The Journal of Neuroscience, July 30, 2003, 23(17):6768-6777
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Gap Junctional Coupling Underlies the Short-Latency Spike Synchrony of Retinal 
Ganglion Cells
Edward H. Hu and
Stewart A. Bloomfield
Departments of Ophthalmology, Physiology and Neuroscience, New York
University School of Medicine, New York, New York 10016
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Abstract
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We examined whether coupling between neighboring
-type ganglion cells
(-GCs) in the rabbit retina underlies their synchronous spike activity.
Simultaneous recordings were made from arrays of -GCs to determine the
synchrony of both spontaneous and light-evoked spike activity. One cell within
each array was then injected with the biotinylated tracer Neurobiotin to
determine which of the cells were coupled via gap junctions. Cross-correlation
analyses indicated that neighboring off-center -GCs maintain
short-latency (
2.5 msec) synchronous spiking, whereas the spontaneous
spike activities of on-center-GC neighbors are not correlated. Without
exception, those off-center-GCs showing synchronous spiking were found
to be tracer coupled to both amacrine cells and neighboring
off-center-GCs. In contrast, on-center -GCs were never tracer
coupled. Furthermore, whereas spikes initiated in an off-center -GC
with extrinsic current injection resulted in short-latency synchronized
spiking in neighboring off-center -GCs, this was never seen between
on-center -GCs. These results indicate that electrical coupling via gap
junctions underlies the short-latency concerted spike activity of neighboring
-GCs.
Key words: retina; gap junctions; spike synchrony; ganglion cells; electrophysiology; coupling
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Introduction
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As the output neurons of the retina, ganglion cell responses represent the
final, integrated visual signals conveyed to higher brain centers. Much of our
knowledge of ganglion cell physiology stems from single-unit recordings in
which the light-evoked activity of individual neurons are analyzed
sequentially. Following this strategy, investigators have described over 10
physiological classes of ganglion cell, including those displaying simple,
concentric center-surround receptive fields or complex trigger features such
as direction and orientation selectivity
(Kuffler, 1953
;
Barlow et al., 1964;
Levick, 1967;
Caldwell and Daw, 1978).
More recently, multielectrode recordings have been used to study the
distributed activity among ganglion cell arrays. Neighboring ganglion cells in
a number of species show concerted firing patterns in both their spontaneous
and light-evoked discharges (Arnett and
Spraker, 1981; Mastronarde,
1983a,b,c,
1989;
Meister et al., 1995;
Brivanlou et al., 1998;
DeVries, 1999). This concerted
spike activity ranges from a relatively loose synchrony, reflected in broad
cross-correlograms spanning tens of milliseconds, to narrowly synchronized
spiking with latencies of <3 msec. The findings that concerted firing may
account for up to one-half of all of the retinal spike activity, coupled with
the high temporal precision in which retinal signals are conveyed to central
targets, suggest that synchronous spikes play an important role in encoding
visual information (Castelo-Branco et al.,
1998; Rager and Singer,
1998; Schnitzer and Meister,
2003).
Synchronous spiking can arise from a number of synaptic circuits, including
common excitatory or inhibitory inputs to neighboring cells. Mastronarde
(1983c) first described
synchronous activity between neighboring Y-type ganglion cells [the
physiological substrate of -type ganglion cells (-GCs)], showing
that an antidromically evoked spike in one cell leads to the generation of a
spike in a neighboring Y-cell with a latency of 0.5-1.0 msec. Antidromic
activation precluded common synaptic inputs as the cause of the spike
synchrony, whereas the relatively short latency argued against the involvement
of chemical synaptic transmission. Mastronarde thus reasoned that
short-latency concerted firing must reflect direct electrical coupling between
neighboring cells. Nearly 10 years later, Vaney
(1991) showed that -GCs
are tracer coupled to one another and to amacrine cells, thus providing the
substrate for electrical coupling. Interestingly, Brivanlou et al.
(1998) reported that
short-latency synchrony between neighboring ganglion cells in salamander
retina survived the presence of cadmium chloride, presumed to block chemical
transmission, supporting a role for electrical coupling. However, synchronous
activity could not be abolished with gap junction blockers, nor was it
established whether the recorded ganglion cells in fact maintained gap
junctions.
In this study, we measured the concerted activity of -GC arrays and
subsequently labeled them with the tracer Neurobiotin to determine their
coupling patterns. Our results indicate unequivocally that short-latency
synchronous spiking generated spontaneously or evoked with light or extrinsic
current occurs only in neighboring -GCs that are tracer coupled.
