph.
Rabbit ganglion cells were divided into classes based on three
functional properties: the mean effective stimulus time course when
stimulated with a checkerboard pattern, the autocorrelation function
shape, and the response to a light pulse (DeVries and Baylor
1997). The functional properties of ON
and
ON brisk transient cells are compared because some of their
characteristics are similar. Figure
3A shows the superimposed mean
effective stimulus time courses of ON
(black) and
ON brisk transient (gray) cells from the same retina. Cells
in the two classes have similar time courses with peaks at
142 and
135 ms, respectively. Figure 3B plots the autocorrelation
functions for the same cells. The minimum interspike interval was
shorter for the ON brisk transient cells (2.0 ms) than for
the ON
cells (2.8 ms), and the interval between the
first and second peaks was longer for ON
cells.
ON
cells could be distinguished readily from
ON brisk transient cells by their poor response to a
diffuse light pulse (Fig. 3C) and by receptive field width.
At the onset of the stimulus, ON brisk transient cells
responded with a burst of spikes the rate of which typically exceeded
100 spikes/s. ON
cells fired throughout the stimulus at
a rate that rarely exceeded 10 spikes/s. The receptive field widths of
ON
cells were one-half to one-third that of neighboring
ON brisk transient cells (Fig. 3D). ON
cells were distinguished from ON sluggish and
ON brisk sustained cells by their mean effective stimulus
time course and autocorrelation shape and from ON DS cells
by their nonselective response to a sinewave grating drifting in one of
four orthogonal directions (DeVries and Baylor 1997
).
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In Fig. 3D, the spatial profiles of ON cells
and the ON center ganglion cells in three classes were
fitted with a generalized Gaussian surface and the centers and 1
ovals are shown. All plots were from the same retina. ON
cell profiles demonstrate a 2
center-center spacing,
similar to that previously reported for most classes of ganglion cells
in rabbit retina (DeVries and Baylor 1997
). The profiles
of cells in the three classes also were distributed with this
characteristic spacing, which would be corrupted if ovals from any two
classes were combined. In the mammalian retina, the receptive fields
and dendritic trees of ganglion cells in the same class often are
observed to evenly tile retina. The dendritic trees of ganglion cells
in different classes appear to form independent mosaics (Wassle
and Boycott 1991
).
ON cells may correspond to the "hybrid" ganglion
cells recognized by Caldwell and Daw (1978)
. Hybrid
cells were described as having properties intermediate between those of
ON brisk transient, ON DS, and ON
sluggish cells. Alternatively, the ON
cell could correspond to a type of ON sluggish cell because two
sluggish types were identified by Caldwell and Daw
(1978)
but only one type was recognized in a previous study
(DeVries and Baylor 1997
). In addition, there is a
possibility that the ON
cell could be a type of
displaced amacrine cell.
Mechanisms of correlated firing
The shape of a cell's cross-correlation function may provide
clues about the retinal circuits that produce the correlated spikes
(Brivanlou et al. 1998; Mastronarde
1983a
,b
; Meister et al. 1995
). Figure
4A shows the cross-correlation
functions for several pairwise combinations of ON brisk
transient and sustained cells in one retina. The functions have a
prominent central peak the temporal width of which at half-height was
5-9 ms. Single peaks in the cross-correlation function also were
characteristic of ON brisk transient-ON
pairs, ON brisk sustained pairs, ON
pairs,
ON brisk sustained-ON
pairs, and
OFF brisk transient-OFF brisk sustained cell
pairs (Table 2 and Fig.
5). One way in which a single central
peak might occur is if both ganglion cells are excited simultaneously
by a spike or another type of transient depolarization occurring in a
common presynaptic interneuron. The circuit for this "divergent"
mechanism is illustrated by the diagram in Fig. 4A. An
excitatory mechanism involving a presynaptic voltage transient followed
by a excitatory postsynaptic current is consistent with the narrow
temporal width of the central peak and assumes only a modest firing
rate in a common presynaptic cell (Mastronarde 1983a
).
