Presynaptic Mechanism for Slow Contrast Adaptation in Mammalian Retinal Ganglion Cells

Presynaptic Mechanism for Slow Contrast Adaptation in Mammalian Retinal Ganglion Cells
Neuron 50, 453–464, May 4, 2006 ª2006 Elsevier Inc.
DOI 10.1016/j.neuron.2006.03.039
Presynaptic Mechanism for
Slow Contrast Adaptation in
Mammalian Retinal Ganglion Cells
Michael B. Manookin1 and Jonathan B. Demb1,2,3,*
Neuroscience Program
Department of Ophthalmology and Visual Sciences
Department of Molecular,
Cellular and Developmental Biology
University of Michigan
Ann Arbor, Michigan 48105
Visual neurons, from retina to cortex, adapt slowly to
stimulus contrast. Following a switch from high to
low contrast, a neuron rapidly decreases its responsiveness and recovers over 5–20 s. Cortical adaptation
arises from an intrinsic cellular mechanism: a sodiumdependent potassium conductance that causes prolonged hyperpolarization. Spiking can drive this mechanism, raising the possibility that the same mechanism
exists in retinal ganglion cells. We found that adaptation in ganglion cells corresponds to a slowly recovering afterhyperpolarization (AHP), but, unlike in cortical
cells, this AHP is not primarily driven by an intrinsic
cellular property: spiking was not sufficient to generate adaptation. Adaptation was strongest following
spatial stimuli tuned to presynaptic bipolar cells rather
than the ganglion cell; it was driven by a reduced
excitatory conductance, and it persisted while blocking GABA and glycine receptors, K(Ca) channels, or
mGluRs. Thus, slow adaptation arises from reduced
glutamate release from presynaptic (nonspiking) bipolar cells.
The natural environment contains a wide range of possible lighting conditions. To operate across these conditions, the visual system must adapt its sensitivity to
the statistics of the immediate environment. At the first
stage of vision, the retina adapts to the mean intensity,
a process also called ‘‘light adaptation,’’ through intrinsic photoreceptor properties as well as postreceptoral
mechanisms (Pugh et al., 1999; Troy and Enroth-Cugell,
1993; Walraven et al., 1990). The retina further adapts to
the range of intensities relative to the mean, also called
‘‘contrast adaptation.’’ A common hypothesis suggests
that, at low contrast, the retina increases sensitivity to
improve the signal-to-noise ratio, whereas, at high contrast, the retina decreases sensitivity to avoid saturating
the response (Baccus and Meister, 2004; Demb, 2002).
Contrast adaptation is largely absent in photoreceptors
and therefore must arise through either network mechanisms or intrinsic properties of ganglion cells (Baccus
and Meister, 2002; Rieke, 2001; Sakai et al., 1995).
At multiple levels of the visual system, including the
retina, cells adapt to contrast over at least two time
scales: a fast adaptation that acts in tens to hundreds
*Correspondence: [email protected]
of milliseconds, and a much slower adaptation that
acts over several seconds (Baccus and Meister, 2002;
Carandini and Ferster, 1997; Maffei et al., 1973; Movshon and Lennie, 1979; Smirnakis et al., 1997; Solomon
et al., 2004). The slow form of contrast adaptation was
recently demonstrated in the primate magnocellular
pathway in vivo (Solomon et al., 2004). Magnocellular
retinal or thalamic cells were stimulated with high contrast followed by a switch to low contrast or mean luminance. Following the switch, these cells showed a suppressed response that required a recovery period of
over 10 s. To put this time period into perspective, consider that under steady contrast conditions, ganglion
cell responses can be predicted based on the previous
w300 ms of the stimulus (Baccus and Meister, 2002;
Chichilnisky, 2001; Kim and Rieke, 2001; Zaghloul et al.,
2003). Thus, a 10 s period of adaptation is relatively long.
Slow contrast adaptation in magnocellular neurons
required a high-contrast stimulus effective at driving
the cell (Solomon et al., 2004). This apparent activity dependence suggested that the mechanism for adaptation
is intrinsic to the ganglion cell. Indeed, there is good precedent for this hypothesis: some cortical neurons express an intrinsic property for slow contrast adaptation
(Sanchez-Vives et al., 2000a). When a cortical cell is
stimulated strongly, either by a visual stimulus or direct
current injection, the cell depolarizes and fires spikes. At
the offset of stimulation, the cell hyperpolarizes and this
afterhyperpolarization (AHP) recovers slowly over seconds. The AHP suppresses spiking responses to lowcontrast stimuli (Carandini and Ferster, 1997; SanchezVives et al., 2000a, 2000b). Ion substitution experiments
demonstrated that the cortical AHP was caused largely
by a potassium conductance sensitive to intracellular
sodium (Sanchez-Vives et al., 2000a). This potassium
conductance can apparently be activated by sodium influx caused by either synaptic input in the absence of
spiking, or by spiking in the absence of synaptic input
(Carandini and Ferster, 1997; Sanchez-Vives et al.,
2000a, 2000b; Vidyasagar, 1990). Thus, for a cortical
cell, spiking is sufficient but not necessary to drive adaptation. A retinal ganglion cell might express a similar
intrinsic mechanism for slow adaptation. However, ganglion cells also show adaptive effects to small stimulus
patches, implicating a possible mechanism in presynaptic bipolar cells (Brown and Masland, 2001). Cortical
cells in area MT also express adaptation that is primarily
caused by a presynaptic mechanism (Kohn and Movshon, 2003, 2004).
Previous studies of slow contrast adaptation in mammalian retina used extracellular recording (Brown and
Masland, 2001; Chander and Chichilnisky, 2001; Smirnakis et al., 1997; Solomon et al., 2004). Here we studied
slow adaptation in mammalian ganglion cells using intracellular recording. We show that slow adaptation results from a slowly recovering AHP, as shown in cortical
cells and salamander ganglion cells (Baccus and Meister, 2002; Carandini and Ferster, 1997; Sanchez-Vives
et al., 2000b). However, slow adaptation in ganglion cells
does not arise primarily from an intrinsic property of the
Figure 1. OFF Ganglion Cells Show Reduced
Spiking following High Contrast Due to
a Slow Afterhyperpolarization
(A) An OFF cell was stimulated with a drifting
sine wave grating with contrast alternating between high (100%, 10 s) and low (20%, 20 s).
Following high contrast, spiking was suppressed and required 7.0 s to recover to the
baseline level (dashed line). Trace at top
shows one cycle of the loose-patch, extracellular recording; poststimulus time histogram
(psth) at bottom shows the average firing
rate across four repeats (bin size, 500 ms).
Grating spatial frequency was 6.7 cycles
mm21 and drifted at 6 Hz (stimulus trace
does not show 6 Hz).
