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The Journal of Neuroscience, June 1, 2002, 22(11):4693-4701
Segregation and Integration of Visual Channels: Layer-by-Layer
Computation of ON-OFF Signals by Amacrine Cell Dendrites
Ji-Jie
Pang,
Fan
Gao, and
Samuel M.
Wu
Cullen Eye Institute, Baylor College of Medicine, Houston, Texas
77030
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ABSTRACT |
The visual system analyzes images through parallel channels, and
our data suggest that the first set of parallel representations of the
visual world is embodied in the inner plexiform layer (IPL) of the
retina, in which light-evoked excitatory inputs of the ON and OFF
bipolar cells to amacrine cells (ACs) are organized in a layer-by-layer
manner. Approximately 30% of ACs have narrowly monostratified
dendrites in 1 of the 10 strata of the IPL, and they receive segregated
bipolar cell inputs: the light-evoked excitatory cation current,
IC, in strata 1, 2, and 4 is OFF
(predominantly mediated by the OFF bipolar cells), the current in
strata 3 and 7-10 is ON (predominantly mediated by ON bipolar cells),
and the current in strata 5 and 6 is ON-OFF (mediated by both ON and
OFF bipolar cells). The remaining 70% of ACs have broadly
monostratified, multistratified, or diffuse dendrites, and they
integrate bipolar cell signals through layer-by-layer summation: ACs
with dendrites ramified in multiple strata exhibit
ICs that are sums of
ICs of individual strata. The
light-evoked inhibitory chloride current, ICl, in strata 1, 2, and 4-6 is
ON-OFF (mediated predominantly by ON-OFF ACs or ON ACs plus OFF ACs),
and the ICl in strata 3 and 7-10 is ON
(mediated predominantly by ON ACs). This indicates that the
amacrine-amacrine inhibitory synaptic circuitry in the IPL is
asymmetrical in favor of the ON channels.
Key words:
retina; amacrine cells; bipolar cells; inner plexiform
layer; dendritic stratification; ON and OFF visual channels; layer-by-layer signal computation; signal segregation and
integration
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INTRODUCTION |
The visual system encodes and
analyzes images of the outside world through parallel channels:
different types of neurons are devoted to processing different
attributes of visual images, such as brightness, shape, color,
contrast, and dynamics, to segregated regions in the brain
(Enroth-Cugell and Robson, 1966
; Hubel and Wiesel, 1977; Hubel and
Livingstone, 1987). The first set of parallel representations of the
visual world is embodied in the inner plexiform layer (IPL) of the
retina, in which axon terminals of bipolar cells carrying different
attributes of visual stimuli stratify at different levels (or strata)
(Wu et al., 2000; Roska and Werblin, 2001). It has been established in
many vertebrate species that the ON and OFF bipolar cell inputs are
segregated in the IPL: synapses of ON cells ramify at the proximal half
(sublamina B) and those of the OFF cells ramify at the distal half of
the IPL (sublamina A) (Famiglietti and Kolb, 1976; Nelson et al.,
1978). In mammalian retinas, one type of rod bipolar cell and 9-10
types of cone bipolar cells with different patterns of axon terminal stratification have been identified (Boycott and Wassle, 1991, 1999;
Euler and Wassle, 1995), but the light response characteristics of
these cells have not been studied systematically. By comparing light
responses and the cell morphology, a recent study has shown that
bipolar cells in the salamander retina can be divided into at least 12 types, each of which carries a unique set of light response attributes
and bears axons that stratify at different strata of the IPL (Wu et
al., 2000). These stacked layers of axon terminals form a highly
organized three-dimensional structure that innervates the third-order
retinal cells [amacrine cells (ACs) and ganglion cells] in the IPL.
It is not clear, however, how third-order retinal cells process these
laminated bipolar cell inputs, and what computational algorithms ACs
and ganglion cells use to generate responses from the layer-by-layer
representation of bipolar cell signals in the IPL.
ACs are the primary interneurons in the inner retina, and they receive
excitatory synaptic inputs from bipolar cells and inhibitory synaptic
contacts from other ACs (Wong-Riley, 1974; Marc and Liu, 2000). AC
morphology has been studied by using Golgi staining and a fluorescent
"photofilling" technique, and they display extreme morphological
diversity (Ramon y Cajal, 1893; Vaney, 1990; MacNeil and Masland, 1998;
MacNeil et al., 1999). It is not certain, however, how AC morphology is
correlated with their light responses and synaptic inputs, and whether
ACs with dendrites stratifying in different strata of the IPL carry
different sets of light response attributes. Here we report a
systematic study on the light response characteristics of 164 morphologically identified ACs in the salamander retina. Light-evoked
current responses at various holding potentials were recorded under
voltage-clamped conditions so that excitatory and inhibitory synaptic
inputs could be separated, and the pattern of dendritic stratification
in the IPL of each recorded cell was examined by Lucifer yellow
fluorescence with a confocal microscope. Based on the patterns of
excitatory and inhibitory light-evoked ON and OFF responses of the
narrowly monostratified ACs, we identify rules and layer-by-layer
computational algorithms used by broadly monostratified and
multistratified ACs to generate their light responses.