Furthermore, whereas neighboring off-center -GCs were tracer coupled
and showed spike synchrony, on-center -GCs were never coupled nor
displayed short-latency concerted activity. These results indicate a
fundamental difference in the electrical coupling and resulting activity of
the -GC subtypes in the mammalian retina.
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Materials and Methods
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Preparation. The experimental procedures used in this study have
been described extensively previously (Hu
et al., 2000). Briefly, adult New Zealand White rabbits
(Oryctolagus cuniculus) (1.5-2.5 kg body weight) were anesthetized
with an intraperitoneal injection of 40% ethyl carbamate (2.0 gm/kg body
weight) and a local injection of 2% lidocaine hydrochloride to the eyelids and
surrounding tissue. The eye was then removed under dim red illumination and
hemisected 1 mm posterior to the ora serrata. The vitreous humor was
removed with ophthalmic sponges, and the resultant retina eyecup was everted.
Four radial cuts, 4 mm in length, were made peripherally in the retina
eyecup in a Maltese cross-configuration to flatten it before placement in a
superfusion chamber. The chamber was then placed in a light-tight Faraday cage
and superfused at a flow rate of 20 ml/min with a mammalian Ringer's solution
(Bloomfield and Miller, 1982).
The superfusate was kept at a constant temperature of 34°C, with
oxygenation and pH 7.4 maintained by bubbling with a gaseous mixture of 95%
O2-5% CO2. Retinas were maintained in complete darkness
for 1 hr before initiation of experimentation. After enucleations, animals
were killed with an intracardial injection of ethyl carbamate (5 ml of a 40%
solution).
The superfusion chamber was mounted on the stage of an upright light
microscope (BX501WI; Olympus Optical, Tokyo, Japan). A 900 nm cutoff filter
allowed transmission of infrared (IR) light from below the stage and then up
through a condenser and the glass coverslip mounted in the superfusion chamber
base. An IR-sensitive CCD camera (VE-1000; Dage-MTI, Michigan City, IN)
captured the retinal image that was displayed on a video monitor outside the
Faraday cage. Still video frames of neurons in the ganglion cell layer (GCL)
were acquired using a video capture card (Snazzi; Dazzle Multimedia, Fremont,
CA). A motorized drive operated by foot pedals allowed for fine focal
adjustments to be made from outside the cage. On either side of the stage,
triple-axis motorized micromanipulators (DC3001R; World Precision Instruments,
Sarasota, FL) were operated by controllers (MS314; Märzhäuser,
Wetzlar, Germany) from outside the cage. One micromanipulator supported the
headstage of an isolated AC differential amplifier (DAM80i; World Precision
Instruments) for extracellular recordings. The body of a high acceleration
linear piezo stepper (Inchworm motor IW-700; controller ULN 6000; Burleigh
Instruments, Fishers, NY) was attached to the other micromanipulator. An
adapter fitting on the tip of the Inchworm motor allowed connection to either
a second extracellular headstage or to the headstage of a high-impedance
amplifier for intracellular recordings (Axoprobe 1A; Axon Instruments, Foster
City, CA). Thus, simultaneous dual extracellular and intracellular recordings
could be made from neighboring cells. Data were digitized online with an
analog-to-digital board (Digidata 1200; Axon Instruments) and stored on a
personal computer.
Visualization of ganglion cells. To visualize
cells, superfusion of the retina was temporarily halted, and three to five
drops of 0.1% Azure B (dissolved in modified Ames medium with 25 mM
sodium bicarbonate without glucose) were placed on the retinal surface. After
60-90 sec, the superfusion was resumed, and the Azure B was suctioned off the
retina and discarded. Optimal staining density occurred within 10-15 min, and
cells remained visible for the entire duration of the experiment (up to 10
hr).
Intracellular and extracellular recordings. Intracellular
recordings were obtained from neurons using microelectrodes fashioned from
standard borosilicate glass tubing (outer diameter, 1.2 mm; inner diameter,
0.6 mm). For most studies, electrodes were filled at their tips with 4%
N-(2-amino-ethyl)-biotinamide hydrochloride (Neurobiotin) (Vector
Laboratories, Burlingame, CA) in 0.1 M Tris buffer, pH 7.6, and
then backfilled with 4 M potassium chloride. Final DC resistances
of these electrodes ranged from 350 to 450 M
. After physiological
characterization of a cell, Neurobiotin was injected into the cell, with a
combination of sinusoidal (3 Hz; 0.8 nA; peak-to-peak) and DC current (0.4 nA)
applied simultaneously; this method allowed for passage of tracer through the
microelectrode without polarization.
