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MS 10203 Received 8 October 1999; accepted after revision 17 July 2000.
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ABSTRACT |
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INTRODUCTION |
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Polymorphism of colour vision is common among New-World primates (Mollon et al. 1984; Jacobs, 1996; Kremers et al. 1999). In most species, there is only one X chromosome locus with three alleles coding for opsins in the middle-to-long wavelength range. This makes males obligatory dichromats, whereas females, if their two X chromosomes code for different opsins, achieve trichromacy. The callitrichids (such as the marmoset) show alleles with spectral absorption maxima at ca 543, 556 and 563 nm, whereas larger species (the cebids, such as the squirrel monkey) possess alleles with maxima at ca 535, 550 and 563 nm. This had been thought to be a universal pattern, but several exceptions are now known (Jacobs et al. 1993, 1996; Jacobs, 1998). This variety in colour vision in New-World monkeys offers the opportunity to test hypotheses concerning the origin of red-green trichromacy and its physiological substrates.
Trichromacy not only requires two middle-to-long wavelength opsins, but also suitable post-receptoral mechanisms to utilize the cone signals. Anatomically, retinae of New-World primates contain neurons with all the features found in Old-World primates, although some quantitative differences are present (Silveira et al. 1994; Ghosh et al. 1996; Goodchild et al. 1996; Wilder et al. 1996; Yamada et al. 1996a,b; Chan et al. 1997; Silveira et al. 1998b). The retinae of trichromats and dichromats have as yet been indistinguishable anatomically. The pioneering physiological and behavioural studies of Jacobs and co-workers (Jacobs, 1974, 1983a,b), together with microspectrophotometric measurements (Mollon et al. 1984), were the first to suggest a sexual dimorphism, but since the New-World polymorphism has been well established, its physiological consequences have only been investigated in the relay cells of the marmoset lateral geniculate nucleus (LGN) (Yeh et al. 1995b; Kremers & Weiss, 1997; Kremers et al. 1997; Kremers & Lee, 1998; White et al. 1998). In trichromats, cell properties of the magnocellular (MC) and parvocellular (PC) pathways were qualitatively similar to those found in the LGN of macaques. In dichromats, PC and MC cells again had the usual properties (e.g. low and high achromatic contrast sensitivity, absence and presence of contrast gain control) but lacked cone opponency.
We pursue here the physiology of the New-World primate visual pathway into the retina of the capuchin monkey, Cebus apella. The capuchin belongs to the second group of platyrrhines, the cebids. The eye of this species is of a size which permits direct recording from the ganglion cells. Through close quantitative comparison of ganglion cell behaviour in capuchin and macaque we wished to further establish the homology between PC and MC pathways in New- and Old-World primates, and obtain clues as to whether their trichromacy had a common origin.
By recording from dichromats, we confirmed that the capuchin conforms to the usual New-World primate polymorphic pattern. Analysis of the three allelic forms of the X-linked gene showed that the spectral tuning substitutions are identical to those of the squirrel monkey (Hunt et al. 1998). For trichromats, we recorded responses of PC and MC cells using chromatic stimulus protocols, so as to be able to compare cell behaviour with similar data from the macaque. Lastly, we examined the temporal response and contrast sensitivity of cells from dichromats and trichromats, again for comparison with macaque data.
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METHODS |
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Cebus apella were obtained from breeding colonies at the Centro Nacional de Primatas in Belem; recordings were performed at the Federal University of Para. Also, three male capuchin were studied in Göttingen. Males were selected from the colony at random. Following completion of recordings, animals were killed by an overdose of barbiturate and a sample of liver was excised for genetic analysis. In order to screen females, blood samples were taken from six individuals maintained in the animal quarters of the Federal University of Para; trichromatic females were identified as described below.
Electrophysiological procedures
Care and maintenance of animals in Brazil and Germany followed procedures approved by the Animal Care Committee of the State of Lower Saxony. Animals were initially anaesthetized with an intramuscular injection of ketamine (ca 20 mg kg-1). Later, anaesthesia was maintained by intravenous infusion of sufentanyl (0·5-4 µg kg-1 h-1). EEG and ECG were continuously monitored to ensure adequate depth of anaesthesia and analgesia. Muscular paralysis was achieved by infusion of 5 mg kg-1 h -1 of gallamine triethiodide, and the animal was respired with O2 to which ca 1-2 % CO2 had been added. End-tidal PCO2 was kept between 4 and 5 % and body temperature maintained within the normal limits. The capuchin eye was prepared in a similar way to the eye of the macaque (Lee et al. 1989b), and recording of ganglion cell activity was performed as in that species. In some animals, we implanted stimulation electrodes in the optic chiasm to test antidromic activation latencies, using a stereotaxic atlas prepared from Nissl-stained sections obtained after perfusion of two males from early experiments. However, inter-animal variability in stereotaxic co-ordinates was so large that consistent electrode placements, and consistent retrograde activation, were difficult to obtain.
Visual stimuli were presented through a Maxwellian view system with red, green and blue diodes (LEDs) as light sources (Lee et al. 1990; Yeh et al. 1995b). The system could be rotated about the pupil so as to centre the stimulus on a cell's receptive field. The temporal waveforms for the LEDs were generated by a computer through 12-bit digital-to-analog converters. The LEDs were driven by means of a frequency-modulated pulse train which provided a highly linear relationship between driving voltage and light output. The spectral energy distribution of the LEDs was measured using a spectroradiometer (Model pro-703/PC, Photo Research, Burbank, CA, USA). The dominant wavelengths of the LEDs were 638, 554 and 460 nm. The blue LED was used mainly for testing blue-on cells (Silveira et al. 1999). For other neurons, the red and green LEDs alone were normally used. We chose to set the relative luminances of the LEDs equal by means of heterochromatic flicker photometry (HFP) by an observer whose luminosity function matched the 2 deg Judd (1951) spectral sensitivity. This seemed a convenient choice, since the 535 and 563 nm opsins resemble in spectral sensitivity those of Old-World primates; however, we would not wish to suggest that the cone ratios - and thus the luminosity function - of trichromatic capuchins with those opsins is like that of humans. Mean chromaticity was 595 nm. Retinal illuminance levels (Westheimer, 1966) for the red and green LEDs together was ca 2000 Td, but because of the small capuchin eye (Silveira et al. 1989) the retinal flux per troland is about 2 times that in humans, and about 1·1 times that in macaques.