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MATERIALS AND METHODS |
Larval tiger salamanders (Ambystoma tigrinum)
purchased from Charles D. Sullivan, Co. (Nashville, TN) and KON's
Scientific Co. Inc. (Germantown, WI) were used in this study. The
procedures of dissection, retinal slicing, and recording have been
described in previous publications (Werblin, 1978; Wu, 1987).
Dissection and recording were done under infrared illumination with a
dual-unit Fine-R-Scope (FJW Industry, Mount Prospect, IL) and Nitemare
infrared scopes (Meyers, Redmond, WA). Oxygenated Ringer's
solution was introduced continuously to the superfusion chamber, and
the control Ringer's solution contained (in mM):
108 NaCl, 2.5 KCl, 1.2 MgCl2, 2 CaCl2, and 5 HEPES, adjusted to a pH of 7.7. All
chemicals were dissolved in control Ringer's solution. A
photostimulator was used to deliver light spots (600-1200 µm
diameter) to the retina via the epi-illuminator of the microscope. The
intensity of unattenuated (log I = 0) 500 nm light was 2.05 × 107
photons · µm
2 · sec1.
Voltage-clamp recordings were made with an Axopatch 200A amplifier
connected to a DigiData 1200 interface and pClamp 6.1 software (all
from Axon Instruments, Foster City, CA). Patch electrodes of 5 M
tip resistance when filled with internal solution containing (in
mM): 118 Cs methanesulfonate, 12 CsCl, 5 EGTA, 0.5 CaCl2, 4 ATP, 0.3 GTP, 10 Tris, 0.8 Lucifer
yellow, adjusted to a pH of 7.2 with CsOH, were made with patch
electrode pullers from Narishige (Tokyo, Japan) or Sutter Instruments
(Novato, CA). The chloride equilibrium potential,
ECl, with this internal solution was
approximately 
60 mV. Estimates of the liquid junction potential at
the tip of the patch electrode before seal formation varied from 9.2
to 9.6 mV. For simplicity, we corrected all holding potentials by 10 mV.
Three-dimensional cell morphology was visualized in living retinal
slices (250-300 µm in thickness) through the use of Lucifer yellow
fluorescence with a confocal microscope (Zeiss 510; Zeiss, Thornwood,
NY). Images were acquired with a 40× water immersion objective
(numerical aperture, 0.75), using the 458 nm excitation line of
an argon laser and a long-pass 505 nm emission filter. Consecutive
optical sections were superimposed to form a single image using Zeiss
Laser Scanning Microscope-PC software, and these compressed
image stacks were further processed in Adobe Photoshop 6.0 (Adobe
Systems, San Jose, CA) to improve the signal-to-noise ratio. Because
signal intensity values were typically enhanced during processing to
improve the visibility of smaller processes, the cell bodies and larger
processes of some cells appear saturated because of their larger volume
of fluorophore. Although the background images of the retinal slices
were acquired simultaneously with the fluorescent cells, they were
imaged using transmitted light. The level at which dendritic processes
stratified in the IPL was characterized by the distance from the
processes to the distal margin of the IPL (Fig.
1A). We selected for
cells in the AC layer with somas situated beneath the surface of the
slice, and they usually had relatively intact processes (assessed by
rotation of the stacked images).
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Figure 1.
A, A stacked confocal fluorescent
image of an AC (a), a sketch of the same cell on
a schematic background of the retina with marked divisions of 10 strata
of the IPL (b), and the light-evoked current
responses to a 0.5 sec light step [500 nm, 3.3 (3.3 log unit
attenuation)] at various holding potentials (c).
CB, Cell body; D, dendrites;
R, rod; C, cone; PRL,
photoreceptor layer; OPL, outer plexiform layer;
INL, inner nuclear layer; GCL, ganglion
cell layer. Scale bar, 25 µm. B, Distribution of
dendritic stratification levels in the IPL of 164 ACs. These cells are
grouped as narrowly monostratified, broadly monostratified, narrowly
bistratified, broadly bistratified, narrowly tristratified, broadly
tristratified, and diffuse cells (definitions are given in
Results). Each vertical column represents an AC,
and the short vertical bars in individual strata
indicate the presence of dendritic processes in that stratum. The
WD of each of the 164 cells is given at the
bottom of each panel in B,
as follows: N, Narrow field; M, medium
field; W, wide field (definitions are given in
Results).