Extracellular recordings were obtained from neurons using carbon fiber
microelectrodes constructed with 10 µm diameter fibers with 25 µm
exposure (World Precision Instruments). The compact construction of these
electrodes (total length, 40-50 mm) provided maximum stability and could be
fitted easily below the water-immersion objectives. After extracellular
recordings, neurons were impaled with an intracellular microelectrode and
labeled with Neurobiotin for subsequent morphological identification. It
should be noted that the carbon electrodes effectively recorded spikes only
from relatively large ganglion cells and only when placed directly atop their
somata. Thus, there was no ambiguity about the identity of the ganglion cell
being recorded.
A green light-emitting diode (
max, 468 nm) focused onto
the retinal surface provided a low scotopic (4.7 Rh* · rod-1
· sec-1), full-field light stimulus to the dark-adapted
retina. During recordings of light-evoked spike activity, the frequency of
stimulation was 1 Hz with a 0.5 sec duration.
Histology. After a physiological experiment, the retina was fixed
immediately in a cold (4°C) fixative solution of 4% paraformaldehyde-0.1%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 12 min. The
retina was then detached, trimmed, fixed onto a gelatinized glass coverslip,
and left in fixative overnight at 4°C. Retinas were washed for 4-5 hr in
10 mM sodium PBS (9% saline; pH 7.6) and then reacted with the
Elite ABC kit (Vector Laboratories) and 1% Triton X-100 (Sigma, St. Louis, MO)
in PBS overnight at 4°C. Retinas were processed for peroxidase
histochemistry using 3-3'diaminobenzidine (DAB) with cobalt
intensification, and then dehydrated, cleared, and flat-mounted in Permount
(Fisher Scientific, Houston, TX).
To determine the level at which dendritic processes stratified in the inner
plexiform layer (IPL), we examined Neurobiotin-labeled cells in flat-mount
under a 100x objective. The borders of the IPL were determined by the
location of amacrine and ganglion cell bodies using Nomarski interference
contrast optics. The position of the outer margin of the IPL next to the
amacrine cell bodies was defined as 0, whereas the vitreal border of the IPL
was defined as 100. The position of cellular processes in the IPL was
determined using a precision micrometer and given a value from 0 to 100.
Multiple measures were made for a single cell to elucidate any variations in
stratification throughout its extent.
Measurements of soma diameters were made using a 100x oilimmersion
objective coupled with an eyepiece graticule. An unexpected advantage of using
Azure B was that all of the cells maintained a bluish label even after
histological processing for Neurobiotin. This allowed us to visualize and
measure the soma diameter of both tracer-coupled and uncoupled ganglion
cells.
Analysis of spike activity. The spike trains were sorted and
timestamped off-line (off-line spike sorter; Plexon, Dallas, TX). The
cross-correlation function between two spike trains was computed by
histogramming all of the time differences between a spike from one cell and a
spike from the other cell (NeuroExplorer; Plexon). Significance was determined
by the 99% confidence limits.
For light-evoked spiking, a shift predictor correction procedure
(Perkel et al., 1967)
separated the features of the cross-correlation functions that are related to
neuronal interactions from those resulting from the coactivation of the cells
by the light stimulus. The shift predictor was generated in the same manner as
a normal cross-correlation function except that one of the spike trains was
shifted over one or more stimulus intervals. Here, the shift predictor was
created as the arithmetical mean of all of the possible interval shifts. The
shift predictor was then subtracted from the original cross-correlation
function to generate the shift-predicted function, which included only those
correlations independent of the light stimulus.
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Results
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The data presented here represent 1,166 recordings made from -GCs of
which 120 were injected intracellularly with Neurobiotin and analyzed
morphologically. Recordings were made throughout the retina; cell
eccentricities ranged from 0.4 to 4.0 mm ventral to the optic disk.
Identification of ganglion cells
To visually target -GCs for electrophysiological recordings it was
necessary to unequivocally identify them in the superfused rabbit retina
eyecup. As reported previously, Azure B was found to stain somata within the
GCL of the living retina (Hu et al.,
2000). These included a mosaic of cells easily identified by their
regular spacing and particularly large somata when viewed under IR
illumination (Fig.
