doi: 10.1038/nature06150
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Light adaptation in cone vision involves switching between receptor and post-receptor sites
Felice A. Dunn1, Martin J. Lankheet4, & Fred Rieke2, 3
1Program
in Neurobiology and Behavior
Hughes Medical Institute
3Department of Physiology and Biophysics
University of Washington
Seattle, WA 98195, USA
4Functional Neurobiology and Helmholtz Institute
Utrecht University
3584 CH Utrecht, The Netherlands
2Howard
Supplementary Methods
Isolation of cone-mediated signals
The mixing of rod and cone signals occurs at several locations within the retina26. Rod and cone photoreceptors
themselves are coupled through gap junctions, causing light responses in cones to contain contributions from signals
originating in both rod and cone outer segments (Fig. S1a, bottom panel)27. Rod input to L cones was minimal in
responses to long-wavelength light (Fig. S1a, top panel). Rod-mediated responses of postsynaptic cells (ON cone
bipolar and ganglion cells) were still apparent, though reduced, for long-wavelength light (e.g., Fig. S1c, top panel);
thus rod-mediated responses in these cells were dominated by mixing occurring within the retinal circuitry, presumably from input to cone bipolar cells generated by the rod bipolar pathway.
Cone-mediated responses in ganglion cells were isolated through a combination of long-wavelength flashes and rodadapting backgrounds (Fig. S1b and c). Responses of both midget and parasol ganglion cells to long-wavelength
flashes in darkness had two temporal components. Two aspects of the slow component indicated it was mediated by
the rods. First, its time course matched the response to a dim short-wavelength flash (Fig. S1b and c, bottom panels).
Second, the ratio of the sensitivity of the slow component to short- and long-wavelength flashes matched almost
exactly the predicted ratio of 560 based on the rod spectral sensitivity (dotted lines in Fig. S1b and c, top panels).
Dim, short-wavelength backgrounds suppressed the rod component of the ganglion cell response to long-wavelength
flashes without affecting the fast (cone-mediated) component (Fig. S1b and c, top panels).
Rod-mediated signals in ON cone bipolar cells did not have as clear a temporal signature as those in ganglion cells,
making an approach similar to Figure S1 difficult for these cells. Instead rod-mediated signals in ON cone bipolar
cells were minimized by blocking the AMPA receptors required for transmission from the rod bipolar cell to the AII
amacrine cell, thus eliminating input to ON cone bipolar cells from the rod bipolar circuit. To check for unanticipated
effects of the pharmacology, we also used rod-adapting backgrounds to isolate cone-mediated responses in cone
bipolar cells (Fig. S2). Due to the difficulty distinguishing rod- and cone-mediated components of the cone bipolar
cell responses, we used backgrounds sufficient to suppress rod-mediated responses in midget ganglion cells. Conemediated signals isolated pharmacologically or by rod-adapting backgrounds had a similar dependence on background light (Fig. S2).
Supplementary Discussion
Comparison with past work
Our work builds on previous studies of retinal adaptation. In amphibians and fish, adaptation contains contributions
from the cones and the retinal circuitry (reviewed by ref. 28-30). Post-receptor adaptation in turtle includes a contribution from horizontal cell feedback to cones31, 32. The most complete picture of adaptation in mammalian retina
comes from studies in primate. Adaptation has been examined in cone photoreceptors33, 34, horizontal cells35-37, ganglion cells38, 39, and in human psychophysics38 (reviewed by ref. 40). Previous studies demonstrate that at least two
sites contribute to adaptation of cone-mediated signals (see reviews by ref. 29, 40-42).
Cone signals characterized here resemble past work in some respects but not in others. Cone amplitudes were reduced to half their value in darkness for backgrounds near 5,500 absorbed photons per cone per second (P* s-1; approximately 290 monkey trolands43), comparable to 1,200 P* s-1 from massed potential measurements44 and 8,700
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P* s-1 in previous recordings from single cones34. The kinetics of our cone responses differ in two respects from past
single cone recordings. First, cone responses showed a much more pronounced speeding with increasing backgrounds than observed previously. Similar speeding with background has been reported in massed recordings45 and
in the cones of other species46, 47 but is absent in past single primate cone recordings33, 34. Second, while previous
primate cone recordings exhibited strongly biphasic responses, our responses were monophasic (i.e., did not undershoot) or weakly biphasic at all light levels tested, more similar to recent cone recordings48, 49. We do not know the
reason for these kinetic differences.
One synapse after the cones, primate horizontal cells exhibit cone-type specific, spatially localized adaptation35 at
backgrounds within a factor of two of the cone photoreceptor adaptation probed with brief flashes36. Thus horizontal
cells appear to inherit most of their adaptation from the cones. The horizontal cell responses sped with background,
unlike past cone measurements but consistent with those reported here. Considered collectively, the cone and horizontal cell measurements indicate that cone adaptation is prominent only for backgrounds exceeding 1,000 P* s-1.
