We used albino guinea pigs weighing 300-700 g. An animal was overdosed with anesthetic (ketamine 100 mg/kg; xylazine 20 mg/kg and pentobarbital 50 mg/kg) and both eyes were removed and hemisected. The eyecup was cut radially and flattened onto a membrane filter with ganglion cells up. This preparation was placed into a chamber on a microscope stage (BX50WI, Olympus America, Melville, NY) and superfused with oxygenated (95% O2 and 5% CO2) Ames medium (Sigma, St. Louis, MO) containing sodium bicarbonate (1.9 g/l) and glucose (0.8 g/l) at ~5-7 ml/min. The chamber temperature was maintained between 34-37 C°. The retina was dark adapted for 0.5 h before measurements were made.
To help find particular retinal regions during the experiment, holes were cut in the membrane filter (). When the tissue was flattened onto the filter, the small central hole (0.8-1 mm diameter) was positioned over the optic disk and the filter was oriented so that the other two elongated holes were located above or below the optic disk. The middle of each region was aligned along the superior-inferior axis of the tissue through the optic disk (3 mm superior or inferior to the optic disk). Recordings were made mainly from within these regions. In the first experimental stage of the study (defined below), recordings were also made from regions in superior retina closer to the optic disk. Retinal locations of the recording sites were measured relative to the optic disk.
S/M opponent cells are present in superior and inferior retina
Ganglion cells were identified for recording by observing the tissue through the infrared differential interference contrast (DIC) optics of the microscope () with a 60X (0.9 NA) objective. Cells were recorded extracellularly with loose patch electrodes (tip impedance = 2 - 4 MΩ). Responses were amplified (NeuroData IR-283, Cygnus Technology, Delaware Water Gap, PA), digitized at a 5 kHz sampling rate with 12-bit precision (DigiData 1200, Molecular Devices, Sunnyvale, CA), and stored for later analysis (AxoScope, Molecular Devices, Sunnyvale, CA). Spikes were identified using a threshold detection method. Spike rates were calculated for ~15 ms bins.
Searching for S/M opponent cells
To search for S/M opponent cells, we went through four experimental stages for a total of 66 experiments, adopting different search strategies in each stage. In the first stage (n = 17 experiments), we began by patching ganglion cells in superior retina and testing every cell for opponency (see the following section for a description of the opponency test.) In targeting cells for patching we excluded brisk-transient ganglion cells, which could be identified because of their large soma size, but otherwise patched all cells encountered. Later analysis revealed that the patched cells generally had soma sizes between 10-15 μm, with a few as large as 18 μm (; soma size was typically estimated along the minor axis.) We patched more than 145 cells in the first stage and encountered three opponent cells. Prior to the first stage, in experiments conducted as part of a separate study, one additional opponent cell was encountered near the superior region of the transition zone, where some M cones coexpress S opsin (Röhlich et al., 1994
). We include that cell here (; morphology in the third panel from top in .)
Dendrites of S/M opponent cells were located in the IPL's ON stratum
For the opponent cells encountered in the first stage, we observed certain anatomical and physiological properties in common. For example, they tended to have a medium soma size and high maintained discharge rate in light. We took those properties as predictive of whether a ganglion cell would be opponent in stage two of the experiments.
In stage two (n = 26 experiments), we searched for ganglion cells in superior retina that matched the properties described above and only took the time to test for opponency when a cell matched these qualitative selection criteria. As we encountered more opponent cells (n = 9 more cells in stage two, out of more than 1085 cells patched), we gradually refined the qualitative selection criteria. These observations led us to develop quantitative screening criteria for identifying S/M opponent cells in superior retina: (a) medium soma size, ranging between 10-14 μm and (b) high maintained discharge rates to a uniform background (main background, see below; >15 spikes/s), and also moderate to high maintained rates in the dark (> 5 spikes/s). All opponent cells encountered in this study satisfied these criteria (; maintained rates in dark are not shown, as they were not measured for some of the opponent cells in the first stage.) Criterion (a) was based on anatomy and was met by a high percentage of ganglion cells (visual observation; Do-Nascimento et al., 1991
). Criterion (b) was based on physiology and was met only by a very small percentage of ganglion cells (see ). In our experience, most ganglion cells had a maintained rate to the main background of ≤10 spikes/s. We also studied more than 41 cells from inferior retina in stage 2, without encountering an opponent cell.