1A,C). After histological processing and counterstaining
with hematoxylin, it was evident that these cells displayed the very largest
cell bodies (Fig. 1B).
Although a comparison of Figure 1,
A and B, would suggest that Azure B stained only
a subset of cells, examination under higher magnification indicated that all
of the cell bodies were indeed labeled, but some smaller somata were very
lightly stained and were thus invisible in the video micrograph. In
preliminary experiments, the largest cells were targeted for intracellular
injection with Neurobiotin to determine their soma-dendritic architecture
(Fig. 1C). We found
the morphological features of these cells consistent with those described for
-GCs in a number of mammalian species, including the rabbit
(Boycott and Wässle, 1974;
Wässle et al., 1975,
1981;
Peichl et al., 1987;
Peichl, 1991). These features
included a large, ovoid soma that emitted four to six stout, primary dendrites
that branched successively in a radiate manner to form a large, circular
dendritic field with acutely branched processes that rarely overlapped
(Fig. 1D). The
dendritic arbors unistratified in either sublamina a or b of
the inner plexiform layer (IPL), which was subsequently found to correspond to
the off- or on-center receptive fields of the cells, respectively
(Famiglietti et al., 1977;
Nelson et al., 1978;
Peichl and Wässle, 1981;
Bloomfield and Miller, 1986).
Indicative of a single class of cells, we found a decrease in cell density at
higher retinal eccentricities coupled with a dramatic enlargement of both the
soma and the dendritic arbor of individual cells. However, despite differences
seen in size, the overall dendritic architecture of cells within this class
was preserved across the retina.
Interestingly, we found that the somata of some -GCs lay more
vitreal than others and were labeled a bit more darkly with Azure B. Although
similar in size and shape, the dark and light -GCs appeared to form
regular, but independent, arrays. Subsequent physiological experiments
indicated that the more darkly labeled -GCs showed off-center
physiology, whereas the lighter cells were on-center. These findings confirm a
previous finding that on- and off-center -GCs achieve a uniform and
independent coverage of the retina
(Wässle et al.,
1981).
Together, the (1) regular mosaics, (2) distinctly large somatic size, and
(3) characteristic dendritic architecture provided strong support for the
positive identification of these neurons as -GCs. The prominence of the
-GC somata in the retinal preparation indicated that they could be
easily targeted for physiological studies.
Physiology of ganglion cells
The -GCs have been identified as the morphological equivalent of the
physiological, brisk-transient Y-cells in the cat retina
(Cleland et al., 1975;
Peichl and Wässle, 1981;
Saito, 1983;
Fukuda et al., 1984;
Stanford and Sherman, 1984).
Extracellularly recorded -GCs in the rabbit showed transient
light-evoked spike activity, consistent with the data from cat
(Fig. 2A,B). The
-GCs could be divided into on- and off-center categories on the basis
of the transient burst of spike activity at light onset or offset,
respectively. Intracellular recordings showed some sustained slow potential
components in the responses of both on- and off-center cells, but these were
not translated into any sustained spike discharges
(Fig. 2C,D). We rarely
recorded antagonistic surround-mediated responses from -GCs because of
the fact that retinas were maintained in the dark-adapted state when surround
activity is lost (Barlow et al.,
1957; Peichl and Wässle,
1983; Muller and Dacheux,
1997).
On- and off-center -GCs displayed very different patterns of
spontaneous spike activity. The spontaneous spikes of on-center -GCs
occurred predominantly as spike doublets with irregular frequency
(Fig. 3A).
Autocorrelation functions showed that these spike doublets had interspike
intervals of 3-5 msec (Fig.
3B). In contrast, spontaneous activity of off-center
-GCs consisted of single spikes spaced at least 10 msec apart
(Fig. 3C,D).
Correlated spike activity between neighboring ganglion
cells
The primary objective of this study was to determine whether electrical
coupling underlies the synchronous spike activity between neighboring
-GCs. To this end, dual, simultaneous recordings were made from 523
pairs of -GCs in 62 arrays, after which the central cell within a
recorded array was injected with Neurobiotin to determine which cells were
tracer coupled.
Cross-correlation analysis of the light-evoked spike activity from
neighboring on-center -GCs typically showed a single peak at time 0
with a width of 40-80 msec (Fig.
4A). In contrast, cross-correlations of the light-evoked
activity recorded from pairs of neighboring off-center -GCs
characteristically showed two peaks with a trough at time 0 indicative of
reciprocal interactions; the peaks showed latencies of ±2.0-2.5 msec
(Fig. 4B). These peaks
were superimposed on the same broad peak seen for on-center -GCs. As
expected, the light-evoked activity of neighboring on- and off-center
-GCs was negatively correlated.
