Manoel da Silva Filho

Morphology and physiology of primate M- and P-cells

Catarrhines and platyrrhines, the so-called Old- and New-World anthropoids, have different cone photopigments. Postreceptoral mechanisms must have co-evolved with the receptors to provide trichromatic color vision, and so it is important to compare postreceptoral processes in these two primate groups, both from anatomical and physiological perspectives. The morphology of ganglion cells has been studied in the retina of catarrhines such as the diurnal and trichromatic Macaca, as well as platyrrhines such as the diurnal, di- or trichromatic Cebus, and the nocturnal, monochromatic Aotus. Diurnal platyrrhines, both di- and trichromats, have ganglion cell classes very similar to those found in catarrhines: M (parasol), P (midget), small-field bistratified, and several classes of wide-field ganglion cells. In the fovea of all diurnal anthropoids, P-cell dendritic trees contact single midget bipolars, which contact single cones. The Aotus retina has far fewer cones than diurnal species, but M- and P-cells are similar to those in diurnal primates although of larger size. As in diurnal anthropoids, in the Aotus, the majority of midget bipolar cells, found in the central 2 mm of eccentricity, receive input from a single cone and the sizes of their axon terminals match the sizes of P-cell dendritic fields in the same region. The visual responses of retinal ganglion cells of these species have been studied using single-unit electrophysiological recordings. Recordings from retinal ganglion cells in Cebus and Aotus showed that they have very similar properties as those in the macaque, except that P-cells of mono- and dichromatic animals lack cone opponency. Whatever the original role of the M- and P-cells was, they are likely to have evolved prior to the divergence of catarrhines and platyrrhines. M- and P-cell systems thus appear to be strongly conserved in the various primate species. The reasons for this may lie in the roles of these systems for both achromatic and chromatic vision.

Developmental sources of conservation and variation in the evolution of the primate eye

Conserved developmental programs, such as the order of neurogenesis in the mammalian eye, suggest the presence of useful features for evolutionary stability and variability. The owl monkey, Aotus azarae, has developed a fully nocturnal retina in recent evolution. Description and quantification of cell cycle kinetics show that embryonic cytogenesis is extended in Aotus compared with the diurnal New World monkey Cebus apella. Combined with the conserved mammalian pattern of retinal cell specification, this single change in retinal progenitor cell proliferation can produce the multiple alterations of the nocturnal retina, including coordinated reduction in cone and ganglion cell numbers, increase in rod and rod bipolar numbers, and potentially loss of the fovea.

Flicker ERGs representing chromaticity and luminance signals.

PURPOSE To test the hypothesis that electroretinograms (ERGs) reflect luminance activity when measured at high temporal frequencies and chromatic activity when measured near 12 Hz. METHODS The authors measured the responses to stimuli in which the output of red and green light-emitting diodes was modulated in counterphase at different ratios, varying the luminance content in the stimulus while keeping the red-green chromatic contrast and its phase constant. RESULTS The high temporal frequency electroretinography was determined mainly by the luminance contrast. At 12 Hz, electroretinographic response amplitudes and phases primarily reflected the red-green chromatic content of the stimulus. Control experiments, performed with a deuteranopic subject and with stimuli that silenced the rods and S-cones, excluded an explanation based on intrusion from rod- and S-cone-driven responses. CONCLUSIONS It now is possible to perform noninvasive measurements of basic electrophysiological properties of the luminance and chromatic pathways on a retinal level, and their disease-related changes, in human observers.

Amplitude of the transient visual evoked potential (tVEP) as a function of achromatic and chromatic contrast : Contribution of different visual pathways