For each cell, we recorded responses to four different stimulus sets. To clarify the relation between the different stimuli, diode modulation waveforms are sketched in Fig. 1. (1) Responses to 200 ms pulses were measured to assess the time course of responses; pulses were incremental or decremental in luminance, and redward or greenward chromatic perturbations (Fig. 1A). (2) To assess cells' spectral sensitivity and search for signs of cone opponency, we measured responses to sinusoids. In a modified HFP protocol, relative modulation depths of the red and green diodes were varied while keeping mean chromaticity and luminance constant. Modulation depths of both diodes were varied, as shown in Fig. 1B. Non-opponent cells show a null or response minimum at some 638/554 nm ratio, giving a cell's relative sensitivity to the two wavelengths. (3) Relative phase of the 638 and 554 nm lights was varied, with modulation amplitude held constant (Fig. 1C). A phase of ±180 deg corresponds to chromatic modulation and of 0 deg luminance modulation. The protocol provides a means of assessing cone opponency and its temporal properties. A formal description of this stimulus can be found in Lindsey et al. (1986), and the way in which cell data can be modelled is described in detail by Smith et al. (1992). For MC cells, 20 or 50 % modulation contrast was employed, for PC cells 50 or 100 % contrast. Six temporal frequencies were measured. (4) Responses to sinusoids as a function of temporal frequency and contrast were measured, usually at twelve temporal frequencies from 0·61 up to 78 Hz at multiple contrasts. Luminance modulation, with the LEDs in phase, was employed for all cells, and for opponent cells responses to chromatic modulation, with the LEDs out of phase, were also measured. For luminance modulation, luminance contrast was calculated as (Lmax - Lmin)/(Lmax + Lmin). For chromatic contrast, cone contrast was calculated in a similar manner, but using cone absorptions. For all sinusoidal modulation conditions, about 6 s of activity was averaged for each condition, and first- and second-harmonic amplitudes and phases were extracted.
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Continuous line, 636 nm LED; dashed line, 554 nm LED. A, to determine responses to step changes, 200 ms pulses (incremental and decremental luminance, and redward and greenward chromatic steps) were used. B, for the HFP protocol, 19 different ratios of 638/554 nm modulation depths were used, ranging from 0·1 (ten times the modulation in the 554 nm light than in the 638 nm light) to 10 (vice versa). As indicated, to achieve this range the modulation depths of both LEDs were modified through the series of ratios. C, for the phase protocol, modulation depth was held constant and the relative phase of the two LEDs was varied. | ||
Genetic analysis
DNA was isolated from liver tissue using a standard method of proteinase K digestion, followed by phenol-chloroform extraction. The absorption maxima (563, 550 or 535 nm) of the capuchin middle-to-long wavelength visual pigments are largely determined by a substitution at codon 180 in exon 3 of the opsin gene, and by substitutions at codons 277 and 285 in exon 5 (Hunt et al. 1998). The polymerase chain reaction (PCR) was used to amplify regions within the two exons that include these three tuning sites, using the following primer pairs and annealing temperatures: 5'-ATGACGGGTCTCTGGTCCCTG-3' and 5'-CTCCAACCAAAGATGGGCGG-3' at 55°C for exon 3, and 5-GAATTCCACCCAGAAGGCAGAG-3' and 5'-GTCGACGGGGTTGTAGTAGTGGC-3' at 55°C for exon 5. Each reaction contained 0·05 U of Taq polymerase, 200 ng each of dATP, dCTP, dGTP and dTTP, 200 ng of the respective primers, 1·5 mM MgCl2, and approximately 20 ng of genomic DNA in 50 µl of reaction buffer. Products of the amplification were viewed by ethidium bromide staining (0·5 µg ml-1) after electrophoresis in 1·5 % agarose using a buffer containing 0·02 M Tris-acetate and 0·5 mM EDTA pH 8·0. Amplified fragments were eluted from the gel and direct sequenced using FS cycle sequencing (Perkin Elmer) and an ABI 373a sequencer. Genotypes of female monkeys were scored on the basis of heterozygosity/homozygosity and base sequence at codons 180, 277 and 285.
Brief reports of some of these results have appeared elsewhere (Lee et al. 1996; Silveira et al. 1996, 1998a).
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RESULTS |
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Photopigments of Cebus apella
Earlier flicker ERG measurements on male Cebus apella had identified animals with pigment maxima near 550 and 563 nm, i.e. two of the three phenotypes of the typical New-World pattern (Jacobs & Neitz, 1987), but the pigment sequences were unknown. In preliminary experiments, we recorded from two males, which we provisionally identified as possessing pigments with maxima at ca 535 and 563 nm using the HFP and phase protocols sketched in the Methods section and further described below. Tissue from the third male phenotype (550 nm; identified through microspectrophotometry) was kindly provided by J. Bowmaker, and tissue from all three animals was subjected to genetic analysis. Sequencing revealed that allelic variation in the X-linked opsin gene of the capuchin at the critical spectral tuning sites is identical to that in the squirrel monkey (Hunt et al. 1998). As pigment spectra, we therefore used microspectrophotometric measurements from squirrel monkey cones (J. M. Bowmaker, personal communication). To provide smooth spectra, these were fitted with the pigment template provided by Lamb (1995). It was unnecessary to correct for macular pigment, since recordings were parafoveal, and lens correction is minor for the 554 and 638 nm diodes.
General observations
We recorded from five further male animals which were identified as possessing either the 535 (1 animal) or 563 nm (4 animals) opsins. Of six females screened genetically, five were trichromats. Four possessed the 535 and 563 nm opsins, and one the 535 and 550 nm opsins. The final animal was a homozygote with the 563 nm opsin. Data were obtained from two 535/563 nm trichromats and one 535/550 nm trichromat, and from the dichromat. The experimenters did not have prior knowledge of the animals' genotypes.