An alternative explanation for the shape of the cross-correlation
function is that the ganglion cells receive tonic inhibition from a
common interneuron. Periodic gaps in the tonic inhibition would allow
both cells to fire at nearly the same time.
-Aminobutyric acid
(GABA) and glycine are the two main inhibitory transmitters in the
retina. However, an inhibitory mechanism involving a GABAergic
interneuron appears unlikely because 20-100 µM picrotoxin increased
the strength of rapid correlations in two retinas (4 ON
brisk transient pairs, mean change = 47%; 2 ON brisk
transient-brisk sustained pairs, 11%; 1 OFF brisk
transient pair, 55%). The role of glycinergic transmission could not
be assessed because strychnine dramatically increased activity in nearly all ganglion cells.
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Figure 4B shows the cross-correlation functions for three pairs of OFF brisk transient cells recorded at the same time as the cells shown in Fig. 4A. The distinctively shaped functions have two peaks separated by a trough at 0 ms. The apices of the peaks are at 2.1 ± 0.2 ms. In a larger sample consisting of 10 OFF brisk transient pairs from different retinas, peaks were displaced from the center by 2.27 ± 0.49 ms. A cross-correlation function consisting of two peaks centered about a trough at 0 ms could result if the two ganglion cells activated each other reciprocally. Thus a spike in one ganglion cell might trigger a spike in its neighbor after a delay of ~2 ms and vice versa. The circuit is illustrated by the diagram in Fig. 4B. If the distance between somas is taken as the distance that a spike must travel before it can be detected by the array, then the 2 ms interval indicates a propagation velocity of ~100 mm/s.
The results in Fig. 4 suggest that reciprocal correlations predominate
in OFF brisk transient cell pairs. Although the presence of
reciprocal firing in ON-center cells cannot be excluded, it is possible to place an upper limit on the percentage of correlated spikes that might occur through such a mechanism. Figure 5A
shows a range of peak shapes. On the left is a typical central peak shape for an ON-center cell pair. On the right is the shape
of the cross-correlation function for an OFF brisk
transient cell pair with a well-defined trough (obtained from Fig.
4B, middle). In between are the central peak shapes that
would result if 10-30% of the correlated events were produced by a
reciprocal mechanism, the remainder being generated by a divergent
mechanism. The shape of the central portion of a peak was described by
its peak ratio, which is the average height of the peak at ±2.25 ms
divided by the height at 0 ms. The peak ratios obtained when different
percentages of reciprocal and divergent spikes occur together are shown
to the left of each peak in Fig. 5A. Figure 5B
shows four cross-correlation functions obtained from cell pairs in
different retinas demonstrating a range of peak shapes. The two
functions on the left were from an OFF brisk
transient-OFF brisk sustained pair and an ON
brisk transient-ON pair, whereas the two on the right
were from OFF brisk transient cells. Because the
cross-correlation function peaks for pairs of OFF brisk
transient cells were not always centered at ±2.25 ms, the peak ratio
tends to underestimate the true extent of splitting. Figure
5C shows the peak ratios obtained from the cross-correlation
functions of >100 pairs. With the exception of OFF brisk
transient cell pairs, peak ratios were similar for all pairings and had
a mean value slightly less than one, consistent with a minor
contribution from a reciprocal mechanism. In contrast, about half of
the OFF brisk transient cells pairs had distinct splitting
(peak ratio >1.6), although, not all pairs showed troughs as
distinctive as those in Fig. 4B. In the other half of the
sample, the central trough was partially or fully obscured. Although a filled-in trough might suggest that divergent and reciprocal mechanism coexist in OFF brisk transient cell pairs, the central
trough may be obscured by mechanisms that do not involve divergent
spikes. These mechanisms will be addressed further in the
DISCUSSION.