It is plausible that the concerted spiking seen for -GC neighbors
arises independently, the result of the activity of each cell being
synchronized to the common light stimulus. To eliminate this possible
mechanism, we performed a shift prediction analysis on the light-evoked spike
trains of neighboring -GCs (Perkel
et al., 1967). After this analysis, the robust unimodal
distribution for on-center cells remained but now fell below the 99%
confidence level, thus indicating that the synchrony was linked to the light
stimulus, consistent with common excitatory synaptic activation via bipolar
cells. In contrast, the bimodal distribution for off-center cells remained the
only components above the 99% confidence interval, indicating that these
correlations reflected synchrony independent of the light stimulus
(Fig. 4C,D).
A more striking difference was found between the cross-correlation
functions of the spontaneous spike activities of on- and off-center -GC
pairs maintained in complete darkness. The spontaneous activity of neighboring
on-center -GC pairs showed no correlated spike activity
(Fig. 5A-C). In
contrast, the spontaneous activity of the neighboring off-center -GC
pair showed the same bimodal cross-correlation distribution seen for
light-evoked activity, consisting of a trough at 0 msec and peaks with
latencies of ±2-2.5 msec (Fig.
5D-F). No correlations were found between the spontaneous
activities of on- and off-center -GCs.
Tracer-coupling patterns
After physiological recordings, 120 individual -GCs were injected
with Neurobiotin to determine their tracer-coupling patterns. We found that
the coupling for on- and off-center -GCs differed dramatically. The
tracer-coupling pattern of off-center -GCs typically showed an
extensive array of over 100 small somata lying within both the GCL and
proximal inner nuclear layer and a local ring of three to eight ganglion cells
with large somata similar in size to that of the injected -GC
(Fig. 6A). The number
of coupled ganglion cells in the local ring decreased with eccentricity
because of the decrease in overall -GC density. Previous studies have
shown that the small cell bodies belong to at least two classes of amacrine
cells, including one with long-range dendritic arbors extending beyond 1 mm
(Vaney, 1991,
1994;
Dacey and Brace, 1992;
Penn et al., 1994;
Xin and Bloomfield, 1997).
Unfortunately, Neurobiotin usually failed to label the dendritic arbor of
coupled ganglion cells, and so it was not possible to unequivocally identify
these coupled neurons as -GCs. However, several pieces of evidence
indicate that -GCs are indeed coupled to neighboring -GCs.
First, both the injected and tracer-coupled ganglion cells formed a regularly
spaced array indicative of cells within a single morphological class. Second,
like the injected -GC, the somata of the tracer-coupled ganglion cells
were larger than those of any neighboring cells in the GCL
(Fig. 7A). Third, the
soma sizes of the tracer-coupled ganglion cells, which were generated from
injections made within 0.4-4.0 mm of the visual streak, all fell within the
range previously described for -GCs in the rabbit retina
(Peichl et al., 1987)
(Fig. 7B). Finally, on
occasion, the proximal dendrites of a tracer-coupled ganglion cell could be
visualized and displayed a radiate branching pattern consistent with that of
an -GC. Our conclusion that -GCs in rabbit retina are coupled
homologously to each other is consistent with tracer-coupling patterns
established for -GCs in a number of mammalian retinas
(Vaney, 1991;
Dacey and Brace, 1992,
Penn et al., 1994;
Xin and Bloomfield, 1997)
In striking contrast, on-center -GCs never showed evidence of tracer
coupling after injection of a single cell with Neurobiotin
(Fig. 6B). Despite the
clear difference in coupling pattern, the completeness and intensity of
Neurobiotin labeling was identical for on- and off-center -GCs.
Furthermore, the different coupling patterns were found even for on- and
off-center -GCs labeled in the same retina under identical dark-adapted
conditions (Fig. 6C).
Thus, the different tracer-coupling patterns of on- and off-center -GCs
could not be explained by differences in the adaptational state of the retina
nor any technical problems related to Neurobiotin labeling.
Synchronous spike activity correlates to tracer coupling of
ganglion cells
The data described above clearly indicate a general correlation between
coupling and spike synchrony across the -GC population: off-center
-GCs show short-latency spike correlations and tracer coupling, whereas
on-center cells show uncorrelated spontaneous spiking and are uncoupled. To
study this correlation further, we determined whether spike synchrony between
pairs of off-center -GCs occurred only for those that were tracer
coupled.