In stages one and two we were focused on finding opponent cells and developing a sense of their general properties. We did not in these exploratory stages keep explicit records of every non-opponent cell patched. The reported numbers (145 for superior retina in stage one, 1085 for superior retina in stage 2, 41 for inferior retina for stage 2) are the number of cells explicitly documented, and represent a lower bound on the number of cells actually patched.
In the third stage (n = 18 experiments), we applied the screening criteria developed in stage two for experiments conducted in both superior and inferior retina. We only patched ganglion cells with appropriate soma size. Of all the cells patched in superior retina (327 cells, 1 opponent), we tried to study in detail those that had appropriate maintained rates and were opponent (). Of all the cells patched in inferior retina (1392 cells, 3 opponent), we tried to study every cell with appropriate maintained rates, except those that responded weakly or not at all to contrast (). We occasionally studied cells whose maintained rates fell below the cutoffs we established and none of those cells were opponent.
The final experimental stage (stage four) was designed to check whether the physiological screening criteria might have excluded a major population of opponent cells. In this stage (n = 6 experiments), we tested every cell patched in superior retina for opponency, and studied all of the opponent cells encountered (n = 3, out of 122 cells patched). All of the opponent cells identified in this stage satisfied the criteria established in stage two (). We also patched 15 cells in inferior retina during this stage, none of which were opponent.
Characterizing chromatic response properties of ganglion cells
Chromatic response properties of ganglion cells were characterized by recording responses to spots of different spectral composition. Flicker experiments measured responses to spots on a spatially uniform background. These spots were produced by modulating mixtures of two primary lights. This method provided an efficient way to assess whether a cell was opponent, and to estimate the spatial extent of its receptive field. We used this method, as described above, to identify S/M opponent cells. Flash experiments were designed to provide a quantitative characterization of a cell's opponency. These experiments measured responses to flashed monochromatic spots on a spatially uniform background. This method was slow but allowed direct characterization of the spectral properties of a ganglion cell. We used this method not only to verify that a cell was opponent, but also to measure the relative strengths of M-cone, S-cone and rod inputs (M and S and rod photopigment weights).
We used the same two optical systems as in our previous study (Yin et al., 2006
). Briefly, (1) LCD system
, light from an LCD projector (PowerLite 730c, Epson America, Long Beach, CA) produced images that subtended ~2.4 × 3.2 mm on the retina, with each pixel corresponding to ~3.1 × 3.1 μm. (2) Lamp system
, light from a xenon lamp (HLX 64642, Osram, Germany), collimated and fed through a tunable narrowband filter (VariSpec, Cambridge Research & Instrumentation, Woburn, MA) and an adjustable aperture produced a uniform spot of monochromatic light (~10 nm bandwidth) on the tissue. The temporal profile of the spot was controlled by a mechanical shutter (VS25S2T1 shutter and 122-BP controller, Uniblitz, Rochester, NY). The outputs of the two optical systems were combined through a beamsplitter and delivered to the tissue through the camera port on the microscope and the objective (4X). Full details of the configurations of the two systems, and on spectral characterization and calibration, are reported in Yin et al. (2006)
. As the original lamp system did not produce sufficient light intensity at 430 nm for this study we expanded the lamp system with another channel, which was fed through a narrowband interference filter of 430 nm.
The flicker experiments used the LCD system alone to modulate a spot (~100-1600 μm diameter) around the background at 2-4 Hz (sinusoidal temporal waveform). The main background produced nearly equal isomerization rates in M and S cones (4.06-4.17 and 4.01-4.12 log10 Rh*/photoreceptor/s) and a somewhat higher rate in rods (4.65-4.76 log10 Rh*/photoreceptor/s). The exact background intensities are noted in the text where necessary. In the earlier experiment (described above, one opponent cell), a CRT instead of the LCD projector was used in the optical system. The background used in that experiment produced isomerization rates of 3.99 (M), 3.53 (S), 4.44 (Rod) log10 Rh*/photoreceptor/s.
Reported isomerization rates were computed as described in Yin et al. (2006)
, based on parameters estimated for intact guinea pig retina. In our in vitro
preparation, the basic orientation of the receptors with respect to the incident light was the same as for intact retina, but detailed differences in orientation could produce deviations between computed and actual isomerization rates. We did not correct for any such differences. Note that our analyses of cell properties are not sensitive to a change in scaling of the computed isomerization rates, since these were based on contrast.