Receptive field profiles of OFF brisk transient ganglion cells
The ability of spikes to flow between OFF brisk
transient cells suggests that a light stimulus that produces a spike in
one cell, the spike donor, could cause a spike in a neighboring
OFF brisk transient cell, the spike recipient. Because
potential spike donors are arrayed circumferentially around a central
recipient, the recipient cell might be expected to display an extended
receptive field "skirt." To search for this peripheral skirt, the
receptive field profiles of OFF brisk transient cells,
which have reciprocal correlations, were compared with those of
ON brisk transient cells, which lack reciprocal
correlations. This comparison is useful because the anatomic substrates
of the ON and OFF brisk transient cells, the
ON and OFF cells, have similarly sized
dendritic trees at the same eccentricity (Peichl et al.
1987
), and thus their receptive fields would be expected to
have similar profiles.
CHECKERBOARD STIMULUS.
Previous results (DeVries and Baylor 1997) suggested
that the receptive field profiles of OFF brisk transient
cells differed from those of cells in other classes in that they were
fitted poorly by a single generalized Gaussian surface. As shown in the next paragraph, the OFF brisk transient cell
receptive field profiles were better fitted by a sum of two Gaussian
surfaces.
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SINEWAVE GRATING STIMULUS.
Determining the mean effective stimulus provided a rapid method to find
the size and shape of many receptive fields. The generality of the
results obtained with this method were tested by stimulating with a
series of contrast reversal spatial sinewave gratings
(Enroth-Cugell and Robson 1966) the spatial period of
which at the retinal surface varied incrementally between 57 µm and
infinity. Previously, the profile centers of ON brisk
transient, ON brisk sustained, and OFF brisk
sustained cells were found to have similar widths when measured with
either spatial sinusoids or the checkerboard pattern (DeVries
and Baylor 1997
). Figure 7 plots
the area under the peak at the fundamental temporal frequency of the
stimulus (0.5 Hz) as a function of the spatial frequency of the
sinusoid (Hochstein and Shapley 1976
) for two
ON and two OFF brisk transient cells in the
same retina. Center and surround widths were determined by fitting the
results with an equation specifying the difference between two
unidimensional Gaussians (Eq. 4 in DeVries and Baylor 1997
; Enroth-Cugell and Robson 1966
). The fits
indicate that the centers of the OFF brisk transient cells
(
= 244 and 247 µm) were almost twice as wide as those of the On
brisk transient cells (
= 130 and 132 µm). In five retinas,
OFF brisk transient cell centers were larger than
ON brisk transient cell centers by a factor of 2.08 ± 0.33 (8 ON brisk transient and 11 OFF brisk
transient cells). One of the retinas additionally was bathed in 100 µM picrotoxin; this diminished input from the inhibitory surround and
permitted an adequate fit with a single Gaussian. The center widths of
two OFF brisk transient cell profiles were each 289 µm,
whereas that of a single ON brisk transient cell was 159 µm.
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Reciprocal firing can blur receptive field boundaries
Occasionally, pairs of OFF brisk transient cells had
>30% correlated spikes. When reciprocal firing occurred to this
extent, receptive fields could be extended markedly. Figure
8A shows the receptive field
profile of an OFF brisk transient cell along with the
receptive field center locations of five adjacent OFF brisk transient cells. The percentage of rapidly correlated spikes for the
cell the receptive field profile of which is shown versus the three
adjacent cells was 32.2, 18.4, and 16.9%. Figure 8B plots
the receptive profile of an ON brisk transient cell in the same retina, and the center locations of five adjacent ON
brisk transient cells. It is evident from Fig. 8 that the profile of the OFF brisk transient cell is considerably broader than
that of the ON brisk transient cell. Because the distance
between receptive field centers is similar for ON and
OFF brisk transient cells, the receptive field coverage
must be greater for OFF brisk transient cells. The
equivalent receptive field center was calculated for the ON
and OFF receptive fields shown in Fig. 8 as the cylinder that contained the same overall "volume" as the receptive field profile, with a height equal to the profile's peak. The radius of the
equivalent center for the OFF brisk transient cell was 370 µm whereas that for the ON brisk transient cell was 190 µm. Profile peaks amplitudes were similar. If the density of
ON and OFF brisk transient cells is assumed to
be equal (DeVries and Baylor 1997; Peichl et al.