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RESULTS |
Morphological diversity of amacrine cells
Figure 1A shows the stacked confocal fluorescent
image of an AC (Fig. 1A, a), a sketch of
the same cell on a schematic background of a retina with the IPL
divided into 10 strata (Fig. 1A, b), and
the light-evoked current responses to a 0.5 sec light step at various
holding potentials (Fig. 1A, c). Typical
AC features include a soma (cell body) centered in the proximal half of
the IPL, and dendritic processes ramifying laterally in the IPL. The AC
in Figure 1A is an example of what we termed a
"broadly monostratified" cell (cells with dendrites ramifying
in two to three contiguous strata, in this case in strata 4-6). By
using this protocol, we studied a total of 164 ACs. Forty-nine (30%)
were "narrowly monostratified" (with dendrites ramifying in one of
the ten strata of the IPL), 32 (19%) were broadly monostratified
(dendrites ramifying in two to four contiguous strata), 24 (15%) were
"narrowly bistratified" (dendrites ramifying in two noncontiguous
strata), 8 (5%) were "broadly bistratified" (dendrites ramified in
two bands and at least one band ramifying in two or more contiguous
strata), 4 (2%) were "narrowly tristratified" (dendrites ramified
in three noncontiguous strata), 11 (7%) were "broadly
tristratified" (dendrites ramified in three bands and at least one
band ramifying in two or more contiguous strata), and 36 (22%) were
"diffuse" (dendrites ramified in four or more bands or in five or
more contiguous strata). The distribution of AC dendrite stratification
levels in the IPL is given in Figure 1B. Each
vertical column represents an AC and the short vertical bars in
individual strata indicate the presence of dendritic processes in that
stratum. Cells were arranged in the order of number of their dendritic
strata (narrowly monostratified cells first, within which cells were
arranged in the order of their dendritic strata 1-10). In this
sample of 164 ACs, approximately one-half of the ACs are
monostratified, three-fifths of which are narrowly ramified in a
single stratum. The second largest group is the diffuse ACs (22%),
among which the majority have dendrites distributed contiguously in
strata 2-9. There are no clear patterns of dendritic distribution
within bistratified and tristratified cells in our data pool.
In addition to the difference in levels of dendritic stratification in
the IPL, ACs vary with respect to the width of their dendritic field
(WD). In our sample of 164 ACs, there
are 39 (24%) narrow-field cells [N cells;
WD < 125 µm (MacNeil and Masland, 1998)], 77 (47%) medium-field cells (M cells; 125 µm < WD <400 µm), and 48 (29%)
wide-field cells (W cells; WD > 400 µm)s. The dendritic field width of each of the 164 cells is labeled
in Figure 1B (bottom row). There is no
clear correlation between the dendritic field width and the levels of
dendritic stratification in the IPL. One exception is that we did not
find any wide-field diffuse ACs: all wide-field cells were
monostratified, bistratified, or tristratified. It is important to
note, however, that our voltage-clamp recordings were made from the AC
cell bodies, and therefore we might not have space-clamped all
dendrites of the ACs, especially those of the wide-field cells. Thus
the light-evoked responses of the wide-field cells may be dominated by
the synaptic inputs to the dendrites close to the cell bodies. Another
potential source of inaccuracy in our measurements is that we recorded
ACs from retinal slices. Although we chose to record from ACs several
layers under the slice surface (which had more intact dendrites as
assessed by image rotation with the confocal microscope; see Materials and Methods), some dendrites may still be cut off during slicing. Therefore, we may have underestimated the AC dendritic field size, especially the WD of the wide-field cells.
Light-evoked current responses of morphologically identified
amacrine cells
Figure 2 shows stacked confocal
fluorescent images (Fig. 2A,C) and
light-evoked current responses at various holding potentials (Fig.
2B,D) of eight ACs. Cells a-d
are narrowly monostratified at strata 1, 3, 7, and 9, respectively;
cell e is broadly tristratified at strata 3, 6, 7, and 9;
cell f is narrowly bistratified at strata 3 and 8; cell
g is broadly monostratified at strata 9 and 10; and cell
h is diffuse across strata 1-9. Cells b,
e, and f are narrow-field cells; cells
a, c, and h are medium-field cells; and cells d and g are wide-field cells. To a 0.5 sec light step measured at 60 mV, the excitatory light-evoked cation
current response (IC) of cell
a was a transient inward current at light offset (OFF
response), the ICs of cells
b-d, f, and g were transient inward
currents at light onset (ON response), and the
ICs of cells e and
h were transient inward currents at both light onset and
offset (ON-OFF response). The inhibitory light-evoked chloride current
responses (ICls) measured at 0 mV
of cells a and f-h were ON-OFF transient
outward currents, and those in cells b-e were ON transient
outward currents. The current responses measured at +40 mV and 100 mV
in each cell were mediated by the light-evoked cation and chloride
conductance changes (gC and
gCl, respectively).