The dichromatic female was readily identified by the lack of frequency-doubled and phase-shifted responses (see below) in MC cells, and the lack of any recordings from red-green cone-opponent neurons. Since ganglion cell behaviour of the dichromatic female was very similar to that of male dichromats, data from males and the female dichromat have been combined.
Cell receptive fields were plotted on a tangent screen. Most fields (78 %) were within 10 deg of the fovea. Initial cell classification was attempted with flashed spots of different colours. Cells with excitatory short wavelength-sensitive (S) cone input were clearly distinguished by their vigorous response to short-wavelength light. In macaques, these neurons have been identified as small bistratified ganglion cells (Dacey & Lee, 1994). A detailed comparison of the anatomy and physiology of this cell class in New-World primates and the macaque is presented elsewhere (Silveira et al. 1999).
Cells with high achromatic contrast sensitivity and transient responses were tentatively identified as MC cells, whereas cells with low contrast sensitivity and sustained responses were identified as PC cells. In trichromatic females, such cells were frequently red-green colour selective. Further support for these classifications was provided by quantitative analysis as described below.
Determination of spectral sensitivity of non-opponent cells: dichromats
Before describing manifestations of cone opponency, we first describe non-opponent cells' spectral sensitivities. We used the HFP test with the 638 and 554 nm light sources, and also the phase protocol where the relative phase of the two lights was varied (see Methods).
In dichromats, these tests gave an immediate indication of the phenotype. For the HFP protocol, the 554 and 638 nm LEDs were modulated in counterphase and their relative modulation depth manipulated, without change in mean chromaticity or luminance. Cone modulation as a function of relative modulation depth was calculated on the basis of the pigment spectral sensitivities and is plotted in the templates shown in the upper panel of Fig. 2A, relative modulation depth being plotted on a logarithmic axis. The discontinuity in the template at a ratio of one is a consequence of modulation depths of both LEDs being varied through the series of ratios tested.
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A, in the HFP protocol, the relative modulation depths of counter-phase modulated 554 and 638 nm LEDs were manipulated. A relative amplitude of unity corresponds to the human luminosity spectral sensitivity function, where a standard observer sees minimum flicker. Based on cone pigment spectra, the expected cone signals' amplitudes were predicted. The discontinuity in the 535 nm template is because modulation depths of both 554 and 638 nm diodes were manipulated to provide the different ratios. In the lower panels are shown data and fitted curves from the 563 and 535 nm phenotypes (one MC ( | ||
First-harmonic response amplitudes for typical MC and PC cells are shown for two male animals in the lower panels in the left-hand column of Fig. 2. The MC cell data have been shifted up by 50 impulses s-1 for clarity, with this level indicated by a line segment around the cell's null. The 554/638 nm ratios at which response nulls occurred are described by the templates, which have been scaled to give the best least-squares fit. When responses are vigorous, away from the null, some indication of response saturation was often observed for MC cells, so that there were deviations between data and template, the former having more pronounced 'shoulders'. Nevertheless, identification of the different phenotypes is unambiguous.
In the phase protocol, the modulation depths of the two lights are held constant and their relative phase is altered. In-phase modulation of the two lights corresponds to luminance modulation, and counterphase modulation corresponds to chromatic modulation. Response amplitude and response phase templates, calculated from the pigment spectra, are given in the upper panels of Fig. 2B and C. This technique has two advantages. Spectral sensitivity can be derived from response phase data, which is usually robust even if response amplitude is weak or noisy. Secondly, this protocol provides useful information as to the temporal properties of the mechanisms providing input to the receptive field, especially if opponent input is present (Smith et al. 1992). In the lower panels of Fig. 2B and C are shown examples of fits of templates to the data from the two phenotypes, for an MC and a PC cell. The amplitude templates have been scaled and the phase templates have been translated vertically to provide the best least-squares fits. The data again provide an unambiguous confirmation of the phenotype.
Spectral sensitivity of non-opponent cells: trichromats
Detailed measurements were obtained from 32 non-opponent ganglion cells in 535/563 nm trichromats. Responses of such cells were usually transient with high contrast sensitivity, and the cells were tentatively identified as cells of the MC pathway. We were specifically interested in the spectral sensitivity of this cell group, since, due to X-inactivation, patchiness of the cone array might occur in New-World primates (Born et al. 1976; Nagy et al. 1981; Jordan & Mollon, 1993). If MC cells were to draw their input from a cone mosaic in which distribution of the opsins were patchy, a greater variability in spectral sensitivity might be expected than in Old-World primates, in which it is currently thought that the cone mosaic is random (Mollon & Bowmaker, 1992). This variability should be reflected in variability of cone weightings in the HFP and phase protocols.
When measuring spectral sensitivity of non-opponent cells with these protocols, we usually analysed 19·5 Hz data, since this frequency is high enough in the macaque to avoid complications due to a chromatic input to the receptive field surround, or rod input. Figure 3 shows results from such measurements. Figure 3A shows results using the phase protocol and Fig. 3B using the HFP protocol. Cell spectral sensitivity was intermediate between that observed in the 535 or 563 nm dichromats. This can be seen in Fig. 3B, where the cell minimum lies between the expected minima for the two opsins seen in Fig. 2A. We fitted HFP data assuming linear summation of inputs from cones with the two opsins. There were two free parameters, an amplitude scaling term and a cone weighting term. However, response saturation was often so marked that we also incorporated a Naka-Rushton saturation function. This was done for the fit in Fig. 3B. For the phase protocol, we used the analysis of the surround mechanism of excitatory S-cone cells described in Smith et al. (1992), where fit equations may be found. In this earlier work we first fitted the phase and then the amplitude data. Here, we fitted data in the complex plane to simultaneously provide fitted curves for amplitude and phase. Of the three free parameters, a cone weighting term determined the slope of the phase curve and the shape of the amplitude plot while the other parameters were a response amplitude scaling term and a phase term which determined the ordinate position of the phase curve and was dependent on temporal frequency. Fits to the data were satisfactory.