1987
; unpublished observation), the OFF brisk
transient cells would have a coverage factor that is 3.8-fold greater
than that of the ON brisk transient cells. In the retina
shown in Fig. 8, the distance between the receptive field centers of
OFF brisk transient cells was ~1.2-fold greater than that
between ON brisk transient cells. The slight increase in
spacing reduces the coverage factor ratio to 3.1-fold. The increased
overlap suggests that reciprocally correlated spikes can significantly
extend the receptive field center of an OFF brisk transient
cell.
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DISCUSSION |
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While reciprocal firing should expand a ganglion cell's receptive
field center, an expansion due to this mechanism has not previously
been recognized. In rabbit retina, reciprocal spikes were observed in
OFF brisk transient ganglion cells but not in ON brisk transient cells or in the other classes of
ON and OFF center cells. Because ON
and OFF brisk transient cells (the rabbit cells) have
dendritic trees of comparable widths at a given eccentricity
(Peichl et al. 1987
), their receptive field profiles can
be usefully compared. In this comparison, the receptive field centers
of ON brisk transient cells were well fitted by a single Gaussian surface, whereas the profiles of OFF brisk
transient cells were wider overall and had two components, a central
Gaussian, similar in width to that of an ON brisk transient
cell's, and a peripheral skirt. The peripheral skirt is most likely
produced by the centripetal flow of signals from neighboring
OFF brisk transient cells.
The comparison between the receptive field profiles of rabbit
ON and OFF brisk transient cells rests on the
assumption that their anatomic substrates are the ON and
OFF cells described by Peichl et al.
(1987)
. In a recent study, rabbit ganglion cells with the
physiological properties of ON and OFF brisk
transient cells were injected with tracer (Muller and Dacheux
1997
). The injected cells had the morphological characteristics
of the previously described rabbit
cells (Peichl et al.
1987
). In the cat retina, it has been shown conclusively that
ganglion cells with the physiological properties of ON and
OFF Y cells are the same as morphological
cells
(reviewed in Wassle and Boycott 1991
). In common with cat Y cells, rabbit ON and OFF brisk transient
cells produce a burst of spikes during a step change in light intensity
(DeVries and Baylor 1997
). In addition, rabbit
ON and OFF brisk transient cells have a strong
second harmonic response during stimulation with inverting sinusoidal
gratings that have a high spatial frequency (DeVries and Baylor
1997
; unpublished observation), a defining characteristic of
cat Y cells (Hochstein and Shapley 1976
). Finally, the
occurrence of reciprocally correlated spikes in OFF brisk transient cells is characteristic of Y cells in the cat
(Mastronarde 1983a
,c
). Based on the anatomic and
physiological results in the rabbit and the similarities with cat Y
cells, it is likely that the rabbit ON and OFF
brisk transient cells correspond to the morphological ON
and OFF
cells.
Peichl et al. (1987) used Lucifer yellow injections to
compare the dendritic tree widths of rabbit ON (inner) and
OFF (outer)
cells. When measured at the same retinal
eccentricity, no systematic differences were found. A difference in
width approaching twofold would have been noticed because it would have
produced a fourfold difference in the anatomic coverage factor, given a
similar percentage of inner and outer cells (44:56%). Rather, the
coverage factor for ON cells was 1.45, whereas that for
OFF cells was 1.85. A study of cat ON and
OFF
cells produced similar results (Wassle et
al. 1981
). A small systematic difference in the widths of
rabbit ON and OFF
cells cannot be excluded.
Indeed, a comprehensive study of midget ganglion cells in human retina
shows that ON cells have tree widths that are a factor of
1.3 greater than those of OFF cells (Dacey
1993
). However, it is unlikely that subtle differences in
rabbit
cell dendritic tree width could account for the observed twofold difference in receptive field profile and the fourfold difference in coverage factor.
Previous qualitative studies did not find a difference in receptive
field width between rabbit ON and OFF brisk
transient cells (Caldwell and Daw 1978; Levick
1967
). A more recent quantitative study (Amthor et al.