(B) Intracellular recording of the same cell
and stimulus shown in (A). Following high
contrast, the membrane potential showed an
afterhyperpolarization (AHP) of 211.4 mV
that required 5.5 s to recover to baseline.
The period of suppressed spiking corresponded to the period of the AHP.
(C) Enlarged area of (B) showing the AHP.
(D) An OFF cell showed a maintained discharge (7.8 Hz) that was suppressed following
a 4 s, high-contrast stimulus (drifting grating,
6.7 cycles mm21, 6 Hz). Trace at top shows
loose-patch record; psth at bottom shows
the average firing rate across four repeats.
The spike rate recovered over 6.5 s.
(E) Intracellular recording of the cell in (D)
shows an AHP following the stimulus that required 4.0 s to return to the resting potential.
(F) Extracellular recording of an ON cell does
not show a prolonged reduction in spike rate
following high contrast (drifting grating, 6.7
cycles mm21, 6 Hz). Following high contrast,
spiking was suppressed for only w100 ms,
evident in the raw trace.
cell. Rather, ganglion cell adaptation arises from a network mechanism: reduced glutamate release from presynaptic (nonspiking) bipolar cells. This presynaptic
mechanism complements intrinsic mechanisms for slow
adaptation found at later stages of the visual pathway.
Demonstration of Slow Contrast Adaptation
in Intracellular Recordings of Mammalian
Ganglion Cells In Vitro
We tested for slow adaptation in brisk-transient (Y-type)
ganglion cells in an intact (retinal pigment epitheliumattached) in vitro preparation of the guinea pig retina
(Demb et al., 1999, 2001a). We targeted Y-type cells by
recording from the largest cell bodies in the ganglion
cell layer (see Experimental Procedures). We focused
on Y-type cells because we could target them routinely
and because they are probably homologous to magnocellular pathway cells (or a subset of these cells), which
show strong adaptation in vivo (Kaplan and Shapley,
1982; Levitt et al., 2001; Solomon et al., 2004). A cell
was stimulated with a drifting grating that alternated
between high contrast (100%, 10 s) and low contrast
(5%–40%, 20 s; see Experimental Procedures). At the
offset of high contrast, the spiking response in a cell, recorded extracellularly, was completely suppressed and
recovered over a 7.0 s period (Figure 1A). A whole-cell,
voltage recording of the same cell showed that the
period of suppressed spiking corresponded to a period
of membrane hyperpolarization that slowly recovered—
an afterhyperpolarization (AHP; Figure 1B), as reported
in salamander cells (Baccus and Meister, 2002). Across
cells, spiking was suppressed at the offset of high
contrast and took 6.9 6 0.9 s to recover (n = 10; see
Experimental Procedures). Each cell recorded intracellularly showed an AHP during the period of suppressed
spiking (AHP amplitude, 28.8 6 0.7 mV; n = 4).
Many ganglion cells discharge continuously at mean
luminance, and this discharge is suppressed following
a period of high contrast. In one representative cell,
maintained firing took 6.5 s to recover to the baseline
rate (Figure 1D). An intracellular recording of the same
cell showed that the period of suppressed spiking corresponded to the period of an AHP (Figure 1E). Under our
conditions, suppressed spiking following high contrast
was found in nearly all OFF-center Y-type cells but
was weak or absent in most ON-center Y-type cells
(Figure 1F; time to recovery, 0.5 6 0.4 s; n = 6). However,
we observed adaptation in several other types of ONcenter or ON-OFF ganglion cells, suggesting that the
ON pathway did express slow adaptation for certain
cell types. For example, in three direction-selective
ganglion cells (one ON-center type, two ON-OFF types)
Retinal Mechanism for Slow Contrast Adaptation
Figure 2. Longer Periods of High Contrast
Evoke Larger and Longer-Lasting Afterhyperpolarizations
(A) An OFF cell was stimulated with either a
1 s or 8 s grating (100% contrast, 6.7 cycles
mm21; 6 Hz) followed by mean luminance.
Compared to the 1 s grating, the 8 s grating
evoked a larger AHP (26.9 mV versus 210.8
mV) that took longer to recover (2.0 s versus
5.7 s), resulting in a larger AHP integral
(25.5 mV s versus 225.2 mV s; see Experimental Procedures). Recovery was measured as the time required to return 90%
back to the resting potential (see Experimental Procedures).
(B) Longer periods of high contrast increased
the amplitude of the AHP (n = 6 cells). Error
bars indicate SEM across cells.
(C) Longer periods of high contrast increased
the time needed to recover to the resting potential.
(D) Longer periods of high contrast increased
the AHP integral.
recorded intracellularly, the switch from a 4 s grating
(6 Hz; 6.7 cycles mm21; 100% contrast) to mean luminance caused a large AHP (29.5 6 0.1 mV) that recovered over 6.2 6 1.1 s (data not shown).
For periods of high contrast of either 1 or 8 s, the AHP
amplitude increased from 24.0 6 0.6 mV to 27.8 6
0.9 mV and the recovery time increased from 3.6 6
0.8 s to 7.9 6 1.6 s (n = 6 OFF Y-type cells); the AHP integral increased from 25.0 6 0.6 mV s to 220.9 6
2.1 mV s (Figure 2). Thus, longer periods of contrast
evoked larger and longer-lasting AHPs. In the following
studies, we focused on OFF-center Y-type cells to investigate the mechanism for slow adaptation, because we
could target these cells routinely and they showed
strong adaptation as reflected by the AHP.
Spiking Is Neither Sufficient nor Necessary to
Generate the Visually Evoked Afterhyperpolarization
We first tested whether the AHP results from an intrinsic
mechanism (e.g., potassium channel) in the ganglion cell
that is sensitive to sodium influx, as found in cortex;
a cortical cell adapts following either a visual stimulus
or direct current injection (Sanchez-Vives et al., 2000a).
We stimulated the same ganglion cell with either the
grating stimulus or a direct injection of current through
the electrode (Figure 3A). Both the grating and the current step evoked a membrane depolarization and a train
of spikes, and in fact the current step evoked a larger depolarization (8.3 6 0.8 mV versus 7.0 6 0.7 mV; a difference of 1.3 6 0.6 mV, p < 0.05) and a higher spike rate
(27.9 6 4.7 spikes s21 versus 15.7 6 1.8 spikes s21; a difference of 12.2 6 3.3 spikes s21, p < 0.005). However,
only the grating evoked a large and long-lasting AHP
(Figure 3B). Across cells, the AHP amplitude w300 ms
after stimulus offset was 25.8 6 0.6 mV following the
grating versus 22.3 6 0.2 mV following the current step
(difference of 3.4 6 0.5 mV, p < 0.001; n = 9). Four sec-
onds after stimulus offset, the AHP persisted following
the grating (21.9 6 0.3 mV) but was nearly back to baseline following the current step (20.30 6 0.15 mV; difference of 1.6 6 0.3 mV; p < 0.001; sampling window, 1.0 s).