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Figure 2.
Stacked confocal fluorescent images (A,
C) and current responses evoked by a 0.5 sec light
step (500 nm, 3.3) at various holding potentials (40, 0, 60, and
100 mV) (B, D) of eight
(a-h) ACs. The ten strata of the IPL in each image are
marked by scales on the right. Scale bars, 25 µm. The
excitatory light-evoked cation current response
(IC) of each cell was measured at
ECl (60 mV), and the inhibitory
light-evoked chloride current response
(ICl) was measured at
EC (0 mV).
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Layer-by-layer analysis of excitatory and inhibitory light-evoked
ON and OFF signals in narrowly monostratified amacrine cells
To determine the stratum-by-stratum excitatory and inhibitory
synaptic inputs, we analyzed the IC
and ICl of narrowly
monostratified ACs, because these cells make synaptic contacts
with bipolar cells and other ACs in one IPL stratum. Figure
3 shows the
IC and
ICl (Fig. 3, bottom
panel) of 10 (marked as cells 1-10, corresponding to the
stratum each cell ramifies) narrowly monostratified cells whose
dendrites ramified in each of the 10 strata of the IPL. The top
panel of Figure 3 shows sketches (based on stacked confocal fluorescent images; same procedure as shown in Fig.
1A) of the AC bodies and a portion of the narrowly
monostratified dendrites near the soma (extended portions of dendrites
of medium- and wide-field cells were cut off to save space) on a
schematic background of the retina. The dendritic thickness (in the
dimension parallel with the photoreceptor axes) of the majority
(75-80%) of the narrowly monostratified cells is approximately
one-tenth the thickness of the IPL, whereas ~10% are slightly wider
than one-tenth of the IPL and the rest are somewhat thinner than
one-tenth of the IPL. At 60 mV, cells 1, 2, and 4 exhibited an OFF
IC; cells 3 and 7-10 gave rise to
an ON IC; and cells 5 and 6 gave an
ON-OFF IC. This pattern of
excitatory light responses was consistent in all narrowly
monostratified ACs within a given stratum, regardless of their
dendritic field size (for example, cells a-d in Fig. 2).
The average ± SD peak amplitudes of
IC in various types of narrowly
monostratified ACs are given in Table 1,
and they suggest that the excitatory inputs to strata 1, 2, and 4 are
predominantly mediated by hyperpolarizing bipolar cells (HBCs);
those to strata 3 and 7-10 are predominantly mediated by the
depolarizing bipolar cells (DBCs); and those to strata 5 and 6 are mediated by both HBCs and DBCs.
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Figure 3.
Top, Sketches based on stacked
confocal fluorescent images (same procedure as shown in Fig.
1A) of 10 (cells 1-10, corresponding to the
stratum each cell ramifies) narrowly monostratified ACs. Extended
portions of dendrites of medium- and wide-field cells were cut off to
save space. PRL, Photoreceptor layer;
OPL, outer plexiform layer; INL, inner
nuclear layer; GCL, ganglion cell layer.
Bottom, Current responses evoked by a 0.5 sec light step
(500 nm, 3.3) at holding potentials of 60 mV
(IC) and 0 mV
(ICl).
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Table 1.
The average ±SD peak amplitudes, time-to-peak
of the rising phase ( 0), and decay time constant
(D, fitted by single exponentials) of
IC and ICl in
various types of narrowly monostratified ACs
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At 0 mV, cells 1, 2, and 4-6 showed ON-OFF
ICls and cells 3 and 7-10
exhibited ON ICls, indicating that
ACs making inhibitory synaptic contacts with cells 1, 2, and 4-6
(those with OFF ICs) gave rise to
ON-OFF light responses, and ACs making inhibitory synaptic contacts
with cells 3 and 7-10 (those with only ON
ICs) gave ON light responses. This
pattern of inhibitory light responses is also consistent in all
narrowly monostratified ACs within a given stratum, regardless of
their dendritic field size (for example, cells a-d in Fig.
2). The average ± SD peak amplitudes of
ICl of various types of narrowly
monostratified ACs are also listed in Table 1, and they suggest that
the inhibitory inputs to strata 1, 2, and 4 (strata with OFF
ICs) and strata 5 and 6 (strata with ON-OFF ICs) are mediated
predominantly by ON-OFF ACs, and that those to strata 3 and 7-10
(strata with ON ICs) are mediated predominantly by ON ACs.