In these experiments, recordings were made continuously from a central
reference off-center -GC while a second electrode was moved
systematically to record the activity of other offcenter -GCs in the
array. This resulted in a series of simultaneous, dual recordings from a local
array of -GCs, all referenced to a particular cell. The reference
-GC was then injected with Neurobiotin to determine which of the
-GCs in the array were tracer coupled to it.
Figure 8A shows an
array of off-center -GCs in which the reference cell (cell 1) was
injected with Neurobiotin after physiological recordings. Dual extracellular
recordings of the spontaneous spike activity were made between cell 1 and four
nearby off-center -GCs (cells 2-5). The cross-correlation functions
were subsequently generated for each cell pair
(Fig. 8B). The darkly
tracer-coupled cells 2 and 3 showed spike activity that was highly
synchronized to that of the reference cell (cell 1) as indicated by the
short-latency (±2.5 msec) bimodal peaks in the cross-correlation
functions (Fig. 8B).
Cells 4 and 5 were not tracer coupled to cell 1, nor did they show any
correlated spontaneous spike activity with cell 1.
The spontaneous spike activity of a total of 102 pairs of off-center
-GCs within 20 arrays was recorded simultaneously in dark-adapted
retinas. We computed the cross-correlation function comparing the spike
activity for each pair. We found 85% (47 of 55) of nearest neighbor pairs to
have significant (above the 99% confidence level) correlated spike activity.
Without exception, we found that every pair of nearest neighbor -GCs
that showed synchronized spiking were also tracer coupled. In contrast, we
found that second-tier -GC neighbors in the dark-adapted retina were
never tracer coupled, nor did their cross-correlation functions show any
synchrony between their spontaneous spike activities.
Synchrony of spikes evoked with extrinsic current injection
In a final set of experiments, we determined whether spikes generated with
extrinsic current into an -GC could evoke synchronous spikes in a
neighboring -GC. In these experiments, the activity of on-center and
off-center -GC pairs was monitored with intracellular and extracellular
recording electrodes (Fig.
9A,B). Spikes were then evoked in one of the -GCs
by injection of current through the intracellular electrode. Although positive
current injection effectively induced spiking in -GCs, we found that
injection of negative current consistently generated single anodal break
spikes whose latencies could be compared with those of spikes in a neighboring
-GC (Fig.
9C,D). We found that 22% (121 of 550) of anodal break
spikes generated in off-center -GCs resulted in a spike in a
neighboring off-center -GC with a latency of <5 msec. In contrast,
only 0.04% (8 of 200) of anodal break spikes in on-center -GCs were
paired with a spike in a neighboring -GC with a latency of <5 msec.
Moreover, a comparison of the short-latency synchronized spiking of off-center
cells after current injection showed a peak latency of 2.4 msec
(Fig. 9E), thus
matching those of the cross-correlation functions described above for the
spontaneous and light-evoked activities of off-center cells. No similar
pattern was found for on-center cells, suggesting that the few spike pairs
with short latencies occurred simply by chance
(Fig. 9F).
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Discussion
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Tracer-coupled cells show short-latency spike synchrony
A number of studies across several species have described concerted spike
firing between retinal ganglion cell neighbors
(Arnett and Spraker, 1981;
Mastronarde,
1983a,b,c,
1989;
Meister et al., 1995;
Brivanlou et al., 1998;
DeVries, 1999). Three basic
types of correlated spike activity exist, each occurring on a different time
scale. Two are characterized by either a medium or broad cross-correlation
function with a single peak, suggesting that the neighboring cells are
synchronized by excitation derived from a common presynaptic input. The other,
which was extensively studied here, is recognized by a narrow bimodal
cross-correlation function with a trough at 0 msec and peaks at
±0.5-2.5 msec. The bimodal distribution and short latency of these
peaks suggests reciprocal activity resulting from direct electrical coupling
(Mastronarde, 1983c). The
finding of extensive tracer-coupling patterns for many ganglion cells,
including -GCs, in mammalian retina provides the morphological
substrate for this electrical coupling (Vaney
1991,
1994;
Dacey and Brace, 1992;
Penn et al., 1994;
Xin and Bloomfield, 1997). By
combining the physiological and morphological approaches of these previous
studies, we showed directly, for the first time, that only tracer-coupled
-GCs show short-latency synchrony of spontaneous, light-evoked, or
current-evoked spike activity.