Ganglion cells were adapted continuously throughout the recording. To screen a cell for opponency, a sequence of flickering spots modulated along M-cone and S-cone isolating directions as well as in M+S and M−S color directions was repeated (typical stimulus parameters: ~800 μm in diameter, temporal frequencies of 2 or 4 Hz, duration of 8 sec for each direction). The spike traces and voltage pulses marking the start and end of each modulation cycle were visualized using AxoScope (Molecular Devices, Sunnyvale, CA). From the relative response amplitudes and phases to modulations along the four color directions, we could determine whether a cell was opponent. The characteristics of S/M opponent cells were that (a) they responded to modulations along both M- or S-cone isolating directions, but with opposite response polarities and (b) they responded strongly to modulation along the M−S opponent direction but weakly to the M+S non-opponent direction (). Non-opponent ganglion cells responded with the same polarity to modulation along both M-cone and S-cone isolating directions, and more strongly to modulation along the M+S non-opponent direction than the M−S opponent direction. In some experiments, to test cells for opponency as efficiently as possible, the flickering stimulus was presented directly through the 60X objective instead of 4X objective. This reduced the size of the stimulus. We did not perform a separate spectral calibration for the 60X objective.
S/M opponent cells characterized by flickering and flashed monochromatic spots
We recorded the spike responses to flickering spots of ganglion cells identified as opponent by the screening procedure (typical stimulus parameters: 800 μm in diameter, temporal frequency of 4 Hz, duration of 32 sec for each direction). The number of contrasts presented for each color direction (M-isolating, S-isolating, M+S, M−S) varied from cell to cell. For some cells, we also recorded area summation data in the form of responses to flickering spots of diameters between 100 μm and 1600 μm (Figs. and , and Supplemental Fig. 1
). For quantitative analysis, the response was taken as the signed fundamental response amplitude (F1) of the peristimulus time histogram (PSTH), with the sign of the response indicating whether the fundamental was best described as in-phase or out-of-phase with the stimulus modulation (after accounting for response delay). Responses during the first and last stimulus cycles were excluded from the analysis.
S- and M- cone receptive field components derived from DOG model
The flash experiments used monochromatic spots from the lamp system on a background produced by the LCD system. The main background was the same that used for the flicker experiments, and cells were adapted continuously throughout the recording. A sequence of flashed monochromatic spots of wavelengths between 430-620 nm was presented. We refer to the presentation of a series of flashed monochromatic spots at one wavelength as a trial, and to the presentation of a sequence of trials for different wavelengths as a block; stimulus parameters: 800-900 μm diameter, flash duration of 300 ms at 1 s intervals (500 ms for two cells), 12 to 32 repetitions per trial, and 19 to 30 trials per block.
The spike traces and voltage traces marking the flash onset and offset were visualized using AxoScope (Molecular Devices, Sunnyvale, CA) and fed to an audio-speaker for real time monitoring. From the response polarities to monochromatic flashes at short, middle, and long wavelengths we could decide whether a cell was opponent. The characteristics of S/M opponent cells were that (a) they had spectral neutral points, that is a wavelength range where they responded only weakly to monochromatic flashes and (b) they responded to monochromatic flashes of wavelengths longer or shorter than spectral neutral points with opposite response polarities.
As the PSTHs were noisy, we derived fits to each individual PSTH using a template method and extracted the response amplitudes from the fits (Appendix A
). Responses were taken as the peak of spike rate increment from baseline at either the flash onset or offset (). We report photopigment weights derived from spectral measurements for seven opponent ganglion cells and one possibly opponent ganglion cell (see Results
; ). We report spectral neutral points for five additional cells where full spectral characterization was not possible (see Results
; ). Stimulus parameters for these cells were the same, except that one was studied with a 2000 μm diameter spot.
Photopigment weights of S/M opponent cells vary across retina and differ from those for horizontal cells and other types of ganglion cells
Spectral neutral points of S/M opponent cells vary across retina
Estimating photopigment weights
From the responses to monochromatic spots, we could estimate the relative strength of M, S, and rod inputs to a ganglion cell. Appendix B
describes the linear non-linear (LNL) model we used to estimate contrast-response non-linearities, action spectra, and photopigment weights. The responses of all nine ganglion cells where we had sufficient flash data to perform this analysis were well-described by the LNL model. For these cells, the maximal cone or rod contrasts presented (across wavelengths) were less than 120% for three cells, 150% for three cells, 200% for one cell, 250% for one cell and 350% for one cell.