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Spectral sensitivity of 563-535 nm trichromat achromatic (MC) cells was measured using the same protocols as in Fig. 2. A, amplitude and phase data for the phase protocol were fitted with a model assuming simple summation of cone signals. Stimulus parameters as in Fig. 2. B, flicker photometric protocol on the same cell. The null is close to unity, indicating that the 562:535 nm cone weighting for this cell was ca 2:1 as for a standard human observer. C, distributions of cone weights over the cell sample (32). Comparison macaque data were taken from Kaiser et al. (1990). Distributions of cone weightings are similar in the two species, arguing against significant patchiness in the cone array. Details of the physiological measurements as in Fig. 2. | ||
Cone weighting parameters obtained with the two protocols were similar. Spectral sensitivity was intermediate between that expected of the two pigments. Figure 3D summarizes the distribution of cone weighting parameters compared with estimations of the cone weightings of MC cells of the macaque (Kaiser et al. 1990). Distributions for the two species are similar. If the spectral sensitivity of MC cells is simply determined by sampling from the cone array covered by the dendritic tree, this result would argue against a strongly non-random cone distribution in the retina of the trichromatic capuchin.
MC cell properties resemble those of macaque
Macaque MC cells show evidence of chromatic input to the receptive field surround, which is responsible for two phenomena in their responses to full-field stimulation. Firstly, a frequency-doubled response to chromatic modulation (Lee et al. 1989a) appears to arise from a rectified chromatic signal (Lee et al. 1993). Secondly, a phase shift with the phase protocol is apparent at low temporal frequencies (Smith et al. 1992). Both these phenomena were found in MC cells of trichromatic capuchin monkeys and were lacking in those of dichromats.
Responses of an MC cell which showed a pronounced frequency-doubled response are shown in Fig. 4A and B. Three histograms illustrate the response at 9·76 Hz to the extremes of the HFP range tested and to a point close to the first-harmonic null. With the latter 554/638 nm modulation ratio, a second-harmonic response appears. In Fig. 4B are plotted first- and second-harmonic amplitudes as a function of 554/638 nm ratio; the second-harmonic amplitudes rise to a peak close to the first-harmonic minimum. This behaviour resembles that seen in the macaque (Lee et al. 1989a). The phase of the second-harmonic, relative to the first-harmonic responses above and below, is as in the macaque, with the second-harmonic peaks occurring midway between those in the upper and lower histograms. The second-harmonic response was not present with modulation close to a tritan confusion line (not shown). Finally, also as in the macaque, the non-linearity appears to be localized to the receptive field surround. Figure 4C and D shows two sets of data with a 4 deg and a 0·5 deg field. The second-harmonic response is absent with the smaller stimulus. Although all these features match macaque findings, second-harmonic amplitudes were generally smaller than those found in macaques. Six of 28 cells showed no obvious second-harmonic response. Such cells are rare in the macaque at the retinal illuminance (2000 Td) used here.
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A, averaged responses of MC cell to two cycles of counterphase modulation of the 554/638 nm lights at relative modulation depths as indicated at left. The first harmonic null is associated with a response at twice the modulation frequency. The phase of the frequency-doubled response is such that peaks lie midway between those in the upper and lower histograms. B, plots of 1st ( | ||
When the phase protocol (Fig. 1) is tested on macaque MC cells, at temporal frequencies below 10 Hz the minimum amplitude of responses occurs not at ±180 deg relative diode phase (chromatic modulation) but is shifted (Smith et al. 1992). This corresponds to psychophysical data (Lindsey et al. 1986); when human observers view the same stimulus, minimum flicker is perceived not at ±180 deg but is shifted to the quadrant where the 638 nm leads the 554 nm light. Such shifts were also seen in many capuchin MC cells. Amplitude and phase data for an on- and an off-centre cell are shown in Fig. 5, for three of the six frequencies tested. The points indicate measured values and the continuous curve represents the fit of the model described by Smith et al. (1992). At 1·22 Hz the phase of least response is where the 638 nm light is ca 90 deg phase advanced relative to the 554 nm light. This phase shift diminishes with increasing frequency. For the macaque, the model previously used consisted of a centre mechanism which summed middle- (M) and long-wavelength (L) cone inputs and a chromatic (|M - L|) input to the surround. Only a model of this sort gave an adequate account of the data; models relying on, for example, a difference in timing of the M- and L-cone signals were not satisfactory. There are five free parameters, which are attributed to the following physiological substrates: centre-surround balance, centre response phase, surround response phase, a cone balance term for the surround and an amplitude scalar. The data were fitted in the complex plane using a least-squares criterion. This model also gave a satisfactory fit to the capuchin data, as seen in Fig. 5.
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A, amplitude and phase of 1st harmonic response for an on-centre cell at three temporal frequencies as indicated. B, similar data for an off-centre cell. At low temporal frequencies the amplitude of minimum response is displaced away from ±180 deg. As frequency increases, this effect diminishes. The continuous lines show the fit of a model described by Smith et al. (1992). Modulation depths were 50 % for the on-centre, 20 % for the off-centre cell. Six-second activity averaged for each point; other details as in Figs 2-3. | ||
The physiological phase shift in the macaque disappears if small spots are used as stimuli (Smith et al. 1992; Kremers et al. 1994), thus implicating the receptive field surround. This was also observed for capuchin MC cells. This is illustrated in Fig. 6, which shows data from a MC off-centre cell stimulated at 1·22 Hz. With a 4 deg field, a typical phase shift is observed; the minimum response occurs near 80 deg on the relative diode phase axis. With a 0·5 deg field the phase shift disappears; the minimum response is now at ±180 deg on the diode phase axis. Response phase for a stimulation phase (relative diode phase) of zero remains relatively unchanged but the slope of the phase plot rotates around this point. The fit parameters indicated that the response was largely driven by the centre in this condition (see legend to Fig. 6). The physiological phase shift in the macaque also becomes smaller or vanishes if stimuli are presented upon a short-wavelength background (B. B. Lee, V. C. Smith & J. Pokorny, unpublished observations). This also occurred with capuchin MC cells, as is illustrated in the lower panels of Fig. 6.