Both parasol and midget ganglion cell spike responses, recorded in vivo, exhibit adaptation at backgrounds well below 1,000 P* s-1 {38, 39}. Thus cone adaptation alone cannot account for midget or parasol ganglion cell adaptation.
The work summarized above leaves several open questions which form the basis of our work. First, comparisons
made between studies (e.g., between cones and horizontal cells and between cones and ganglion cells) are based on
different recording conditions and often different stimuli and are further subject to uncertainty in light calibrations
(see, e.g., discussion of ref. 36). Our work compares the different cell types directly using consistent experimental
conditions. Second, rod- and cone-mediated signals were not well separated in some past ganglion cell recordings.
Hence adaptation of rod-mediated signals could account for some of the differences between cone and ganglion cell
adaptation. Third, comparison of adaptation in midget and parasol ganglion cells in past work is difficult either because of differences in rod contributions39 or differences in stimuli used to probe the two ganglion cell types38. Finally, the locus of adaptation within the retina remained a mystery until the midget and diffuse cone bipolar cells,
connecting the cones to ganglion cells, could be recorded directly.
Comparison of ganglion cell response kinetics to previous work
The kinetics of the excitatory synaptic inputs we measure in ON midget and parasol ganglion cells are largely similar
to spike responses measured in other work, with a few notable differences.
The time to peak and duration of the midget ganglion cell synaptic inputs are similar to estimates from responses to
sinusoidal gratings for a wide range of backgrounds39 and to the spike-triggered average from noise stimuli at backgrounds near 20,000-30,000 P* s-1 {50}. The time to peak and duration of the parasol ganglion cell synaptic inputs at
backgrounds exceeding 3,000 P* s-1 are similar to estimates from grating responses39, 51 and to the spike-triggered
average52; for dimmer backgrounds the synaptic inputs were briefer than estimates from gratings39, possibly reflecting a contribution of rod input to the grating responses.
Both midget and parasol input currents were less biphasic than spike responses measured in other work, and correspondingly the amplitude spectra for the input currents had less attenuation of low temporal frequencies. Attenuation
of high temporal frequencies appears similar, though the grating measurements provide superior resolution beyond
10-20 Hz. Inhibitory synaptic inputs (absent in our measured excitatory synaptic currents), the dynamics of spike
generation, and differences in stimuli may all contribute to the greater low frequency attenuation. Inhibition and the
dynamics of spike generation could similarly cause the adaptation we measure to differ from previous measurements
of spike responses.
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Supplementary Figures and Legends
a
L cone photoreceptor
b
Midget ganglion cell
c
Parasol ganglion cell
pA per P*
640 nm flash
Dark
-1
46 Rh* s
-1
153 Rh* s
0.005
scaled dark
470 nm flash
-1
0.0
-0.5
-2
0.000
0
470 nm flash
0.10
pA per Rh*
0
0
0.010
500
1000
Dark
-1
46 Rh* s
-1
153 Rh* s
0.05
500
Time (ms)
1000
-10
-15
800
0
scaled dark
470 nm flash
0
400
500
800
0
-2000
Dark
-1
1.4 Rh* s
-1
2.8 Rh* s
-1
5.6 Rh* s
-1000
0
400
500
0
-500
0.00
0
-10
0
0
-5
0
400
Time (ms)
800
-4000
-6000
-8000
Dark
-1
1.4 Rh* s
-1
2.8 Rh* s
-1
5.6 Rh* s
0
400
Time (ms)
800
Figure S1 Isolation of cone-mediated responses in cones and ganglion cells.
a, b, c, Average responses of an L cone photoreceptor (a), a midget ganglion cell (b), and a parasol ganglion cell
(c) to a 640 nm flash (top panels) and a 470 nm flash (bottom panels) in darkness (black) and on three 470 nm
backgrounds (gray traces; numbers indicate background intensities in absorbed photons per rod per second (Rh* s1)). Insets in b and c (top panels) show isolated cone responses on brightest backgrounds. Dotted lines in b and c
(top panels) represent the response to the 470 nm flash in darkness (bottom panels) scaled by the ratio of the rod
sensitivity to the 470 and 640 nm LEDs (a factor of 560). Flashes delivered at time 0 and were 10 msec in duration.
Responses are normalized by the flash strength (P* for cone photon absorptions and Rh* for rod photon absorptions). Bandwidth 0-100 Hz. Differences in response amplitudes in b and c reflect different gains of cone signals
(top panels) and rod signals (bottom panels) reaching the ganglion cells. 470 and 640 nm flashes produced different
response shapes, violating the principle of univariance and providing evidence for the involvement of more than one
photopigment (i.e., rhodopsin and the L cone pigment).