Dye staining, immunostaining, and quantitative morphology
After recording, some ganglion cells were penetrated with a sharp electrode and stained with Lucifer yellow (3%, in 0.1 M LiCl solution; Invitrogen, Carlsbad, CA) to visualize their morphology. To verify that the cell stained was the one recorded, DIC views of the cell soma were captured before the loose patch electrode was released and after the sharp electrode was positioned near the soma. After staining, the tissue was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 1 h in dark and store in PB in 4 C° before immunostaining.
After a brief wash in PB, the retina was peeled from the pigment epithelium and incubated in blocking buffer (10% normal goat serum, 5% Triton X-10 in PB with 5% sucrose) for 1 h to reduce non-specific binding. We reacted the retina with primary antibodies to Lucifer yellow to enhance the staining of the dendrites of the ganglion cell, and to S opsin to reveal the overlaying S cone mosaic. For Lucifer yellow we used rabbit anti-Lucifer yellow (Invitrogen, Carlsbad, CA) at a dilution of 1:500 and for S opsin we used rabbit anti-S opsin (Chemicon, Temecula, CA) at a dilution of 1:600. The reaction was carried out initially at room temperature for 2 h, then at 4 C° for 35 h. After several washes in PB with 5% sucrose (SPB), we reacted the retina with secondary antibodies to visualize the primary antibody staining (4 h, at room temperature). Both the dendrites of the ganglion cell and S opsin were tagged with FITC or CY3 (Invitrogen, Carlsbad, CA). After several washes in SPB, we reacted the tissue with SYTO 61 or SYTO 13 nucleic acid stains (Invitrogen, Carlsbad, CA) to visualize the amacrine cell layer (ACL) and the ganglion cell layer (GCL), which outlined the boundaries of the IPL. Finally after several washes in SPB, we mounted the retina on a glass slide with the ganglion cell side up and covered it with mounting medium (Vectashield, Vector Laboratories, Burlingame, CA) and a coverslip.
The tissue was examined using a confocal microscope (Leica TCSNT, Leica Microsystems, Germany) with 10X (0.3 NA), 40X (1.25 NA) and 100X (1.4 NA) objectives. Image stacks were obtained at two z-depths, with one covering IPL, including the stained ganglion cell and another one covering the photoreceptor layer, including the S cone outer segments. To quantify the dendritic morphology of a ganglion cell: (a) we estimated the diameter of the dendritic field from the tangential view at 20X (10X objective with 2X magnification; 500 × 500 μm). We drew a polygonal contour connecting the most distal dendritic tips and then fit the polygon with an ellipse using the NIH image software (http://rsb.info.nih.gov/nih-image/
). The estimated diameter was the average length of the major and minor axis of the fitted ellipse. (b) We measured the length of the ON or OFF dendrites from the tangential image stack at 40X (250 × 250 μm) by tracing all dendrites in 3D using the Volocity software (Improvision, Lexington, MA). The density of the ON dendritic processes was calculated by dividing the length of ON dendrites by the area of the polygon containing all the ON dendrites in the same view. This density is one of the parameters that proved effective for morphological classification of ganglion cells in a cluster analysis reported by Kong et al. (2005)
. (c) We measured the relative depths of distal dendrites within IPL at several locations from the tangential image stack at 40X and sometimes 100X (250 × 250 μm), also using the Volocity software. The depth of dendritic stratification was expressed as the percentage of the total depth of the IPL, starting from 0% at the boundary with the amacrine cell layer and ending with 100% at the boundary with the ganglion cell layer. To generate radial views, we re-sliced a section of ~250 × 60 μm within the tangential image stack using the NIH image software (Figs. and ).
Bistratified ganglion cells did not show explicit opponency
We quantified the dendritic membrane area of opponent ganglion cells using a procedure similar to that described by Xu et al. (2008)
. First, we divided the tangential image stack obtained at 40X magnification into sub-regions: a circular region of 30 μm diameter centered around the cell soma and a series of outward expanding concentric rings with equal widths of 25 μm. All of the image stacks analyzed had x- and y- resolutions of 0.24 μm and a z-resolution of 0.49 μm. One image stack (that shown in ) was excluded because its z-resolution was larger than the diameters of typical distal dendrites. Second, after smoothing the images with 3 × 3 median filter, we used the Volocity software to recognize and reconstruct the dendrites contained in each of the ring sub-regions. Finally, we measured the dendritic membrane area from the reconstructed dendrites and calculated the dendritic membrane density by dividing the dendritic membrane area by the retinal area of corresponding ring.