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Shown are response amplitude and phase, and fits, for an on-centre MC cell. A, effect of stimulus size: 4 deg and 0·5 fields (2·44 Hz, mean chromaticity 595 nm). For the 4 deg field, the response minimum is shifted away from ±180 deg to ca 80 deg. This shift is absent with small stimuli. B, the phase effect is also absent on adding a 460 nm, 1000 Td background. These effects are also found in macaque MC cells. | ||
However, not all capuchin MC cells displayed this effect. Seven MC cells were dominated by rod input at low frequencies even at 2000 Td. This was revealed by response amplitude being largely independent of relative diode phase, and response phase lying on the 45 deg diagonal in the phase plot. This means that only the 554 nm diode was driving the response, which is consistent with rod input. Excluding these cells, Fig. 7 compares the mean and standard deviations of phases of minimum amplitude for capuchin and macaque. On- and off-centre cells did not differ and have been combined. Although data are similar for the two species, error bars for capuchin are much larger. Inspection of individual cells showed this variability to be due to intrusion of rod input at low temporal frequencies. This caused a shift in the phase of minimum response in the opposite direction to that usually observed (Lee et al. 1997). Rod input at such high retinal illuminance is very rare in the macaque; in our original sample (Smith et al. 1992) only one such cell was found.
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The phase of minimum response was obtained for each cell and condition by fitting with a cosine function, and the data are plotted here against temporal frequency. Mean and 95 % confidence limits are shown. Comparison macaque data were obtained from Smith et al. (1992). A similar shift is present in both species, although variability is greater in capuchin. | ||
Thus many features of responses to chromatic stimuli are shared by MC cells of the capuchin and those of the macaque, although some differences in detail are present.
Cone-opponent cells in the trichromatic capuchin
Cells with tonic responses and low achromatic contrast sensitivity were recorded from both dichromatic and trichromatic animals. All cells from dichromats showed, as expected, no trace of cone opponency except in those cells which received S-cone input. Of 15 such cells recorded from trichromats and studied in detail, 12 were strongly cone opponent, one was weakly cone opponent, and two were non-opponent off cells. Responses of cone-opponent cells strongly resembled PC cells from the macaque retina.
The phase protocol is a sensitive means of revealing opponency. Figure 8 shows first-harmonic amplitudes and phases for a red on-centre (+563-535 nm) and a green on-centre (+535-563 nm) cell. The continuous curve is the fit of the model described below. At low frequencies, the cells give maximum response to chromatic modulation (±180 deg) and a minimum response to luminance modulation (0 deg). As frequency increases, the phase of minimum responses moves away from zero, in opposite directions for the two cell types. This behaviour is also characteristic of opponent cells of the macaque. The phase of minimum response of macaque PC cells and capuchin cone-opponent cells is compared in Fig. 9A as a function of temporal frequency. Red-on (and green-off cells; filled squares) show opposite behaviour to green-on cells (and red-off cells; filled triangles).
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A, response amplitudes and phases for a +M - L cell. B, similar data from a +L - M cell. At low temporal frequencies, there is a maximum response to chromatic and a minimum response to luminance modulation. As frequency increases, the phase of minimum response shifts, in opposite directions for the two cell types. The continuous lines show the fit of a model described by Smith et al. (1992). Modulation depths were 50 % for both cells. | ||
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A, phases of minimum response were obtained by fitting a cosine function for each cell and conditions. L- (5) and M-cone (7) centre cells show shifts in different directions. A difference in latency of the opponent cone mechanisms by a few milliseconds is responsible for the shift. Data resemble those of macaque. B, cone weightings in opponent cells derived from model fits; comparison of capuchin and macaque. A value of 0·5 gives an equal cone balance, with no response to luminance modulation (and a vigorous response to chromatic modulation). Data from both species cluster around this value. | ||
Responses of opponent cells can be described by a simple model (Smith et al. 1992) which was also fitted to the current data. There are four free parameters: an amplitude scaling term, a cone weighting term, a frequency-dependent phase term and a phase delay between the two opponent mechanisms, corresponding to a centre-surround delay. This model adequately described the current results (Fig. 8), with a similar range of parameters. In particular, the cone weightings of 12 of the 15 cells were almost equal, and independent of temporal frequency. This is illustrated in Fig. 9B, which compares cone weightings of opponent cells of capuchin and macaque; the macaque data have been replotted from Smith et al. (1992). Both sets of weightings cluster around 0·5, which indicates an equal cone balance, implying little response to luminance modulation and maximal response to chromatic modulation. Both sets of weightings were largely independent of temporal frequency up to 19·5 Hz.
These data are consistent with a strong homology between cone-opponent cells in capuchin and macaque.
Temporal response and contrast sensitivity in trichromats and dichromats
Figure 10 shows response histograms to step changes in luminance and chromaticity of PC and MC cells from a trichromatic capuchin. PC and MC cells differed in achromatic contrast sensitivity and response time course. MC cells delivered a transient response to step changes in luminance down to low contrasts. PC cell responses were more sustained, and achromatic contrast sensitivity was lower. With chromatic perturbations, MC cells gave little response, but the responses of PC cells were vigorous and sustained.
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A, responses of MC cell. B, responses of +M - L PC cell. Percentages are modulation depths; 100 % chromatic modulation corresponded to a mean cone modulation of near 45 %. MC cells give a phasic response to luminance increments and decrements even at low contrast, and little or no response to red or green chromatic perturbations. The cone-opponent cell gives a vigorous, sustained response to chromatic perturbations and little response to luminance. Average of 20 stimulus repetitions, 16 ms binwidth. | ||
Responses to the same stimuli from ganglion cells of the dichromatic female are shown in Fig. 11. Putative MC cells gave transient responses down to low contrasts, but presumed PC cells were much less contrast sensitive. There was no response to chromatic perturbations, as expected. We tested if the low achromatic contrast sensitivity of dichromat PC cells was due to surround inhibition by flashing small spots of different sizes on the tangent screen, but responsivity did not obviously increase.
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The same format is followed as in Fig. 10. There is no chromatic response from the PC cell, but its luminance response is also weak. | ||
With flashed stimuli, data of trichromats thus strongly resembled those from equivalent measurements in macaque retina, and data of dichromats showed similar features except for the lack of cone opponency. In order to further explore the parallel to macaque ganglion cells, we employed sinusoidal luminance and chromatic modulation at different frequencies and contrasts.