The finding that not all of the -GCs showed tracer coupling was
fortuitous, in that the uncoupled -GC population formed an internal
control to test for spike synchrony. In fact, a previous report from our
laboratory described variability in -GC tracer-coupling patterns, but
it was unclear whether this reflected different subpopulations of -GCs
or light-induced coupling changes related to alterations in adaptational state
(Xin and Bloomfield, 1997).
The present results indicate that the variability in -GC coupling is
related to their on- and off-center physiology: off-center -GCs are
tracer coupled, whereas on-center -GCs are not. Uncoupled on-center
-GCs and coupled offcenter -GCs were found side-by-side in the
same retinas under identical adaptational conditions. Thus, the difference in
coupling suggests a fundamental dichotomy in the organization of the on- and
off-center subtypes, whereby the former simply lack gap junctions. Support for
this notion comes from a recent finding of GABA accumulation, presumably
resulting from permeation through gap junctions made with GABAergic amacrine
cells, in off-center, but not on-center, -GCs
(Marc and Jones, 2002).
Consistent with the difference in tracer-coupling patterns of -GCs,
we found that off-center -GCs displayed short-latency spike synchrony,
whereas on-center -GCs did not. Spontaneous, light-evoked, and
current-evoked spiking of off-center -GCs showed narrow synchrony with
latencies of 2.0-2.5 msec. The bimodal distribution of their cross-correlation
functions suggested reciprocal activation. In contrast, neighboring on-center
-GCs showed no correlated spontaneous spike activity. However, the
correlations for light-evoked spike activities of neighboring on-center
-GCs did show a broad distribution with a single peak at 0 msec. Our
finding that the shift predictor brought this peak below the 99% confidence
level is consistent with previous assertions that this type of correlation
reflects common synaptic inputs (Brivanlou
et al., 1998; DeVries,
1999). Interestingly, the light-evoked activity of off-center
-GC pairs also showed this same broad correlation beneath the bimodal
short-latency peaks, indicating two modes of spike synchrony.
We found that only the nearest neighbor -GCs were tracer coupled and
displayed short-latency concerted spike activity. These data suggest then that
the synchronous networks formed by coupled -GCs are highly localized,
composed of a central -GC and its three to eight nearest -GC
neighbors. However, the limits in the spread of tracer as well as the current
underlying spike synchrony should not be construed as the result of some
physical barrier. Quite the opposite, coupled -GCs and amacrine cells
should be considered a continuous electrical array that spans the entire
retina. However, the conductance of the intercellular gap junctions must be
relatively low, whereby tracer and current spread is restricted to nearest
neighbors.
Comparison with other studies
Mastronarde
(1983a,c)
reported short-latency spike synchrony between neighboring on- and off-center
Y-cell pairs. These data suggest that, in cat retina, both on- and off-center
-GCs (the morphological counterpart of Y-cells) are coupled via gap
junctions. The discrepancy between these data and the present study could
reflect species differences. In fact, on- and off-center parasol cells, the
primate homolog of -GCs, are both tracer coupled, indicating a clear
difference with our findings in the rabbit
(Dacey and Brace, 1992).
However, another plausible explanation arises from the fact that Mastronarde
was unable to visualize the cells he recorded and therefore based his
identification strictly on physiological criteria. It is possible that
ganglion cell types other than -GCs were included in his study. In
fact, we found that, at least in rabbit retina, not all of the ganglion cells
physiologically classified as brisk-transient are -GCs.
Our data are consistent with those reported by DeVries
(1999), showing bimodal peaks
of 2.5 msec in the cross-correlation functions for off-center, but not
on-center, brisk-transient ganglion cells in the rabbit retina. Our findings
thus confirm DeVries' hypothesis that off-center, but not on-center,
-GCs are electrically coupled. In contrast, Arnett and Spraker
(1981) reported spike synchrony
between both on- and off-center brisk-transient ganglion cells in the rabbit
retina. However, they admittedly grouped cells into very broad categories, and
it is thus likely that their sample included ganglion cell types other than
-GCs.