In addition to differences in ON and OFF inputs, the kinetics of
IC and
ICl in various types of narrowly
monostratified ACs was different, and the average ± SD
rise time (0) and decay time constant
(D) of IC
and ICl are listed in Table 1. The kinetics of cells 1, 2, and 10 was faster than that of the rest. The
mean decay time constant (D) of cells 1, 2, and 10, for example, ranged from 27 to 56 msec, whereas that of cells
3-9 varied from 150 to 725 msec. Because the axon terminals of
rod-dominated HBCs ramify primarily in strata 1 and 2, and axon
terminals of rod-dominated DBCs ramify primarily in stratum 10 (Wu et
al., 2000), this result suggests that the postsynaptic responses of
rod-dominated bipolar cells in ACs are more transient than the
postsynaptic responses of cone-dominated bipolar cells. It is not clear
why rod-dominated inputs are more transient in ACs. Because the light
responses of rod-dominated bipolar cells are not more transient than
the cone-dominated bipolar cell responses (Yang and Wu, 1997; Wu et al., 2000), the difference in AC response kinetics is probably mediated
by differences in the amacrine-amacrine and amacrine-bipolar synaptic
network. Additional studies are needed to clarify this issue.
Layer-by-layer computation of ON and OFF signals in broadly
monostratified and multistratified amacrine cells
We subsequently examined whether the rules segregating ON and OFF
channels in various IPL strata set forth by the narrowly monostratified
cells are followed by broadly monostratified and multistratified
(bistratified, tristratified, and diffuse) ACs. Figure
4 shows the morphology (top
panel) and ICl and
IC (bottom panel)
of 10 (cells 11-20) ACs. Cell 11 was broadly monostratified in strata
3 (ON stratum) and 4 (OFF stratum), and
ICs and
ICls were ON-OFF. Cell 12 was
broadly monostratified in strata 8 (ON stratum), 9 (ON stratum), and 10 (ON stratum), and ICs and
ICls were ON. Cell 13 was broadly
monostratified in strata 1 (OFF stratum) and 2 (OFF stratum), the
IC was OFF, and the
ICl was ON-OFF, whereas cell 14 was narrowly tristratified in strata 3 (ON stratum), 7 (ON stratum),
and 9 (ON stratum), and ICs and
ICls were ON. Cell 15 was narrowly
tristratified in strata 1 (OFF stratum), 7 (ON stratum), and 9 (ON
stratum), and ICs and
ICls were ON-OFF, and cell 16 was
narrowly bistratified in strata 2 (OFF stratum) and 4 (OFF stratum),
the IC was OFF, and the
ICl was ON. Cells 17 and 18 were
diffuse across all strata (ON and OFF strata), and
ICs and
ICls were ON-OFF. Cell 19 (wide
dendritic field) was broadly monostratified in strata 9 (ON stratum)
and 10 (ON stratum), and ICs and
ICls were ON, and cell 20 was
broadly monostratified in strata 1 (OFF stratum), 2 (OFF stratum), and 3 (ON stratum), and ICs and
ICls were ON-OFF.
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Figure 4.
Top, Sketches based on stacked
confocal fluorescent images of 10 (cells 11-20) broadly monostratified
(cells 11-13, 19, and 20), narrowly bistratified (cell 16), narrowly
tristratified (cells 14 and 15), and diffuse (cells 17 and 18) ACs.
PRL, Photoreceptor layer; OPL, outer
plexiform layer; INL, inner nuclear layer;
GCL, ganglion cell layer. Bottom, Current
responses evoked by a 0.5 sec light step (500 nm, 3.3) at holding
potentials of 60 mV (IC) and 0 mV (ICl).
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In addition, as shown in Figure 2, cell e was broadly
tristratified in strata 3 (ON stratum), 6 (ON-OFF stratum), 7 (OFF
stratum), and 10 (ON stratum), and
ICs and
ICls were ON-OFF. Cell
f was narrowly bistratified in strata 3 (ON stratum) and 8 (ON stratum), and ICs and
ICls were ON-OFF. Cell
g was broadly monostratified in strata 9 (ON stratum) and 10 (ON stratum), and ICs and
ICls were ON, and cell h
ramified diffusely across strata 1-9 (ON and OFF strata), and
ICs and
ICls were ON-OFF. We also examined the correlation between ON-OFF IC
and ICl responses and patterns of
dendritic stratification of the remaining 101 multistratified ACs
listed in Figure 1B. All but five diffuse ACs (which
showed ON instead of ON-OFF IC and
ICl) followed the ON-OFF rules for the IC and
ICl responses: cells with dendrites
in strata 1, 2, and 4 exhibited an OFF
IC and an ON-OFF
ICl; cells with dendrites in strata
3 and 7-10 gave rise to an ON IC
and ICl; and cells with dendrites
in strata 5 and 6 or with diffuse dendrites gave rise to an ON-OFF
IC and
ICl.