MC and PC cells of the macaque are distinguished not only by a difference in achromatic contrast sensitivity but also by the presence (MC cells) or absence (PC cells) of marked response saturation and phase advance as a function of contrast; this is usually attributed to the presence of contrast gain control only in the MC pathway (Benardete et al. 1992; Yeh et al. 1995a). This distinction held also for capuchin ganglion cells, as illustrated in Fig. 12. Response amplitude and phase of an on-centre MC cell from a trichromat are shown as a function of contrast in Fig. 12A. The response to 0·61 Hz is weak but the response to 19·5 Hz is vigorous and is rapidly driven into saturation. This is associated with an advance in response phase. Amplitude data have been fitted by the Naka-Rushton saturation function (Naka & Rushton, 1966). The same pattern of results held for MC cells of dichromats, as indicated in Fig. 12D. For cone-opponent cells of trichromats, responsivity to luminance modulation was weak at low frequencies (Fig. 12C). At 19·5 Hz responsivity is higher. This band-pass character is likely to be due to the centre-surround latency difference, as in the macaque (Smith et al. 1990). The response to chromatic modulation (plotted in terms of cone contrast; Fig. 12D) is more vigorous than the response to luminance modulation at both frequencies. There is little indication of response saturation, and no advance in response phase. Finally, the luminance response of neurons classified as PC cells in dichromats resembled equivalent data from trichromats (Fig. 12E), but such cells gave no response to chromatic modulation.
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A, response amplitude and phase from MC cell of trichromat. Two frequencies, 0·61 and 19·5 Hz, are shown in these and other panels. MC cell responses at 19·5 Hz rapidly saturate accompanied by an advance in response phase. B and C, PC cell responses to luminance and chromatic modulation. Less saturation is evident, and no phase advance. The saturation of response that does occur appeared to be linked to response rectification. For the chromatic modulation, mean cone contrast has been plotted on the ordinate. Responsivity is high even at 0·61 Hz. For luminance modulation, PC cell responsivity is low at low frequency but increases at high frequency due to a centre-surround latency difference no longer causing full cancellation of opponent signals. D and E, response amplitude and phase of MC cells and PC cells of a dichromat. Data resemble trichromat luminance data. Data have been fitted with Naka-Rushton functions. Physiological data collection details as in Fig. 2. | ||
The data from the trichromat of Fig. 12 resemble very closely those from similar measurements from the macaque. An alternative way of plotting responsivity as a function of contrast and frequency is shown in Fig. 13. The top two panels show response amplitude as a function of frequency from trichromat capuchin and macaque MC cells, for different contrasts of luminance modulation. As contrast increases, the shape of the frequency response becomes flatter, the frequency of peak response becomes higher and there is an indication of a sharp peak at 40-50 Hz, followed by a rapid fall in responsivity. At least some of the changes with increasing contrast are usually attributed to a contrast gain control, although detailed behaviour at high temporal frequencies may be better described by a model incorporating a contrast-dependent second-order filter (Nadig et al. 1995). In any event, a similar mechanism appears to be present in capuchin and macaque. In the lower panels, equivalent sets of data are presented for cone-opponent neurons, for chromatic modulation at different cone contrasts. The form of the temporal response is essentially contrast independent, the curves translating upward without significant change in shape as contrast increases.
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Data such as those of Fig. 12 plotted in an alternative form and compared with macaque results. Response amplitude is plotted as a function of frequency at different contrasts. A, for the MC cells (luminance modulation), the frequency of maximum response increases with increasing contrast, and there is an indication of a resonance peak at ca 50 Hz. B, for the PC cells (chromatic modulation), the curves at different contrasts show no such change in curve shape as contrast increases. Macaque data were obtained under identical conditions to the capuchin data (Smith et al. 1990). | ||
The initial slope of the contrast-response curves of Fig. 12 is termed the contrast gain. Contrast gain as a function of frequency is plotted in Fig. 14 for trichromatic and dichromatic capuchin and for macaque for different cell types with luminance and chromatic modulation. For chromatic modulation, data are expressed in terms of mean cone contrast. Results from five well-studied cells were averaged for each curve. Temporal response with different cell types and conditions shows broad similarity, except that capuchin ganglion cells consistently responded up to higher temporal frequencies than observed in the macaque. The retinal illuminance (2000 Td, delivered through the same Maxwellian view stimulator) was the same in both sets of experiments. Although flux per unit retinal area would be 10 % higher in capuchin due to the smaller eye size, this seems unlikely to have much effect on temporal response. In macaque, MC cells responded weakly and PC cells never to the highest frequency available (78 Hz). In capuchin, MC cells often responded vigorously, and PC cells weakly, to this frequency.
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Contrast gain is the initial slope of the Naka-Rushton functions fitted in Fig. 12. Mean data from five well-studied cells was averaged in each case. The form of the curves are similar in different phenotypes and species, but capuchin cells continued to respond to higher temporal frequencies. | ||
We conclude that the temporal response properties of capuchin ganglion cells show most if not all features previously described for the macaque; their responses to luminance modulation do not depend on the opsins present.
Rod input
We noted evidence for rod input to some MC cells even at 2000 Td. Such cells had receptive fields at similar eccentricities to others without rod input. Rod input may also be present in marmoset ganglion cells at relatively high retinal illuminances (Yeh et al. 1995b), although this may be eccentricity dependent (Weiss et al. 1998). We tested for rod input in nine MC cells using the methods described in Lee et al. (1997). As in the macaque, rod input was always present at 200 Td, and at 20 Td MC cells were rod dominated. Thus, rod input may be stronger than in the macaque, although otherwise the scotopic transition appeared similar. We were not able to test for rod input to other cell types.