Furthermore, the AHP integral was about four times
larger following the visual stimulus (218.9 6 2.3 mV s) relative to the current step (24.4 6 1.4 mV s; difference,
14.5 6 0.9 mV s; p < 0.001). We also tried sine-wave current injection at the stimulus frequency (6 Hz, +0.2 nA
amplitude; Figure 3C). In this case (n = 8 cells), current injection evoked a higher spike rate (10.3 6 1.1 spikes s21
versus 7.2 6 1.2 spikes s21), but the grating evoked
a larger AHP amplitude (25.1 6 0.5 mV versus 21.0 6
0.3 mV) and a larger AHP integral (217.3 6 2.0 mV s versus 22.8 6 2.0 mV s). Thus, even though current injection
evoked a larger spiking response, the grating evoked
a larger AHP, indicating that spiking alone is not sufficient to generate the full visually evoked AHP. Apparently
the mechanism for slow contrast adaptation differs between retina and primary visual cortex (Sanchez-Vives
et al., 2000b).
We next asked whether spiking was necessary to generate the AHP. We included QX-314 (5 mM) in the pipette
solution to block voltage-gated sodium channels in the
recorded cell (Connors and Prince, 1982). Under these
conditions, a grating stimulus evoked a membrane
depolarization without spiking (n = 10; Figure 3D). Following stimulus offset, each cell showed an AHP that
was, on average, 24.8 6 0.4 mV in amplitude and required 6.2 6 0.6 s to return to the resting potential (AHP
integral, 214.4 6 2.2 mV s). Thus, spiking was not necessary to generate the AHP.
Spatial Sensitivity of the Afterhyperpolarization
Suggests a Presynaptic, Bipolar Cell Mechanism
We hypothesized that the retinal basis for the AHP might
arise from a presynaptic mechanism, possibly in the
Figure 3. Spiking Is Neither Sufficient nor
Necessary to Generate the Afterhyperpolarization
(A) An OFF cell was stimulated with either a
4 s grating (top; 100% contrast, 6 Hz, 6.7 cycles mm21) or a 4 s step of positive current
(bottom; +0.2 nA). Both stimuli generated
membrane depolarization and spiking. At
the offset of the grating, there was a relatively
large AHP (25.5 mV), whereas at the offset of
the current pulse, there was a smaller AHP
(23.2 mV).
(B) Average subthreshold membrane potential and spike rate for the grating stimulus
and the current step (n = 9 cells; 100 Hz sampling). Membrane depolarization and spiking
responses were larger following the current
step, whereas the AHP was larger and longer
lasting following the grating. Thus, spiking
alone, as evoked by the current step, was
not sufficient to generate the full visually
evoked AHP. Stimulus responses were advanced 46 ms in time to align with the current
responses. Spike rate was binned at 250 ms
here and in (C).
(C) Same format as (B), except the current
stimulus was a 4 s period of 6 Hz sine-wave
stimulation with a peak of +0.2 nA (current
injection protocol shown above response
traces). Current injection evoked a higher
spike rate, whereas the grating evoked
a larger and longer-lasting AHP (n = 8 cells).
(D) An OFF cell was stimulated with a 4 s grating (100% contrast, 6 Hz, 6.7 cycles mm21)
while applying QX-314 (5 mM) through the
pipette to block spiking (top). On average
(bottom, n = 10 cells), the AHP was similar
to control conditions; thus, spiking was not
necessary to generate the AHP.
bipolar cells that release glutamate and excite the
ganglion cell. In the above experiments, we routinely
used a high spatial frequency grating, with bar width
of w80–100 mm. This bar width is much narrower than
the w600 mm receptive field center of the ganglion cell,
but similar to the w100 mm receptive field center of bipolar cells (Dacey et al., 2000; Demb et al., 2001b). We
directly examined the effect of spatial frequency on the
AHP amplitude. A low spatial frequency (0.3 cycles
mm21) evoked a strong response at the 6 Hz drift rate
of the grating (F1 amplitude; Demb et al., 2001b; Hochstein and Shapley, 1976), as expected, but produced
only a small AHP (Figure 4A). A high spatial frequency
(6.7 cycles mm21) evoked a steady depolarization with
a small 6 Hz modulation riding on top, followed by a large
AHP (Figure 4A). Both spatial frequencies evoked a
similar average spike rate over time (F0 component;
Figure 4B), and so, as shown above, the magnitude of
the AHP did not directly correspond to the preceding
spike rate.
For primate diffuse bipolar cells (which synapse onto
magnocellular ganglion cells), the receptive field surround strength is 1 to 1.4 times the strength of the receptive field center (Dacey et al., 2000). Assuming a similar
receptive field profile in guinea pig bipolar cells (presynaptic to the Y-type cell), the surround should reduce
each bipolar cell’s response to the low spatial frequency, relative to the optimal spatial frequency, to
less than 30% of the maximal response (Dacey et al.,
2000). Thus, the reduced ganglion cell AHP following
the low-frequency grating would be explained by the reduced response of each presynaptic bipolar cell. This
explanation implies that individual bipolar cells require
strong stimulation in order to adapt their release rate
and drive the ganglion cell AHP.
We measured grating responses to a total of seven
spatial frequencies. As expected, the F1 amplitude
peaked at low frequencies and gradually declined at
higher frequencies (Figure 4C) whereas the AHP amplitude peaked at high frequencies (Figure 4D). To put
this spatial tuning into context, we made two measurements of the ‘‘nonlinear subunit’’ property of the Y-type
cell receptive field, where the subunits apparently represent presynaptic bipolar cells (Demb et al., 2001a,
2001b; Enroth-Cugell and Freeman, 1987; Enroth-Cugell
and Robson, 1966; Hochstein and Shapley, 1976). The
first subunit measure is the mean level of depolarization
(F0) during the grating, and this showed the same spatial
tuning as the AHP (Figure 4C). The second measure was
the F2 (second harmonic) amplitude to a contrastreversing grating (spike recordings; Figure 4E), and this
also showed similar tuning to the AHP (Figure 4E)
(Demb et al., 2001b). Thus, the spatial tuning of the
AHP matches the tuning of the ‘‘nonlinear subunits,’’
suggesting that strong stimulation of bipolar cells is
required to evoke the AHP.