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doi: 10.1038/nature06150
b
5µM NBQX
Dim blue mean
pA per P*
0.02
0.00
0.00
Dark
-0.01
-1
1,000 P* s
-1
10,000 P* s
-0.02
0
100 200 300
Time (ms)
1
0.1
-0.02
-0.04
c
Amp/Ampdark (2-8 Hz)
a
0
L cone fit
Diffuse cone bipolar current
NBQX (n = 15)
Dim blue mean (n = 8)
100 200 300
Time (ms)
0
100
2
10
-1
IB (P* s )
4
10
Figure S2 Two methods of isolating cone-mediated responses in cone bipolar cells.
a, Average responses of ON diffuse cone bipolar cells perfused with 5µM NBQX to block transmission between the
rod bipolar and AII amacrine cells and eliminate rod signals coming through the rod bipolar pathway (identical to
Fig. 2d, right panel). b, Average responses of ON diffuse cone bipolar cells with a constant 470 nm rod-adapting
background producing 120-500 Rh* s-1 (red). Responses measured with additional backgrounds of 350 (gray for all
backgrounds without specified color), 930 (blue), 3,500, 11,000 (green), 25,000 P* s-1. c, Background-dependence
of response amplitude (mean ± s.e.m.) for diffuse cone bipolar cells in presence of NBQX (open diamonds) and with
a rod-adapting background (closed triangles). Amplitudes (Amp) were measured between 2-8 Hz and normalized by
the amplitude in darkness (Ampdark). Solid line is fit to cone currents from Figure 1b.
a
b
Amp/Ampdark (18-22 Hz)
Midget pathway
Parasol pathway
1
L cone (n = 19)
L cone fit
Diffuse cone bipolar
input current (n = 15)
Diffuse cone bipolar
output voltage (n = 15)
Parasol ganglion cell (n = 11)
L cone (n = 19)
L cone fit
Midget cone bipolar
input current (n = 11)
Midget cone bipolar
output voltage (n = 7)
Midget ganglion cell (n = 18)
Midget ganglion cell
(slice) (n = 3)
0.1
0
100
2
4
10
10
-1
IB (P* s )
0
100
2
4
10
10
-1
IB (P* s )
Figure S3 Background dependence of amplitudes at higher frequencies dominated by receptor
adaptation.
a, b, Average response amplitudes (Amp), measured from 18-22 Hz and normalized by the amplitude in darkness
(Ampdark), show cells of the midget (a) and parasol (b) pathways adapt similarly to the cones. Receptor adaptation
dominates at these higher frequencies, whereas post-receptor adaptation dominated at lower frequencies in Figure
1b. Solid line represents fit of cone currents to equation (1) (Methods) with a, b fixed at 100, 1.3, and c = 0.00012.
Error bars represent ± s.e.m. for the populations of each cell type.
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a
b
mV per P*
0
-2
Dark
-1
1,000 P* s
-1
10,000 P* s
!
100
200
300
Time (ms)
-1
-2
-3
c
Horizontal cell
0
100
200
300
Time (ms)
400
pA
0
-3x10
-3
-4x10
0
NBQX to block feedback
mV per P*
control
control
5 µM NBQX
200
0
0
100 200 300
Time (ms)
Figure S4 Cone photoreceptors show similar adaptation with and without horizontal cell feedback.
a, b, Average voltage flash responses of 5 L cone photoreceptors in a flat mount in darkness (red) and on backgrounds of 1,000 (blue) and 10,000 P* s-1 (green) under control conditions (a) and with AMPA receptors blocked (b)
to eliminate cone transmission to horizontal cells (5 µM NBQX). Cone adaptation measured in slices also persisted
in the presence of NBQX. c, Horizontal cell current responses recorded in a slice to a saturating flash under control
conditions (black) and in the presence of 5 µM NBQX to block AMPA receptors (gray). NBQX eliminated horizontal cell light responses but did not affect cone receptor adaptation. Thus at the tested light levels receptor adaptation
does not require horizontal cell feedback.
Supplementary Notes
26. Bloomfield, S. A. & Dacheux, R. F. Rod vision: pathways and processing in the mammalian retina. Prog Retin Eye
Res 20, 351-384 (2001).
27. Schneeweis, D. M. & Schnapf, J. L. Photovoltage of rods and cones in the macaque retina. Science 268, 10531056 (1995).
28. Naka, K. I., Chan, R. Y. & Yasui, S. Adaptation in catfish retina. J Neurophysiol 42, 441-454 (1979).
29. Shapley, R. & Enroth-Cugell, C. in Progress in Retinal Research Vol. 3 (eds Osborne, N. & Chader, G.) 263-346
(Pergamon, London, 1984).