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DISCUSSION |
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Physiological similarities between capuchin and macaque retinae
In comparison with recordings from PC and MC cells in the LGN of marmoset (e.g. Yeh et al. 1995b), recording directly from capuchin retinal ganglion cells provided a more physiologically and mechanically stable preparation. This enabled us to pursue the possible homology between ganglion cells of capuchin and macaque in greater detail. Ganglion cell behaviour in the capuchin showed most features found in the macaque retina. In particular, the frequency-doubled responses (Fig. 4; Lee et al. 1989a) and phase effects (Figs 5-7; Smith et al. 1992) found in macaque MC cells were also observed in MC cells of the trichromatic capuchin. Both these effects appear to be due to a chromatic input to the receptive field surround, for these reasons. A frequency-doubled component appears as soon as any chromatic component is present in the stimulus, i.e. within quadrants in an L-M-cone excitation space where these cones add, as well as in the opponent quadrants. This is not expected on the basis of a rectification or saturation non-linearity within M-L-cone summation, and requires a non-linearity in a chromatic, opponent input. For the phase protocol, the shift requires a linear, first-harmonic opponent input, as discussed by Smith et al. (1992); cone weighting alone cannot cause this effect. The presence of these phenomena in the capuchin indicates remarkable physiological homology. Also, the chromatic responses of cone-opponent PC cells bore a strong quantitative resemblance to the macaque (Figs 8, 9 and 12-14). Finally contrast-response relationships (Fig. 12), the presence of contrast gain controls only in MC cells (Figs 12 and 13) and temporal tuning (Fig. 14) all showed macaque-like features.
Nevertheless, some quantitative differences were apparent. The frequency-doubled response of MC cells was less apparent in capuchin than in macaque at 2000 Td, and even at this relatively high retinal illuminance some capuchin MC cells showed evidence of rod input, which is rare in the macaque (Lee et al. 1997). These aspects of capuchin MC cell behaviour were more similar to the macaque at 200 Td. However, the limit to the temporal response, the critical fusion frequency (CFF), of all types of capuchin ganglion cells appeared to exceed equivalent macaque measurements, which would argue against the capuchin retina operating as if at a lower effective retinal illuminance.
The rod input observed in capuchin retina at high light levels is similar to marmoset results (Yeh et al. 1995b; Weiss et al. 1998), despite the high cone density in both these species (Goodchild et al. 1996; Wilder et al. 1996; Franco et al. 1998). The reason for this difference from the macaque is unknown, but one possibility is that New-World primates underwent a nocturnal phase during evolution; the owl monkey would then have reverted to a rod-dominated retina (Jacobs et al. 1993; Silveira et al. 2000).
Recording from retinal ganglion cells suffers from the disadvantage that cell classification has to be based on exclusively physiological criteria, while in the lateral geniculate nucleus the lamina of recording provides additional evidence. In the trichromatic capuchin, cell classification was relatively straightforward, since most cells showed clear cone opponency, or showed the transient responses and high contrast sensitivity of MC cells. However, we did record some cells from trichromats (20 %) with low contrast sensitivity and sustained responses, much like putative PC cells in dichromats. This suggests a similar situation to the marmoset, where some cells recorded in the parvocellular layers lack cone opponency (Yeh et al. 1995b). In the dichromatic capuchin, cone opponency was absent, and classification more difficult. Nevertheless, most cells either had high contrast sensitivity and transient responses or low contrast sensitivity and sustained responses.
In the dichromat recordings, cell classification was more difficult. Two main criteria were used in cell classification. Cells with high contrast sensitivity and transient responses were classified as MC cells, while cells with low contrast sensitivity and sustained responses were classified as PC cells. In later analysis of contrast/response functions, the former class almost always showed evidence of contrast-gain control while the latter class did not. However, the classification was ambiguous in a few cells.
Spectral sensitivity, contrast sensitivity and temporal response
The spectral sensitivity of MC cells of trichromatic capuchin showed a degree of scatter similar to that found in the macaque. Although cones in Old-World monkey (Mollon & Bowmaker, 1992) and human are thought to be randomly arrayed, some degree of patchiness in the cone mosaic has been suggested in the retinae of human female carriers of congenital colour vision defects (Born et al. 1976; Nagy et al. 1981). A similarly patchy distribution, due to X inactivation (Lyon, 1972), could potentially occur in the trichromatic New-World primate retina. However, in a microspectrophotometric examination (Mollon et al. 1984), small arrays of squirrel monkey cones showed no obvious regularity or patchiness. In our study, large patches of the same cone would result in MC cells dominated by one cone type or the other. This was not apparent in trichromatic capuchin MC cells, so any patches must be small relative to MC cell centre size. MC cell receptive field centres and parasol cell dendritic trees in the macaque (Lee & Dacey, 1997) and capuchin (Yamada et al. 1996b; Franco et al. 1998) are about eight cones across.
In the macaque, the achromatic contrast sensitivity of PC cells is low compared to their high chromatic cone contrast sensitivity. The low achromatic contrast sensitivity is largely due to mutual cancellation of the opponent cone signals. This also was the case in the trichromatic capuchin (Fig. 14). The functional significance of the low achromatic contrast sensitivity of dichromat PC cells is uncertain. We used large-field (4 deg) stimuli for the contrast measurements in Figs 12-14. We also used small spots in informal tests on a few cells, but no obvious enhancement of sensitivity occurred. As in the marmoset (Yeh et al. 1995b), such cells could best be described as colour-blind versions of cone-opponent PC cells. The role played in dichromats by such a numerous, apparently luminance contrast-insensitive cell class remains uncertain. One possibility is a role in contrast discrimination and brightness estimation for high contrast stimuli; the larger dynamic range for PC cells serves to permit discrimination at contrasts where MC cells have saturated (Silveira, 1996).
A further comparative point of interest is the response of capuchin ganglion cells to higher temporal frequencies than in the macaque, although the slower CFF of PC relative to MC cells was maintained. The presence of a higher CFF in both cell types relative to the macaque suggests it arises early in the retina before PC and MC signals diverge in the outer plexiform layer. An obvious locus would be the cones themselves. Although any agile arboreal omnivore presumably requires a fast response from its visual system, the significance to the capuchin of a higher ganglion cell CFF than the macaque is unknown.