1989a
) also failed to show a systematic difference, although
the sample size was limited. Similarly in cat retina, detailed
measurements have shown that ON and OFF Y cell
receptive fields have the same width at a given eccentricity when
measured under mesopic conditions (Peichl and Wassle 1979
,
1983
). However, receptive fields may enlarge when ambient light
intensity is reduced (Barlow et al. 1957
; see also
Muller and Dacheux 1997
), and this increase may occur
disproportionately for OFF Y cells (Peichl and
Wassle 1983
).
Although previous studies have not recognized a receptive field skirt,
the skirt is not likely to result from a hidden bias in the present
measurement because two different methods were used. In the first
method, the retina was stimulated with a randomly varying checkerboard
pattern and a ganglion cell's mean effective stimulus was computed by
cross-correlation with the spike train. In most cases (41/47), the
receptive field center profile had central and flanking components. The
two-component profile did not vary when the checkerboard square size
was changed over a significant range (100-160 µm per side) when
compared with the receptive field width (1 radius of 100-250 µm)
(DeVries and Baylor 1997
). In the second method, the
retina was stimulated with spatial sinewave gratings, and the
fundamental response was plotted as a function of stimulus frequency.
The receptive field profiles of OFF brisk transient cells
were found to be twice as wide as those of neighboring ON
brisk transient cells. Although the increase in width is consistent
with having two central components, the two central components were not
clearly resolved. In a few cases (6/47), the checkerboard stimulus also
failed to resolve two center components. In these experiments, the
OFF brisk transient cell receptive field center width was
twice that of the neighboring ON brisk transient cells.
There are several reasons why the peripheral skirt may not have been
noted in previous studies. First, with the multielectrode array, it is
possible to compare the receptive fields of adjacent ganglion cells
that are measured simultaneously. Differences in receptive field shape
might be overlooked when receptive fields are measured serially at
slightly different eccentricities in one animal or in different animals
during separate experiments. Second, qualitative measures of receptive
field width were used in older studies (Caldwell and Daw
1978; Levick 1967
). Thus the transition from a
strong field center to a weaker, same-sign skirt might have been
missed. Third, ganglion cells have complex receptive field structures.
For example, detailed measurements of receptive field shape indicate
that most cat Y cell receptive fields have an extensive same-sign
"far periphery" (Derrington et al. 1979
; McIlwain 1966
), along with the classical center and
opponent surround. The dynamic nature and complexity of the Y cell
receptive field suggest that a same-sign skirt could have been present
during previous studies but not recognized.
Although most OFF brisk transient cell receptive fields had
a peripheral skirt, a distinctive split in the cross-correlation function was seen in less than half of OFF brisk transient
cell pairs. In the remaining cases, the trough largely was obscured. Although spikes that are generated through a divergent mechanism could
fill in the trough, other mechanisms also might contribute. For
example, two coupled OFF brisk transient cells could
activate each other reciprocally but also might be activated
simultaneously by a spike in a third OFF brisk transient
cell, the three cells being located at the apices of a triangle. In
effect, a divergent mechanism might exist solely among OFF
brisk transient cells. A second possibility is that the "filling
in" could result from structure within the spike train of the
individual OFF brisk transient cell. Both ON
and OFF brisk transient spike trains consisted of doublets
or triplets in which spikes occurred at intervals of 2.3-2.5 ms
(DeVries and Baylor 1997). If the first spike in an OFF brisk transient cell's train activated a spike in a
neighboring cell with a 2.2-ms delay, the spike in the neighbor is
likely to be temporally coincident with the subsequent spike in the
initiating cell. The coincidence would add to the events in the center
of the cross-correlation function. Occasionally, an OFF
brisk transient cell fired mostly "singlets" as judged by its
autocorrelation function. The most distinctive splits in the
cross-correlation function were observed in such pairs of
OFF brisk transient cells (Fig. 4). Pairs of ON
brisk transient cells that fired singlets showed a very minimal split
in the central peak.