Homologous versus heterologous coupling
Consistent with previous studies, our results suggest that tracer-coupled
-GCs form homologous gap junctions with each other as well as
heterologous junctions with at least two amacrine cell types, including
long-range cells (Vaney 1991,
1994;
Dacey and Brace, 1992;
Penn et al., 1994;
Xin and Bloomfield, 1997). The
obvious question that arises is whether the direct homologous and/or indirect
heterologous coupling is responsible for the short-latency spike synchrony
between neighboring -GCs. In fact, it is currently unclear whether
-GCs are directly coupled to one another: tracer-coupled -GCs
could be the result of -GC-to-amacrine cell-to--GC movement of
Neurobiotin (Jacoby et al.,
1996). This indirect pathway may explain why the latencies for
synchronization of -GCs in rabbit appear somewhat longer than those
reported in cat (Mastronarde,
1983c) and salamander
(Brivanlou et al., 1998).
However, we found that injection of Neurobiotin into a single -GC
results in darker and more extensive labeling of neighboring -GCs than
when a coupled amacrine is injected, thus suggesting direct
-GC-to--GC coupling (our unpublished data). Furthermore, our
finding that spike synchrony between off-center -GCs is limited to the
circumscribed array that are tracer coupled, provides compelling evidence that
ganglion-to-ganglion cell coupling indeed exists and likely mediates this
synchrony. Two additional lines of evidence also argue against
amacrine-to-ganglion cell coupling being responsible for short-latency spike
synchrony. First, within a single array, >100 amacrine cells are typically
tracer-coupled, extending far beyond the circumscribed ring of coupled
-GCs. Second, individual long-range amacrine cells have enormous
dendritic and receptive fields (Bloomfield,
1992; Xin and Bloomfield,
1997). Together, these data suggest that if amacrine-to-ganglion
cell coupling was responsible, it should have imparted spike synchrony to a
much larger array of ganglion cells, far beyond nearest neighbors. Finally, it
is interestingly to note that Long et al.
(2002) have reported a similar
2 msec lag in synchronous spikes of inferior olivary neuron pairs showing
direct, electrical coupling.
Role of local correlated activity
Studies in salamander retina have shown that the receptive fields of the
synchronous spike activity from pairs of ganglion cells are often smaller than
those of individual ganglion cells and are located at their intersection
(Meister et al., 1995;
Meister, 1996). This
synchronous activity may thus serve to preserve high-resolution spatial
signals and compress information for efficient transmission across the limited
capacity of the optic nerve (Meister and
Berry, 1999). Furthermore, these data suggest that synchronous
activity provides additional visual information to the brain that is
multiplexed with the asynchronous signals from individual ganglion cells.
However, this idea has recently been challenged by Nirenberg et al.
(2001).
Synchronous spike activity may also enhance the saliency of visual signals.
Synchronized EPSPs at central targets are more likely to elicit spikes than
EPSPs dispersed over longer time intervals
(Alonso et al., 1996;
Matsumura et al., 1996;
Alonso and Martinez, 1998;
Stevens and Zador, 1998;
Usrey and Reid, 1999). It has
been proposed that concerted activity may provide the temporal precision by
which retinal signals are reliably transmitted to central targets
(Singer, 1999).
A final question raised by our results is the following: Why do off-center
-GCs show short-latency synchrony, whereas on-center cells do not? In
general, the off-center -GCs are optimized to detect targets that are
darker than the ambient background, such as during twilight. Recently,
Tsukamoto et al. (2001)
suggested that rod photoreceptors couple under these conditions to pool
signals, thereby improving response signal-to-noise and increasing
sensitivity. Likewise, coupling between the off-center -GCs may serve
to amplify the saliency of signals encoding dark targets. This would be
particularly important for rabbits to avoid predators, particularly birds,
during dawn or dusk. This scenario would suggest that coupling patterns for
-GCs might vary according to changes in retinal adaptational state. It
will thus be of interest to determine whether synchronous activity between
-GCs is regulated by changes in ambient light conditions.
|
Footnotes
|
|---|
Received Apr. 10, 2003;
revised May. 22, 2003;
accepted Jun. 9, 2003.
This work was supported by National Institutes of Health Grant EY07360
(S.A.B.) and Medical Scientist Training Program GM07308 (E.H.H.) and by
Research to Prevent Blindness, Inc. E.H.H. was a Research to Prevent Blindness
Medical Student Eye Research Fellow. We thank Drs. Esther Gardner and Eric
Lang for helpful discussions.
Correspondence should be addressed to Dr. Stewart Bloomfield, Department of
Ophthalmology, New York University School of Medicine, 550 First Avenue,
MSB181, New York, NY 10016. E-mail:
blooms01{at}med.nyu.edu.
Copyright © 2003 Society for Neuroscience
0270-6474/03/236768-10$15.00/0
|
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Abstract
Introduction