Evolution of trichromacy
In principle, an individual's expression of two opsin genes in the middle-to-long wavelength range need not result in behavioural trichromacy. Up to now, however, no exceptions have been described, and in this paper we have assumed that presence of three opsins results in trichromatic vision. The physiological and genetic analysis presented here and elsewhere (Hunt et al. 1998) confirms an earlier report (Jacobs & Neitz, 1987) suggesting that the capuchin conforms to the standard New-World primate polymorphic pattern. A recent study (Pessoa et al. 1997) purported to show that male capuchin may be behaviourally trichromatic, but these authors themselves admit that other cues may have been used by their observers; Jacobs (1999) recently confirmed this possibility.
There are three possible scenarios for the evolution of trichromatic vision in primates (Mollon, 1991): New- and Old-World primates evolved trichromacy independently; New-World primate polymorphism represents an ancestral pattern from which the Old-World primate model evolved; or both New- and Old-World primates once possessed regular trichromatic vision (with two opsin genes on the X chromosome) but the New-World primates lost regular trichromacy, to be replaced by the polymorphism. It has been argued on a genetic basis that independent evolution of trichromacy is most likely (Hunt et al. 1998), although this view is not universally accepted (Kainz et al. 1998). It is agreed that the acquisition (or reacquisition) of full trichromacy of the howler monkey is a relatively recent genetic event (Hunt et al. 1998; Kainz et al. 1998).
The differentiation of the PC and MC pathways appears to have begun in a prosimian ancestor, prior to trichromacy. In the bushbaby, a laminated LGN receives projections from MC- and PC-like ganglion cells (Yamada et al. 1998). These authors also found cone bipolar cells with midget morphology, implying input from a single cone. This may be associated with the very low cone density in this species (Wikler & Rakic, 1990). If such morphology were present in a nocturnal simian ancestor, a substrate for the single cone connectivity and cone opponency of the midget system would be present if a genetic event were to make appropriate opsins available as the species became diurnal.
It is plausible that the M-L-cone opponent system evolved from a ganglion cell system with small receptive fields, since only then would there be a low enough cone convergence onto a single ganglion cell for a useable cone-opponent signal to become available following the evolution of multiple opsins (Wässle & Boycott, 1991). These authors proposed that a fully developed midget system evolved in a diurnal ancestral species for purposes of visual resolution, although it is unclear why this should be advantageous when the point-spread function of the eye's optics is substantially larger than a single cone. It is currently uncertain if simian ancestors were nocturnal or diurnal (Martin, 1993). It is also unknown how far the acquisition of trichromacy provoked further modification in the retina; for example, a system with small receptive fields and minimal cone convergence could have been further refined into the midget system after the acquisition of multiple opsins. These various scenarios are not mutually exclusive; more physiological and anatomical data from prosimians and other species may clarify these issues.
The present data indicate a strong homology between the physiology of capuchin and macaque retinae, consistent with the anatomical similarities. It could be argued that some common features arose through parallel evolution. For example, the PC cell phase plots of Fig. 9 are attributable to a centre-surround latency difference, which is a standard feature of receptive field organization. Also, the presence or absence of contrast gain controls may have been characteristic of MC and PC cell systems prior to the evolution of trichromacy. On the other hand, the frequency-doubled responses and phase effects of MC cells are derived from chromatic signals, but have no obvious physiological substrate or functional role. There would seem no reason for them to arise independently in the two primate branches. Thus these aspects of our physiological data may support a common origin for New- and Old-World trichromacies. This would appear inconsistent with the molecular data, which indicate that the trichromacy of Old-World primates did not arise by combining two different variants from a pre-existing polymorphism on to a single X chromosome. However, this does not rule out the possibility that a polymorphism existed in this ancestral primate and that the physiological similarities between New- and Old-World monkeys reported here arose at this time.
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We would like to thank Walter de Carvalho, Francinaldo Gomes and Cézar Saito for assistance during the experiments, Dr J. A. P. Muniz of the Centro Nacional de Primatas in Belem for provision of animals, Jill Cowing who carried out the DNA sequencing, and Jim Bowmaker for providing squirrel monkey spectra. Travel support for this work was provided by CNPq/MPG no. 91.0234/94-9 and CNPq/MPG no. 910060/96-7 to B.B.L. and L.C.L.S., CNPq/DAAD no. 91.0248-96.6 to L.C.L.S. and J.K., Australian NHMRC grant 960970 to P.R.M., NATO Collaborative Research Grant 931162 to J.B.T. and B.B.L., and NIH grant EY06669 to J.B.T. Also supported by FINEP/FADESP no. 4.3.90.0082.00, CNPq no. 521640/96-2 and PRONEX/FUJB no. 76.97.1028.00 to L.C.L.S. L.C.L.S. and E.S.Y. have CNPq research fellowships and J.K. is a Heisenberg Fellow of the Deutsche Forschungsgemeinschaft (KR1317/5-1). E.S.Y. is also Fellow of the Pew Foundation (P0199SC). Finally, the Y. Yamada Corporation provided generous logistical support in the field.
Corresponding author
B. B. Lee: Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Email: blee{at}gwdg.de
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), one PC cell (
) in each case, data of the MC cell being shifted up for clarity), shown to match the appropriate templates. B and C, response amplitude and phase templates for phase protocol. The relative phases of the 554 and 638 nm lights was manipulated at fixed modulation depth. Luminance modulation corresponds to a phase of 0 deg, chromatic modulation to a phase of ±180 deg. Again, the fit (one MC, one PC cell) of the templates to the data provided an unambiguous indication of the genotype. Modulation depth was 20 % for MC cells and 50 or 100 % for PC cells. Responses were averaged over 6 s for each point. First-harmonic amplitudes and phases were extracted by Fourier analysis. Measurements were at 19·8 Hz, 4 deg field, 2000 Td.
) as a function of relative 554/638 nm ratio (4 deg field). First-harmonic data fitted as in Fig. 3. The second- harmonic response rises to a peak at the first-harmonic minimum. C and D, data from an MC cell tested with two sizes of spot. The frequency-doubled response almost disappears with small stimuli, and can thus be localized to the receptive field surround. Details of the physiological measurements as in Fig. 2, except that 9·76 Hz modulation was used.