Mechanism of correlated activity
RECIPROCAL.
The injection of the low-molecular-weight tracer neurobiotin into cat
ganglion cells and their likely homologs in monkey (Dacey
and Brace 1992
), rabbit (Vaney 1991
; Xin
and Bloomfield 1997
), and ferret (Penn et al.
1994
) reveals that
cells are dye coupled to amacrine cells
and neighboring
cells. It has been suggested that tracer coupling
between
cells occurs through amacrine cells (Dacey and Brace
1992
; Jacoby et al. 1996
; Vaney 1991
) because tracer stains the local amacrine cells more
intensely than neighboring ganglion cells. In addition, gap junctions
have been observed in ultrastructural studies between monkey parasol cells and the dye-coupled long-range amacrine cells but are apparently rare between parasol cells (Jacoby et al. 1996
). Either
direct
cell coupling by gap junctions or coupling through an
intermediary amacrine cell are consistent with the slow rate of
transmission (100 mm/s) and relatively weak synchrony observed in
rabbit OFF brisk transient cells.
DIVERGENT.
The interneurons that produce the divergent correlations have not
been identified. An excitatory interneuron might make either electrical
or chemical synapses with ganglion cells. Electrical synapses have been
shown to mediate divergent correlations in salamander retina by using
blockers of chemical synaptic transmission (Brivanlou et al.
1998). However in the mammalian retina, the pattern of
connectivity revealed by tracer studies does not match the pattern of
"connectivity" predicted by correlated activity. For example, cat
cells and their likely homologs in monkey and ferret are not dye
coupled to other
cells; nor are they coupled to nearby amacrine or
ganglion cells (Dacey and Brace 1992
; Penn et
al. 1994
; Vaney 1991
). Yet the spikes in cat
X/
cells are correlated with those in neighboring X/
and Y/
cells. In rabbit retina,
ganglion cells are dye coupled
homologously to neighboring
cells but apparently not to other types
of ganglion cells. Yet the spikes in rabbit ON brisk
transient cells are correlated with those in neighboring ON
brisk transient, brisk sustained, and
cells. Thus excitatory
chemical synapses are the likely source of the shared input to
mammalian ganglion cells.
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CONCLUSION |
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The ability of OFF brisk transient cells to exchange
spikes blurs the boundaries between their receptive fields. This
blurring seems counterintuitive because it would degrade the spatial
resolving power of the ganglion cell lattice. However, resolving
spatial detail may be only one of the tasks for which OFF
brisk transient cells are optimized. One possibility is that weak
electrical coupling would ensure that neighboring ganglion cells fire
together when a stimulus covers their receptive fields
(Neuenschwander and Singer 1996). The benefit of
correlated firing in this instance might be that the short-lived
excitatory postsynaptic potentials (5-10 ms) (Jonas et al.
1993
; Zhang and Trussell 1994
) produced by
neighboring ganglion cells could overlap and sum in a postsynaptic
lateral geniculate neuron. Otherwise, a dim uniform stimulus might
produce spikes in neighboring cells that were separated by as much as 50 ms (Mastronarde 1983b
; Nakatani et al.
1991
), and simple summation in a postsynaptic cell might be
precluded. ON and OFF brisk transient cells
both generate rapidly correlated spikes but the mechanisms differ. It
is tempting to speculate that this difference is due to constraints on
retinal wiring rather than differences in ultimate function.
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ACKNOWLEDGMENTS |
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The author thanks Dr. Denis A. Baylor for advice and support and R. Schneeveis for technical assistance.
This work was supported by National Eye Institute Grant EY-05750, the Ruth and Milton Steinbach Fund, National Eye Institute Postdoctoral Fellowship EY-06387, and a fellowship from The Bank of America-Giannini Foundation.
Present address and address for reprint requests: Dept. of Ophthalmology and Visual Science 7.024, University of Texas Houston Health Sciences Center, 6431 Fannin, Houston, TX 77030.
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FOOTNOTES |
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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 19 March 1998; accepted in final form 14 October 1998.
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REFERENCES |
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