Retinal Mechanism for Slow Contrast Adaptation
Figure 4. The Afterhyperpolarization Is
Evoked Most Effectively by High Spatial Frequencies
(A) In an OFF cell, a low spatial frequency
grating (0.3 cycles mm21) evoked a strong response at the 6 Hz drift rate followed by
a small AHP. A high spatial frequency grating
(6.7 cycles mm21) evoked a tonic depolarization plus a small 6 Hz modulation followed by
a large AHP. Traces in the insets show 500 ms
of response with a fitted 6 Hz sine wave (F1
response; y axis: 280 to 240 mV). In both insets, the cell fires 13 spikes (bursts of five,
four, and four spikes to the low-frequency
stimulus and 13 spikes dispersed over time
to the high-frequency stimulus). Dashed line
indicates the resting potential.
(B) A psth for the two conditions in (A) (bin
size, 500 ms); mean firing rate for the two conditions was similar.
(C) The amplitude of the first Fourier harmonic (F1) of the membrane potential peaked
for low spatial frequencies and declined at
high frequencies, whereas the mean membrane potential (F0 component) peaked at
high frequencies, reaching a peak near w5–
7 cycles mm21 (n = 8 cells). Error bars indicate SEM across cells.
(D) AHP amplitude increased with spatial frequency, reaching a peak near w5 cycles
mm21 (n = 8 cells).
(E) Spike recordings to a drifting or contrastreversing grating illustrate the relative size of the overall ganglion cell receptive field center and the nonlinear subunits; the subunits represent
bipolar cell inputs to the ganglion cell (see Results). The F1 amplitude to a drifting grating is a measure of the overall receptive field center. This
amplitude peaks at low frequencies and declines at higher frequencies. The F2 amplitude to a contrast-reversing grating reflects the nonlinear
subunit response. The subunit amplitude peaks at high frequencies, similar to the pattern of sensitivity of the F0 membrane potential in (C) and
the AHP amplitude in (D) (see also Demb et al., 2001b).
The Afterhyperpolarization Corresponds
to a Decreased Inward Current, Consistent
with a Bipolar Cell Mechanism
We considered two hypotheses for how bipolar cells
could drive a slow AHP in a ganglion cell. First, following
the offset of high contrast, bipolar cell glutamate release
might be suppressed and recover slowly. Second, bipolar cells might drive inhibitory amacrine cells to release
GABA and/or glycine, and this inhibition might require
several seconds to subside. To test between these alternatives, we measured membrane currents under voltage
clamp during and after high-contrast stimulation. The
large cells under study have a low input resistance
(37.9 6 2.1 MU, n = 14) (Cohen, 2001; O’Brien et al.,
2002), and so we initially used holding potentials (Vh)
close to the resting potential (Vrest) to minimize errors
in the current measurements (n = 8; see Experimental
During the high-contrast grating, we measured an
inward current with an estimated reversal potential
(223.9 6 6.7 mV; n = 8) that suggests a mix of excitatory
and inhibitory input (Figure 5B). Following grating offset,
there was an outward ‘‘aftercurrent’’ that recovered
slowly back to baseline. This aftercurrent amplitude
was larger at Vh of w280 mV relative to the amplitude
at Vrest (w265 mV); this pattern (negative slope on an
I-V plot) suggests that the aftercurrent is driven by a reduced excitatory conductance, rather than an increased
inhibitory conductance (Figure 5B). Near Vrest, we measured similar aftercurrents using pipette solutions that
were either K+-based (n = 3) with a weak Ca2+ buffer
(0.1 mM EGTA; 92.9 6 11.3 pA) or Cs+-based (n = 5)
with a strong Ca2+ buffer (10 mM BAPTA; 129.0 6 14.0
pA; see Experimental Procedures). Thus, the aftercurrent did not depend on Cs+-sensitive K+ channels or a
Ca2+-dependent mechanism, which is further evidence
that the mechanism was not intrinsic to the ganglion
We performed additional experiments with TEA in the
pipette to improve the ability to clamp the dendrites at
a positive holding potential (see Experimental Procedures). In the most stable recordings (n = 6), we measured a reversal for the aftercurrent that was +10.3 6
10.2 mV (Figures 5G–5I). This reversal is slightly positive
to 0 mV, which is likely explained by an incomplete
space clamp of the dendrites (i.e., in which case, an
w+10 mV potential at the soma might correspond to
an w0 mV potential at the dendrite). The aftercurrent
showed a more sustained time course at the positive
holding potential, relative to the time course measured
near Vrest (Figure 5G, inset), probably caused by an
increased contribution from NMDA-receptor conductances at positive potentials (Cohen, 2000). Because of
a probable NMDA-receptor contribution to the aftercurrent, the linear fits used to estimate reversal potential are
As a control, we measured an outward current that we
did expect to be driven largely by direct inhibition (Demb
et al., 2001a; Zaghloul et al., 2003): the OFF cell’s ‘‘ON’’
response to a bright spot stimulus (Figures 5D–5F).
Figure 5. The Afterhyperpolarization Corresponds to a Reduced Excitatory Postsynaptic Current
(A) An OFF cell was recorded under voltage
clamp and stimulated with a 4 s grating
(100% contrast; 6.7 cycles mm21; 6 Hz; K+based solution) at a holding potential (Vh) =
245 (gray) or 279 (black). Both the inward
current evoked by grating presentation and
the outward aftercurrent at grating offset
declined at the positive holding potential.
Traces are illustrated at 100 Hz sampling.
Traces here and below are leak subtracted.
(B) Current-voltage (I-V) plot of transient response (black circles) and sustained response (gray circles) to the grating and the
aftercurrent (white circles) from the cell in
(A). Grating responses are consistent with
a mixed chloride + cation current with estimated reversal potential (transient: 223.8 6
10.8 mV; sustained: 223.9 6 6.7 mV; n = 8)
between Ecation (w0 mV) and ECl (273 mV).
The aftercurrent response showed a slight
decline at positive holding potentials, consistent with a reduced cation current but inconsistent with a chloride current. Fitted lines
here and below show linear regressions.
(C) Outward currents following the grating
(aftercurrent) at Vh near Vrest (6 5 mV) or
near 280 mV (65 mV). Relative to Vh = Vrest,
Vh = 280 mV increased the aftercurrent. Error
bars indicate SEM (n = 8 cells).
(D) The same cell in (A) was stimulated with
a spot that reversed contrast (square-wave
reversal; 0.6 mm outer diameter, contrast =
100%) at Vh = 245 or 279 mV.
(E) I-V plot of traces in (D) Across cells, outward current during the positive contrast
shows an estimated reversal of 286.4 6
1.4 mV (n = 8 cells), negative to ECl (273 mV).
(F) Outward currents during the ‘‘ON’’ response at Vh near Vrest (65 mV) or near 280
mV (65 mV). The response decreased at Vh
of 280 mV relative to the response at Vh of
Vrest. Error bars indicate SEM (n = 8 cells).