It is clear from these results that the rules correlating ON-OFF
channels and dendritic stratification in the IPL set forth by narrowly
monostratified cells (Fig. 3 and Table 1) are closely followed by all
79 broadly monostratified, bistratified, and tristratified ACs,
and by the vast majority (31 of 36) of diffuse ACs. Although the
amplitudes of the broadly monostratified cells and the responses of the
multistratified ACs did not necessarily correlate with the amplitudes
of narrowly monostratified AC responses, the kinetics of
IC and
ICl was very similar to that of the
narrowly monostratified ACs ramifying in the strata in which the
broadly monostratified and multistratified cells have dendritic
branches. These results suggest that ACs with dendrites in multiple
strata compute their signals in a layer-by-layer (stratum-by-stratum)
manner. At least for the ON-OFF signals, responses of broadly
monostratified and multistratified cells seem to result from summing up
responses of the individual dendritic strata of the cells.
Amacrine-amacrine synaptic circuitry in the IPL
In the salamander retina, the dark membrane potential of ACs is
near 80 mV (Yang et al., 2002), and
ECl values in amphibian ACs are in the
range of 50 to 70 mV (Miller and Dacheux, 1983). Therefore the
light-evoked voltage responses are mediated by both IC and
ICl, and thus the ON-OFF pattern
of voltage responses at 80 mV (V) of each AC is
determined by the ON-OFF pattern of
IC and
ICl (the I-V
relationship of the light-evoked current is approximately linear
between 0 mV and 100 mV) (Yang et al., 2002). Consequently, if all
ACs have dark membrane potentials near 80 mV, then narrowly
monostratified cell types 1, 2, and 4-6 may receive inhibitory inputs
from themselves, from other ACs with an ON-OFF V, or
from at least one type of AC with an ON-OFF V and some
ACs with an ON V. However, narrowly monostratified cell
types 3 and 7-10 can receive inhibitory inputs only from themselves or
from other ACs with an ON V. These amacrine-amacrine synaptic connections in the 10 IPL strata are summarized by a logic
circuit diagram (Tokheim, 1994) in Figure
5. This logic circuit gives all possible
connections made by ACs on narrowly monostratified ACs in each stratum.
The number of possible connections is very large, because for each OR
(any or all) gate of five inputs, there are
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31 possible connections (where
C
is the
number of combinations with k out of five strata), and for
the AND (all or nothing) gate with two inputs from the OR gates,
there are 31× 31 = 961 possible connections. Therefore there are
31 + 961 = 992 possible connections that mediate amacrine cell
inputs (ICl) to the narrowly
monostratified ACs in strata 1, 2, and 4-6 and 31 possible connections
that mediate amacrine cell inputs
(ICl) to the narrowly
monostratified ACs in strata 3 and 7-10. Figure 5 clearly indicates
that the amacrine-amacrine synaptic circuitry is asymmetric in favor
of the ON channels: ACs with ON responses may make synaptic connections
with ACs in all 10 strata, whereas ACs with ON and OFF responses may
make synaptic connections only with ACs in strata 1, 2, and 4-6.
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Figure 5.
Logic circuit diagram of amacrine-amacrine
synaptic connections in the IPL.
IC,
V80, and
ICl are light-evoked excitatory current,
light-evoked voltage response at 80 mV, and light-evoked chloride
current, respectively. Open circles are ON responses,
filled circles are OFF responses, and
half-open/half-filled circles are ON-OFF responses.
 and
 are logic OR
and AND gates respectively.
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The logic circuit in Figure 5 is based on the finding that most ACs in
dark-adapted salamander retina have a dark membrane potential near 80
mV (Yang et al., 2002). However, for those ACs with dark membrane
potentials near 60 mV or above (Vallerga, 1981), the ON-OFF patterns
of the voltage responses of the cell (V) are
different from the V80 listed in
Figure 5, and more complex logic circuits are needed. In addition,
analysis of cells e-h in Figure 2 and cells 11-20 in
Figure 4 shows that the layer-by-layer rule for the ON-OFF responses
applies for amacrine-amacrine inputs
(ICl) in broadly monostratified and
multistratified ACs; therefore ICls
of ACs with dendrites ramified in multiple strata may be computed as
sums of ICls of individual strata.