30. Burkhardt, D. A. Light adaptation and contrast in the outer retina. Prog Brain Res 131, 407-418 (2001).
31. Tranchina, D., Gordon, J. & Shapley, R. M. Retinal light adaptation--evidence for a feedback mechanism. Nature
310, 314-316 (1984).
32. Burkhardt, D. A. Synaptic feedback, depolarization, and color opponency in cone photoreceptors. Vis Neurosci
10, 981-989 (1993).
33. Schnapf, J. L., Nunn, B. J., Meister, M. & Baylor, D. A. Visual transduction in cones of the monkey Macaca fascicularis. J Physiol 427, 681-713 (1990).
34. Schneeweis, D. M. & Schnapf, J. L. The photovoltage of macaque cone photoreceptors: adaptation, noise, and
kinetics. J Neurosci 19, 1203-1216 (1999).
35. Lee, B. B., Dacey, D. M., Smith, V. C. & Pokorny, J. Horizontal cells reveal cone type-specific adaptation in primate retina. Proc Natl Acad Sci U S A 96, 14611-14616 (1999).
36. Smith, V. C., Pokorny, J., Lee, B. B. & Dacey, D. M. Primate horizontal cell dynamics: an analysis of sensitivity
regulation in the outer retina. J Neurophysiol 85, 545-558 (2001).
37. Lee, B. B., Dacey, D. M., Smith, V. C. & Pokorny, J. Dynamics of sensitivity regulation in primate outer retina: the
horizontal cell network. J Vis 3, 513-526 (2003).
www.nature.com/nature
5
doi: 10.1038/nature06150
SUPPLEMENTARY INFORMATION
38. Lee, B. B., Pokorny, J., Smith, V. C., Martin, P. R. & Valberg, A. Luminance and chromatic modulation sensitivity of
macaque ganglion cells and human observers. J Opt Soc Am A 7, 2223-2236 (1990).
39. Purpura, K., Tranchina, D., Kaplan, E. & Shapley, R. M. Light adaptation in the primate retina: analysis of changes
in gain and dynamics of monkey retinal ganglion cells. Vis Neurosci 4, 75-93 (1990).
40. Hood, D. C. Lower-level visual processing and models of light adaptation. Annu Rev Psychol 49, 503-535 (1998).
41. Hood, D. C. & Finkelstein, M. A. in Visual Psychophysics: Its Physiological Basis (eds Boff, K. R., Kaufman, L. &
Thomas, J. P.) (Academic Press, New York, 1986).
42. Walraven, J., Enroth-Cugell, C., Hood, D. C., DIA, M. L. & Schnapf, J. L. in Visual Perception: The Neurophysiological Foundations 53-101 (Academic Press, 1990).
43. Makous, W. L. Fourier models and the loci of adaptation. J Opt Soc Am A Opt Image Sci Vis 14, 2323-2345
(1997).
44. Valeton, J. M. & van Norren, D. Light adaptation of primate cones: an analysis based on extracellular data. Vision
Res 23, 1539-1547 (1983).
45. Seiple, W., Holopigian, K., Greenstein, V. & Hood, D. C. Temporal frequency dependent adaptation at the level of
the outer retina in humans. Vision Res 32, 2043-2048 (1992).
46. Baylor, D. A. & Hodgkin, A. L. Changes in time scale and sensitivity in turtle photoreceptors. J Physiol 242, 729758 (1974).
47. Matthews, H. R., Fain, G. L., Murphy, R. L. & Lamb, T. D. Light adaptation in cone photoreceptors of the salamander: a role for cytoplasmic calcium. J Physiol 420, 447-469 (1990).
48. Hornstein, E. P., Verweij, J. & Schnapf, J. L. Electrical coupling between red and green cones in primate retina.
Nat Neurosci 7, 745-750 (2004).
49. Hornstein, E. P., Verweij, J., Li, P. H. & Schnapf, J. L. Gap-junctional coupling and absolute sensitivity of photoreceptors in macaque retina. J Neurosci 25, 11201-11209 (2005).
50. Benardete, E. A. & Kaplan, E. The receptive field of the primate P retinal ganglion cell, I: Linear dynamics. Vis
Neurosci 14, 169-185 (1997).
51. Benardete, E. A. & Kaplan, E. The dynamics of primate M retinal ganglion cells. Vis Neurosci 16, 355-368 (1999).
52. Chichilnisky, E. J. & Kalmar, R. S. Functional asymmetries in ON and OFF ganglion cells of primate retina. J Neurosci 22, 2737-2747 (2002).
www.nature.com/nature
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