(G) Same format as (A) for a second cell
probed at positive holding potentials (Cs+based pipette solution with TEA). The aftercurrent became inward at Vh = +47 mV. Inset
shows filtered traces (Gaussian filter, SD =
100 ms); axes represent from 4 to 16 s and from 2200 to +200 pA. The time course of the aftercurrent differed at the two holding potentials, probably because of an NMDA receptor component at +47 mV.
(H) Same format as (B), showing a reversal of the aftercurrent near 0 mV.
(I) Same format as (C), showing the average aftercurrent amplitude with Vh = Vrest (265.5 6 5.6 mV) or w+20 mV (+19.8 6 8.3 mV, mean 6 SD)
across six cells.
(J) An ON cell was recorded under the same conditions as in (A). The aftercurrent was briefer than that in OFF cells but still showed a reduction at
the depolarized holding potential.
(K) I-V plot of response to the grating from the cell in (G); same format as in (B).
Indeed, this outward current showed an estimated reversal negative to Vrest, suggesting that it was driven
largely by a direct inhibitory conductance and demonstrating that our protocol was adequate to see existing
direct inhibitory influences.
Following a grating stimulus, ON-center Y-type cells
also showed an aftercurrent, although it was relatively
brief (n = 3; Figure 5J). This brief period of the aftercurrent explains why spikes were suppressed only transiently in extracellular recordings (see above; Figure 1F).
An ON cell’s aftercurrent corresponded to a reduced inward current, with estimated reversal near 0 mV (Figures
5J and 5K). Thus, ON-center Y-type cells showed an out-
ward current similar to OFF-center cells, except that the
recovery was much faster.
The Afterhyperpolarization Does Not Require
Conductances Driven by GABA or Glycine
Receptors, Calcium-Activated Potassium Channels,
or Metabotropic Glutamate Receptors
The AHP does not appear to be driven by GABA or glycine release onto the ganglion cell (Figure 5); however,
GABA or glycine release feeding back onto the bipolar
terminal could play a role in suppressing glutamate
release. This seemed unlikely based on a previous
experiment with extracellular recording, in which either
Retinal Mechanism for Slow Contrast Adaptation
Figure 6. The Afterhyperpolarization Does Not Require Conductances Driven by GABA or Glycine Receptors, K(Ca) Channels, or Metabotropic
Glutamate Receptors (mGluRs)
(A) An OFF cell was stimulated with a 4 s grating (100% contrast; 6.7 cycles mm21; 6 Hz) under voltage clamp (Vhold = 275 mV). The grating response was recorded under control conditions (A1) and after adding antagonists to glycine and GABAA/B/C receptors (strychnine, 2 mM; bicuculline, 100 mM; CGP35348, 100 mM; TPMPA, 100 mM) (A2). The receptor antagonists caused a large increase in responsiveness (note the different
scales in [A1] and [A2]). However, the aftercurrent, following the stimulus, persisted in the presence of the receptor antagonists ([A3], average
traces are leak subtracted). The arrows in (A2) indicate bursts of inward current present during drug application, which probably represents
bursts of glutamate caused by blocking all major inhibitory synapses throughout the retina. The initial inward current in the average trace in
the presence of the drugs (21.9 6 0.53 nA) has been clipped in the figure (A3). Without leak subtraction, the leak current was 250 6 16 pA in
the control condition and 2372 6 41 pA in the drug condition.
(B) Same format as (A), except that the cell was recorded in current clamp and we applied antagonists to two types of calcium-activated potassium channels: apamin (1 mM) to block SK(Ca) channels, and charybdotoxin (20 nM) to block BK(Ca) channels. Relative to control (B1), the channel
blockers caused bursting of the membrane potential and increased the maintained discharge (B2), but did not block the AHP (B3).
(C) Same format as (B), except that we applied antagonists to all major mGluRs: MCPG (1 mM) to block group I and group II mGluRs, and CPPG (1
mM) to block group III mGluRs. The drugs did not block the AHP following the grating stimulus (C2 and C3). However, during a contrast reversal of
a 0.6 mm diameter spot (2 Hz, 10%–20% contrast), the drugs blocked the hyperpolarizing response to light onset in OFF cells ([C3], inset; averaged across five cells; line above indicates time course of the contrast reversal). This was expected, because the hyperpolarizing response to
light onset depends on an inhibitory synapse from an ON amacrine cell, and the ON pathway is blocked by CPPG (Awatramani and Slaughter,
2000; Zaghloul et al., 2003).
strychnine (a glycine receptor antagonist) or picrotoxin
(a GABAA/C receptor antagonist), when applied individually, did not block slow adaptation in spiking (Brown and
Masland, 2001). However, this experiment left open the
possibility that both GABA and glycine are involved in
adaptation, in which case blocking only one class of receptors at a time would not block the total amacrine cell
contribution. To follow up this result, we measured the
aftercurrent, under voltage clamp, while simultaneously
blocking glycine and GABAA/B/C receptors (strychnine,
2 mM; bicuculline, 100 mM; CGP35348, 100 mM; TPMPA,
100 mM, respectively).
The receptor antagonists caused, in most cases, spontaneous ‘‘bursting’’ and strongly increased the response
to the grating (Figure 6A). However, the aftercurrent
persisted and in fact the amplitude increased (control:
74.1 6 13.1 pA; drugs: 219.5 6 20.9 pA). Furthermore,
the antagonists altered the time course of both the grating response and the aftercurrent. The altered time
courses probably arise from the extreme change in bipolar release under these conditions, caused by removing
all inhibition of the bipolar terminal. Therefore, we do
not draw conclusions from these conditions about the
normal time course of glutamate release. However, we
can conclude that the aftercurrent (and the associated
AHP) does not require inhibitory synaptic transmission.
This provides further evidence that the AHP arises from
reduced bipolar cell glutamate release.
We checked two putative mechanisms for adaptation in bipolar cell release. Bipolar cells express Ca2+dependent K+ channels [K(Ca)](Sakaba et al., 1997), which
could contribute to slow adaptation (Llinas and LopezBarneo, 1988). We blocked K(Ca) channels throughout
the retina (bath applied charybdotoxin, 20 nM; apamin,
Figure 7. At Low Mean Luminance, an OFF
Cell Shows a Depolarized Membrane Potential, Increased Membrane Noise, and an Enhanced Afterhyperpolarization
(A) An OFF cell was stimulated with a 4 s
grating stimulus (100% contrast; 6.7 cycles
mm21; 6 Hz). The response was recorded at
two different levels of mean luminance differing by a factor of ten (see Experimental Procedures). The low mean luminance caused
a depolarized Vrest and increased membrane
noise, suggesting increased presynaptic glutamate release. Low mean luminance also increased the amplitude of the AHP from 27.1
to 212.8 mV.