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DISCUSSION |
Bipolar cell inputs to amacrine cells are organized in a
layer-by-layer manner in the IPL: segregation and integration of ON and
OFF signals
A major finding of this study is that the light-evoked excitatory
synaptic inputs from ON and OFF bipolar cells to ACs,
IC, are organized in a
layer-by-layer manner with 10 strata in the IPL. This functional
architecture resembles many parallel information pathways in higher
visual centers, such as the columnar segregation of neurons registering
different orientations in the visual cortex (Hubel and Wiesel, 1968,
1969). Our data show that ~30% of ACs are narrowly monostratified
(Fig. 1A), and they ramify in each of the 10 strata
of the IPL (types 1-10 for strata 1-10). Responses of these cells
demonstrate that the ON and OFF channels are segregated in 10 strata in
the IPL: ICs in strata 1, 2, and 4 are OFF responses (predominantly mediated by the OFF bipolar cells);
ICs in strata 3 and 7-10 are ON
responses (predominantly mediated by ON bipolar cells); and
ICs in strata 5 and 6 are ON-OFF
responses (mediated by both ON and OFF bipolar cells). Among the 12 types of bipolar cells in the tiger salamander retina, only five types
have monostratified axon terminals, whereas the axons of others are
bistratified, tristratified, or pyramidally branching (Wu et al.,
2000). Therefore, some strata in the IPL may contain axon terminals
primarily from one type of bipolar cell, whereas other strata may
contain axon terminals from several types of bipolar cells. What our
data suggest, nevertheless, is that the inputs from bipolar cells to
ACs in each stratum are the same within that stratum: ON, OFF, or
ON-OFF.
Our data suggest that narrowly monostratified ACs carry segregated
bipolar cell signals in the inner retina. In contrast, broadly
monostratified and multistratified cells integrate bipolar cell signals
through layer-by-layer summation: ACs with dendrites ramified in
multiple strata exhibit IC that are
sums of ICs of individual strata
(Fig. 4). This suggests that the ON and OFF bipolar cell inputs to ACs
(monostratified, multistratified, narrowly stratified, or broadly
stratified) in each stratum are very similar, and that
ICs from each stratum are
transmitted to the soma in a parallel (and thus additive) manner. This
is consistent with previous studies that have shown that most bipolar
cells, either ON (DBCs) or OFF (HBCs), have synaptic terminals
ramifying in a restricted stratum (or strata) of the IPL (Wu et al.,
2000). Although it is difficult to match all bipolar cell
light-response attributes with ICs
in ACs, our results demonstrate a clear stratum-by-stratum rule for the
ON-OFF response channels (set forth in Fig. 3 and Table 1) in the IPL.
This rule primarily agrees with the sublamina A/B rule for the OFF-ON
cells established by previous studies in the cat retina (Famiglietti
and Kolb, 1976; Nelson et al., 1978) and salamander retina (Pang et
al., 2002). An obvious exception is that we found that the
IC in stratum 3 (located in the
middle of sublamina A) exhibits ON instead of OFF responses. Previous studies in salamanders and turtles have shown that there are DBCs that
bear axon terminals ramifying in two strata in the IPL, one in
sublamina A and the other in sublamina B (Ammermuller and Kolb, 1995;
Wu et al., 2000). Recent results have shown that antibodies against
G-protein 
-subunit stain DBCs in the salamander retina, and in
addition to heavy labeling in sublamina B, there is a clear band near
stratum 3 in sublamina A (J. Zhang and S. M. Wu,
unpublished results). It is possible, at least in the tiger salamander
retina, that the ON channels mediated by DBCs activate synapses not
only in sublamina B but also in the middle of sublamina A (stratum 3).
This may partially explain why the vast majority of ACs and ganglion
cells in the salamander retina are ON-OFF or ON cells, and only
~5-10% of these third-order cells are OFF cells (Hensley et al.,
1993; Gao and Wu, 1998; Yang et al., 2002).
At first glance, the division of the IPL into 10 strata appears
arbitrary. However, the radial dendritic thickness of the majority of
the narrowly stratified cells (either monostratified or
multistratified) is very close to one-tenth the thickness of the IPL
(only ~10% are slightly wider than one-tenth of the IPL, and
~10-15% are thinner than one-tenth of the IPL). This provides the
anatomical rationale for the 10 strata scheme. In addition, our
physiological data also support this scheme. For example, cells 2 and 4 in Figure 3 (narrowly monostratified in strata 2 and 4, respectively)
are OFF cells, but cell 3, which has dendrites ramified approximately
one-tenth of the IPL thickness away from the dendrites of cells 2 and
4, is an ON cell. Table 1 and Figure 4 show that this clear
physiological difference can be observed in all monostratified and
multistratified cells that bear narrowly stratified dendrites in strata
2-4. Another example is that although cells 9 and 10 (Fig. 3) are both
ON cells, their IC kinetics is
quite different. Table 1 shows that the
IC decay time constant (D) of cells in stratum 9 is 725 ± 78, and that the decay time constant of cells in stratum 10 is 56 ± 11. Therefore, the morphological and physiological data we obtained
support the 10 strata scheme for classifying ACs in the tiger
salamander retina.