(B) Across cells, reducing mean luminance by
a factor of ten had multiple effects: depolarization of Vrest, increased synaptic noise (SD
of Vm measured over 2 s, before stimulus onset), and increased AHP amplitude. However,
the response to the grating was similar under
the two conditions. Error bars indicate SEM
across cells (n = 6).
1 mM), and this condition caused bursting in the ganglion
cell membrane potential and an increased maintained
discharge (n = 5; Figure 6B). However, the AHP following
the grating persisted (control amplitude: 24.7 6 1.0 mV;
drugs: 26.0 6 0.6 mV). This result also rules out a role
for K(Ca) channels in the ganglion cell, consistent with results above showing that the aftercurrent persists under
voltage clamp in the presence of high BAPTA (Figure 5).
Another possible mechanism for adaptation in bipolar
cells is metabotropic glutamate receptors (mGluRs) on
the synaptic terminal, which could create a feedback
signal and reduce release (Awatramani and Slaughter,
2001). To test this possibility, we blocked group I, II,
and III mGluRs simultaneously (MCPG, 1 mM; CPPG, 1
mM) (n = 5; Figure 6C). This condition blocked the hyperpolarization at light onset of a spot response, as expected (Zaghloul et al., 2003) (see Figure 6C3 inset).
However, the AHP following the grating persisted (control amplitude: 26.1 6 0.9 mV; drugs: 26.8 6 0.8 mV).
This result also rules out a role for mGluRs as a postsynaptic mechanism to generate the AHP in ganglion
Evidence that Basal Glutamate Release from Bipolar
Cells Affects the Afterhyperpolarization
During the course of the experiments, we varied the
mean luminance (see Experimental Procedures). At
lower mean luminance, we observed in OFF Y-type ganglion cells two effects: a depolarized resting potential
(low, 263.4 6 1.3 mV; high, 268.0 6 1.1 mV; p < 0.05;
n = 6) and increased voltage noise (low, 1.9 6 0.4 mV;
high, 0.7 6 0.1 mV; p < 0.05) (Figure 7). This pattern
implies that glutamate release from OFF bipolar cells increased at the lower mean, presumably driven by increased glutamate release from cones (Demb et al.,
2004). We hypothesized that the AHP in the ganglion
cell would be related to the level of basal glutamate release from bipolar cells: higher basal release (at low
mean luminance), when suppressed, would produce
a relatively larger AHP in the ganglion cell.
We tested the effect of mean luminance on AHP size,
presenting the same cell with the 4 s drifting grating
(100% contrast; 6.7 cycles mm21; 6 Hz) at two levels of
mean luminance, differing by a factor of ten (Figure 7).
At the two levels of mean luminance, the response to
the grating was similar (low, 7.4 6 0.6 mV; high, 9.9 6
1.3 mV; p > 0.10; n = 6). However, the AHP was about
twice as large at low mean luminance (low, 29.0 6
1.1 mV; high, 24.3 6 0.9 mV; p < 0.05). Thus, increasing the apparent basal glutamate release onto the ganglion cell, by reducing the mean luminance, increased
the AHP.
We have demonstrated a slow form of contrast adaptation in the subthreshold membrane potential of mammalian ganglion cells in vitro. Following a period of high
contrast, spike rate was suppressed and required several seconds to recover; we observed suppressed spiking with both extracellular and whole-cell recordings
(Figure 1). The recovery of the spike rate corresponded
to a period of membrane hyperpolarization (Figure 1),
and this afterhyperpolarization (AHP) depended on the
period of prior visual stimulation (Figure 2). Spiking was
neither necessary nor sufficient to generate the full visually evoked AHP (Figures 3 and 4). However, spiking was
sufficient to generate at least a minor component of the
visually evoked AHP (Figure 3). The AHP was strongest
following high spatial frequency stimuli, tuned to presynaptic bipolar cells (Figure 4). Voltage-clamp analysis
showed that the AHP corresponded to a reduced inward
current, consistent with suppressed bipolar cell glutamate release (Figure 5). Pharmacology experiments
ruled out a role for amacrine cell GABAergic or glycinergic synapses or for calcium-activated K+ channels
or metabotropic glutamate receptors in the bipolar cell
(or elsewhere; Figure 6). Lowering mean luminance
apparently increased basal glutamate release from OFF
bipolar cells (Figure 7). At the lower mean luminance, the
Retinal Mechanism for Slow Contrast Adaptation
AHP increased, suggesting a link between basal release
and the size of the AHP (Figure 7).
Retinal Model for Slow Adaptation following
a Period of High Contrast
Our results support a model where, at the offset of high
contrast, bipolar cell glutamate release drops below the
basal rate, and this drop in release hyperpolarizes the
ganglion cell, causing the observed AHP and suppressed spiking. Glutamate release apparently requires
several seconds to return to the basal level, resulting in
gradual membrane depolarization of the ganglion cell
back to its resting potential (Figure 8). In this model, bipolar cells require strong stimulation in order to suppress their release. So, for example, when a low spatial
frequency grating is presented and stimulates a bipolar
cell weakly (due to the influence of the receptive field
surround; Dacey et al., 2000), there would be no suppression at stimulus offset, and the ganglion cell AHP
would be weak or absent (Figure 4).
Our synaptic model for slow adaptation in ganglion
cells is consistent with the conclusion reached in a previous study, based on extracellular recording (Brown
and Masland, 2001). However, we also found a minor
contribution to slow adaptation from an intrinsic property of the ganglion cell: current injection evoked a depolarization and spiking, followed by an AHP with an integral that was, at most, about 25% of the visually evoked
AHP integral (Figure 3). However, relative to the visual
stimulus, the current injection typically evoked greater
depolarization and more spikes. Thus, taking into account the relatively smaller response to the visual stimulus, the intrinsic mechanism for adaptation in the ganglion cell probably contributes less than 25% of the
visually evoked AHP.
Retinal Model for Slow Adaptation during
a Period of High Contrast
Here, we have focused on the slowly recovering AHP following a grating stimulus, but we could also measure
adaptation during the presentation of the grating itself.
This side of adaptation was reflected by an initially
high spike rate that declined during prolonged contrast
stimulation (Figures 1 and 3). This adaptation during the
stimulus seemed to depend on two components. First,
there was an excitatory synaptic component driving the
response, which is apparent in voltage-clamp recordings near ECl (Figures 5 and 8): inward currents were initially large and then declined during continued grating
presentation. This decline could occur due to a decreasing glutamate release rate during the grating or postsynaptic mechanisms of receptor desensitization caused by
the high release (von Gersdorff and Borst, 2002). Possible presynaptic mechanisms for a decline in release during high contrast include vesicle depletion or auto-feedback at the bipolar terminal (DeVries, 2001; Palmer et al.,
2003a, 2003b; Singer and Diamond, 2006). A second
component driving adaptation during the grating was
a spike-frequency adaptation. This adaptation was apparent during direct current injection, which caused
a high rate of spiking that then declined (Figure 3). Spike
frequency adaptation seems to be a general property
of many types of ganglion cells and at least partially
reflects the effect of sodium channel inactivation (Kim
Figure 8. Working Model for the Mechanism of Slow Contrast Adaptation
(A) Glutamate release increases during periods of high contrast to
a high level initially and then to a medium level for the duration of
the stimulus. Following the offset of high contrast, glutamate release
declines sharply. The release rate takes several seconds to recover
to the initial baseline level.
(B) Time course of adaptation during and following high-contrast
stimulation. Trace shows the average response, under voltage
clamp, of eight cells, where Vhold was near ECl (trace is leak subtracted; 100 Hz sampling). The inward current is suppressed soon
after grating onset causing a decrease in the inward current; gray
line shows a double exponential fit with time constants of 19 and
927 ms. This decline in the inward current could reflect multiple
mechanisms, including a drop in glutamate release during high contrast (see [A], stages 2 and 3) as well as possible postsynaptic receptor desensitization caused by the high glutamate release (see Discussion). At stimulus offset, there is an outward aftercurrent that
slowly recovers back to baseline (dashed line); the gray line shows
a single exponential fit with time constant of 6.2 s. This aftercurrent
presumably reflects suppressed glutamate release that requires
several seconds to recover (see [A], stages 4 and 1).
and Rieke, 2001, 2003; O’Brien et al., 2002). Furthermore, this spike frequency adaptation mechanism may
explain why some cortical cells show a decline in spike
rate during a stimulus with no subsequent recovery period following the stimulus (Sanchez-Vives et al., 2000b).
We examined, for excitatory synaptic currents, the relationship between the time course of adaptation during
a high-contrast stimulus and the subsequent recovery
period following the stimulus (Figure 8B). The onset of
adaptation, during the grating, was well fit by two exponentials, with time constants of 19 ms (87%) and 927 ms
(13%). The aftercurrent, following the grating, was well
fit by a single exponential with a time constant of 6.2 s.
Thus, the decline of the inward current during the stimulus is apparently w10–100 times faster than the recovery time. There are two explanations for this asymmetry
in time course between these two sides of adaptation.
First, as described above, the period during the grating
alone could involve mechanisms of synaptic depression
that relate specifically to periods of high transmitter release (e.g., postsynaptic receptor desensitization; von
Gersdorff and Borst, 2002), and these mechanisms
could shorten the time constant for this period of adaptation relative to the time constant for the aftercurrent.
Second, even without the involvement of postsynaptic
mechanisms, such as receptor desensitization, there
appears to be an asymmetry between the onset and
recovery from depression of presynaptic release. For
example, suppressed release from the rod bipolar
cell shows fast onset (<1 s) with a slow recovery time
(w10 s; Singer and Diamond, 2006). A similar asymmetry
exists at the calyx of Held, where depression can be
induced in <1 s but requires several seconds to recover
(von Gersdorff et al., 1997; Wang and Kaczmarek, 1998).
Furthermore, a similar rapid onset with slow recovery
exists, on a different time scale, for sodium channel
inactivation (Colbert et al., 1997).
Comparison between Guinea Pig and Primate Retina
A recent study based on extracellular recordings in vivo
suggested that the suppressed spiking following high
contrast arises from a postsynaptic mechanism in the
ganglion cell (Solomon et al., 2004). That conclusion
was based on an experiment in which a stationary, contrast-reversing grating was positioned so as to evoke no
response in the ganglion cell (because the border between grating bars was centered over the cell’s receptive field, in a ‘‘null’’ phase). Following this stimulus,
the ganglion cell responses were not suppressed, which
suggests that the ganglion cell must necessarily be
stimulated in order to evoke an adaptive effect. This result would be consistent with an intrinsic mechanism in
the ganglion cell for adaptation, rather than a network
mechanism involving bipolar cells. There are several explanations for the discrepancy between these findings in
primate and ours. Two explanations relate to the different recording conditions (in vivo versus in vitro) and the
different species (primate versus guinea pig). For example, the intrinsic mechanism for adaptation in the in vitro
guinea pig cells was a minor component of adaptation
under our conditions (Figure 3), but this component
might be more prominent in the in vivo primate cells.
Even within primate experiments, there are differences
in slow adaptation between in vitro and in vivo conditions (Chander and Chichilnisky, 2001; Solomon et al.,
2004). Another example of a difference between the
guinea pig and primate studies relates to adaptation in
ON and OFF cells. The primate study found adaptation
in both ON and OFF magnocellular cells, whereas we
found strong adaptation in OFF cells but only weak effects in ON cells; presently we cannot explain this discrepancy (Solomon et al., 2004).
We offer one further explanation for the lack of adaptation following the ‘‘null’’ stimulus in the primate
study (Solomon et al., 2004). Magnocellular ganglion
cells in the previous study were recorded at 5º–25º
eccentricity, which should have dendritic tree diameters
of w40–140 mm (Croner and Kaplan, 1995; Perry et al.,
1984). These ganglion cells probably collect from up
to w30 bipolar cells (Calkins, 1999; Jacoby et al.,
2000), which would correspond to w6 cells across the
width of the dendritic tree. We also assume that each bipolar cell receptive field width is w90–100 mm (Dacey
et al., 2000). Furthermore, the central-most bipolar cells
contribute the largest number of synapses onto the ganglion cell (Kier et al., 1995). Thus, based on this pattern of
convergence, it is likely that the ‘‘null’’ stimulus for the
ganglion cell was also largely ineffective at strongly driving the central-most bipolar cells. Given this weak stimulation, these central-most bipolar cells would not show
an adaptive effect after the stimulus was removed, and
this might explain the lack of adaptation in the ganglion
cell. However, intracellular studies of magnocellular ganglion cells are clearly required to fully resolve this issue.
Nonspiking cells exist in many sensory systems, and
these cells may also express mechanisms of slow adaptation. At present, the most likely mechanism in bipolar
cells is an activity-dependent suppression of glutamate
release. This mechanism apparently does not involve
inhibitory synaptic feedback, K(Ca) channels in the bipolar cell, or mGluRs at the bipolar terminal (Figure 6;
Awatramani and Slaughter, 2001; Sakaba et al., 1997).