Amacrine-amacrine synaptic interactions in the IPL: asymmetric
synaptic circuitry in favor of the ON channels
Our data show that the inhibitory synaptic inputs to ACs are more
complex than the excitatory inputs. Responses of narrowly monostratified ACs suggest that the light-evoked inhibitory inputs, ICl, to strata 1, 2, and 4 (strata
with OFF ICs) and strata 5 and 6 (strata with ON-OFF ICs) are
ON-OFF (mediated predominantly by ON-OFF ACs), and that those to
strata 3 and 7-10 (strata with ON
ICs from DBCs) are ON (mediated
predominantly by ON ACs). This indicates that the narrowly
monostratified cells receiving excitatory inputs from OFF bipolar cells
(types 1, 2, and 4) must receive inhibitory inputs from ON and OFF
channels mediated by both ON and OFF bipolar cells. However, narrowly
monostratified cells receiving excitatory inputs from ON bipolar cells
(types 3 and 7-10) receive inhibitory inputs only from the ON channel mediated by the ON bipolar cells. Therefore, at least in the tiger salamander, the amacrine-amacrine synaptic circuitry in the IPL seems
asymmetric in favor of the ON channels. The logic circuit diagram in
Figure 5 clearly demonstrates this asymmetry: ACs with ON responses may
make connections with ACs in all 10 strata, whereas ACs with ON and OFF
responses may make connections only with ACs in strata 1, 2, and 4-6.
Although it is unclear how such an asymmetrical synaptic network is
organized at the ultrastructural level, the logic circuit in Figure 5
begins to suggest some computational algorithms for amacrine-amacrine
synaptic wiring in the inner retina.
Morphological diversity and classification of retinal
amacrine cells
We have shown that ACs in the tiger salamander, like their
counterparts in other vertebrates, exhibit extreme morphological diversity (Vaney, 1990; MacNeil and Masland, 1998; Masland, 2001). ACs
differ from one another in their shape and size of cell bodies, dendritic width and thickness, patterns of dendritic branching, and
level(s) of dendritic stratification in the IPL. Certain morphological features are closely correlated with different physiological responses (e.g., the levels of dendritic stratification in the IPL of narrowly monostratified cells closely correlate with the
IC ON and OFF responses), whereas
other morphological features are not [e.g., the bistratified cell in
strata 3 and 8 (cell f in Fig. 2) and the monostratified
cell in stratum 7 (cell c in Fig. 2) have very similar light
responses]. In addition, some ACs with dendrites ramifying in the same
strata exhibit very different light responses (e.g., cell 17 and cell
18 in Fig. 4, both with diffuse dendrites ramified in strata 1-10,
exhibit different ICs and
ICls).
From these results, it is evident that AC classification is a complex
task. In addition to levels of dendritic stratification in the IPL and
the polarity and kinetics of IC and
ICl, there are many other
morphological and physiological parameters that define AC types. These
parameters include the receptive field size, presynaptic and
postsynaptic partners, neurotransmitter contents, and patterns of
dendritic branching of the cells. Each of these additional parameters
may "lump" or "split" the various cell types set forth by the
previous parameters (e.g., types based on the different levels of
dendritic stratification in Fig. 1B) into a new set
of AC types. For example, cell 13 and cell 20 in Figure 4 resemble the
dopaminergic (tyrosine hydroxylase-positive) ACs, and cell 12 and cell
19 in Figure 4 and cell g in Figure 2 resemble the serotonin
cells (Watt et al., 1988; Watt, 1992). Therefore the additional
parameter (neurotransmitter content) lumps cells 13 and 20 into one
type and cells 12 and 19 into another type. However, cell types 1 and 2 (Fig. 1B) both contain narrow-, medium-, and
wide-field cells; thus, the addition of receptive field size as a
parameter splits cell types 1 and 2 into six types of ACs. Therefore,
because of the extreme morphological diversity and complex
physiological and neurochemical variations, classification of retinal
ACs is highly dependent on the parameters chosen. It may be impractical
to expect a simple universal classification scheme for such a diverse
population of interneurons in the retina. It is perhaps more useful,
instead, to determine computational rules for individual parameters,
such as the layer-by-layer rule for the ON-OFF bipolar cell inputs
described in this article, and then integrate them into a comprehensive
framework that process various attributes of visual images presented to
the eyes.
| |
FOOTNOTES |
Received Nov. 5, 2001; revised Feb. 26, 2002; accepted March 8, 2002.
This work was supported by National Institutes of Health (NIH) Grant EY
04446, NIH Vision Core Grant EY 02520, the Retina Research Foundation
(Houston, TX), and Research to Prevent Blindness, Inc. We thank Drs.
Bruce Maple and Roy Jacoby for critically reading this manuscript.
Correspondence should be addressed to Dr. Samuel M. Wu, Cullen Eye
Institute, Baylor College of Medicine, One Baylor Plaza, NC-205,
Houston, TX 77030. E-mail: swu{at}bcm.tmc.edu.
| |
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ABSTRACT
INTRODUCTION