Furthermore, voltage recordings from salamander retina
suggest that the AHP in ganglion cells exists in the absence of an AHP in presynaptic bipolar cells (Baccus
and Meister, 2002; Rieke, 2001). Thus, the most likely
mechanism for the ganglion cell AHP is a depressed bipolar cell glutamate release that is not reflected by a hyperpolarization of the bipolar cell membrane potential.
Further studies will be required to elucidate the mechanism for depressed bipolar cell glutamate release, as it
relates to contrast adaptation. This would apparently require a novel preparation in mammalian retina: the ability
to record from pairs of cone bipolar cells and postsynaptic neurons (amacrine or ganglion cell) where the bipolar voltage can be controlled while its release is read
out directly by the postsynaptic neuron. Presently,
such paired recordings, which require routine identification of synaptically connected cells, have only been accomplished in the rod pathway (Singer and Diamond,
2006; Singer et al., 2004).
Experimental Procedures
In each experimental session, a guinea pig was anesthetized with
ketamine (100 mg kg21) and xylazine (10 mg kg21) and decapitated,
and both eyes were removed. All procedures conformed to NIH and
University of Michigan guidelines. The back of the eye (retina, pigment epithelium, choroids, and sclera) was mounted flat in a chamber on a microscope stage. The retina was superfused (w6 ml min21)
with oxygenated (95% O2 and 5% CO2) Ames medium (Sigma,
St. Louis, MO) at 33ºC–35ºC. The retina and electrode were visualized using a cooled CCD camera (Retinga 1300C, Qcapture software; Qimaging corporation, Burnaby, British Columbia). The largest cell bodies in the ganglion cell layer (20–25 mm diameter) were
targeted for recording. A glass electrode (tip resistance, 2–6 MU)
was filled with Ames’ solution for extracellular recording, or intracellular recording solution. Intracellular solutions included K+-based
solution (solution 1; in mM: K-methylsulfate, 140; NaCl, 8; HEPES,
10; EGTA, 0.1; ATP-Mg2+, 2; GTP-Na+, 0.3; titrated to pH = 7.3);
K+-based solution with QX-314 (solution 2), where NaCl was reduced
Retinal Mechanism for Slow Contrast Adaptation
to 3 mM and QX-314-Br was added (5 mM); or Cs+-based solution
(solution 3) where Cs-methane sulfonate (120 mM) replaced K-methylsulfate and BAPTA (10 mM) replaced EGTA; Cs+-based solution
with TEA (solution 4), where TEA-Cl (5 mM) and Lucifer Yellow
(0.1%) were added and QX-314-Br was reduced to 2 mM. The chloride reversal potential (ECl) indicates the reversal of the synaptic
response to GABA or glycine and includes a contribution from bromide; the calculated reversal was w273 mV for solutions 1 through
3 and w267 mV for solution 4. All chemicals were purchased from
Sigma-Aldrich (St. Louis, MO) except for BAPTA (Invitrogen;
Eugene, OR), Strychnine (Fisher Scientific; Pittsburgh, PA), and
(RS)-MCPG and CPPG (Tocris, Bristol, UK).
Membrane potential was amplified, continuously sampled at
10 kHz, and stored on computer using a MultiClamp 700A amplifier
and pClamp 9 software (Axon Instruments, Foster City, CA; Zaghloul
et al., 2005). Junction potential was corrected in all cases. We wrote
programs in Matlab (The Mathworks, Natick, MA) to analyze responses in the spike rate, subthreshold membrane potential, or
membrane currents. For current-clamp recordings, we balanced
the bridge every few minutes in cases where we injected current.
For voltage-clamp recordings, we corrected for an error in the holding potential introduced by the series resistance. The corrected holding potential (Vh_corr) was determined by the formula Vh_corr = Vh 2
(Ileak 3 Rs 3 (1 2 Rs_correct)), where Vh is the apparent holding potential before the stimulus (in mV), Ileak is the leak current (in nA), Rs is the
series resistance (18.2 6 0.3 MU; n = 14) and Rs_correct is the series
resistance compensation (typically 0.4–0.5). For positive holding
potentials (Figure 5B), we allowed an outward current, unblocked
by Cs+ or 5 mM TEA, to inactivate (w30–60 s) before recording the
visual response. Results are from 80 cells: 16 ON cells and 64 OFF
cells. The resting potential (Vrest) of OFF cells was similar between
experimental conditions (K+-based solution, 266.0 6 1.5 mV, n =
20; K+-based solution with QX-314 solution, 265.6 6 1.5 mV, n =
10; Cs+-based solution, 264.8 6 0.7 mV, n = 5; Cs+-based solution
with TEA, 264.2 6 2.2 mV, n = 6).
Visual Stimuli
The stimulus was displayed on a miniature monochrome computer
monitor (Lucivid MR1-103; Microbrightfield, Colchester, VT) projected through the top port of the microscope through a 43 objective and focused on the photoreceptors (mean luminance, w103–
104 isomerizations cone21 s21; resolution, 640 3 480 pixels; 60 Hz
vertical refresh). The relationship between gun voltage and monitor
intensity was linearized in software with a lookup table. Stimuli were
programmed in Matlab as described previously (Brainard, 1997;
Demb et al., 1999; Pelli, 1997). Cell type was determined using
methods described previously (Zaghloul et al., 2005), and cell health
was ascertained by repeated measurements of the responses to
spots, annuli, and gratings.
The grating was either windowed in a circular patch (0.75 mm diameter; in most experiments) or presented over a 3 3 3 mm field (Figure 4). The gratings drifted at 6 Hz and in most cases had a spatial frequency of 5–7 cycles mm21 and contrast of 100%. All stimuli were
centered on the cell body. In some cases, a neutral density filter was
inserted in the light path to change the mean luminance by a factor
of ten.
Except where noted, we measured AHP or aftercurrent amplitude by
averaging over 100 ms centered at times noted in the text. Recovery
time of the AHP was determined by fitting a polynomial function to
the AHP and determining the time required for the fit to return 90%
back to the baseline response level. We used standard fitting routines in Matlab. To determine the AHP integral, we normalized the
trace by subtracting Vrest and measured the integral of the trace,
over an 8 s period, starting at grating offset. Spike poststimulus
time histograms are binned at 500 ms, except where noted. Average
membrane potential traces are shown with the resting potential subtracted. Data are reported as mean 6 SEM.
search to Prevent Blindness Career Development Award (J.B.D.),
an Alfred P. Sloan Foundation Fellowship (J.B.D.), and the NIH
(EY14454 and EY07003; T32-DC005341 and T32-EY13934, support
to M.B.M.).
Received: August 29, 2005
Revised: February 17, 2006
Accepted: March 30, 2006
Published: May 3, 2006
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