We investigated how the stimulation mode influences transient visual evoked potentials (tVEP) amplitude as a function of contrast of achromatic and isoluminant chromatic gratings. The chromatic stimulation probed only responses to the red-green axis. Visual stimuli were monocularly presented in a 5 degrees diameter circle, achromatic and chromatic horizontal gratings, 1 Hz pattern reversal stimulation, and achromatic and chromatic gratings, 300 ms onset per 700 ms offset stimulation. For the achromatic pattern reversal stimulation, a double slope function describes how the P100 amplitude varied as a function of log contrast which had a limb at low-to-medium contrasts and another limb at high contrasts. For the achromatic onset/offset stimulation, C2 amplitude saturated at the highest contrast tested and a single straight line described how it changed along most of the contrast range. Both presentation modes for chromatic gratings resulted in amplitude versus log contrast relations which were well described by single straight lines along most of the contrast range. The results may be interpreted as if at 2 cpd, achromatic pattern reversal stimulation evoked the activity of at least two visual pathways with high and low contrast sensitivity, respectively, while achromatic onset/offset stimulation favored the activity of a pathway with high contrast sensitivity. The neural activity in the M pathway is the best candidate to be the high contrast mechanism detected with pattern reversal and pattern onset/offset VEPs. The activity of color opponent pathways such as the P and K pathways either combined or in isolation seems to be responsible for VEPs obtained with isoluminant chromatic gratings at both presentation modes. When the amplitudes of chromatic VEPs were plotted in the same contrast scale as used for achromatic VEPs, chromatic contrast thresholds had similar values to those of the achromatic mechanism with high contrast sensitivity.

Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model

Quinolinic acid (QA) is an N-methyl-D-aspartate receptor agonist that also promotes glutamate release and inhibits glutamate uptake by astrocytes. QA is used in experimental models of seizures studying the effects of overstimulation of the glutamatergic system. The guanine-based purines (GBPs), including the nucleoside guanosine, have been shown to modulate the glutamatergic system when administered extracellularly. GBPs were shown to inhibit the binding of glutamate and analogs, to be neuroprotective under excitotoxic conditions, as well as anticonvulsant against seizures induced by glutamatergic agents, including QA-induced seizure. In this work, we studied the electrophysiological effects of guanosine against QA-induced epileptiform activity in rats at the macroscopic cortical level, as inferred by electroencephalogram (EEG) signals recorded at the epidural surface. We found that QA disrupts a prominent basal theta (4-10 Hz) activity during peri-ictal periods and also promotes a relative increase in gamma (20-50 Hz) oscillations. Guanosine, when successfully preventing seizures, counteracted both these spectral changes. MK-801, an NMDA-antagonist used as positive control, was also able counteract the decrease in theta power; however, we observed an increase in the power of gamma oscillations in rats concurrently treated with MK-801 and QA. Given the distinct spectral signatures, these results suggest that guanosine and MK-801 prevent QA-induced seizures by different network mechanisms.

Normal and dichromatic color discrimination measured with transient visual evoked potential.

It would be informative to have an electrophysiological method to study, in an objective way, the effects of mercury exposure and other neurotoxics on human color vision performance. The purpose of the present work was to study human color discrimination by measuring chromatic difference thresholds with visual evoked potential (VEP). Six young normal trichromats (24 +/- 1 years old) and one deutan (26 years old) were tested. The stimuli consisted of sinusoidal isoluminant chromatic gratings made from chromaticity pairs located along four different color directions centered on two reference points. Heterochromatic flicker photometry (HFP) protocol was used to obtain the isoluminance condition for every subject and for all chromaticity pairs. Spatial frequency was 2 cycles/deg. Presentation mode comprised onset (300 ms)/offset (700 ms) periods. As previously described, we found a negative deflection in the VEP which was related to the chromatic difference: as chromatic difference increased, amplitude increased and latency decreased. VEP response amplitude was plotted against distance in the CIE 1976 color space between the grating chromaticities and fitted with a regression line. We found color thresholds by extrapolating the fitting to null amplitude values. The thresholds were plotted in the CIE 1976 color space as MacAdam ellipses. In normal trichromats the ellipses had small size, low ellipticity, and were vertically oriented. In the deutan subject, the ellipses had large size, high ellipticity, and were oriented towards the deutan copunctal locus. The VEP thresholds were similar to those obtained using grating stimuli and psychophysical procedures, however smaller than those obtained using pseudoisochromatic stimuli (Mollon-Reffin method). We concluded that transient VEP amplitude as a function of contrast can be reliably used in objective studies of chromatic discrimination performance in normal and altered human subjects.

The Spatial Properties of L- and M-cone Inputs to Electroretinograms that Reflect Different Types of Post-receptoral Processing

We studied the spatial arrangement of L- and M-cone driven electroretinograms (ERGs) reflecting the activity of magno- and parvocellular pathways. L- and M-cone isolating sine wave stimuli were created with a four primary LED stimulator using triple silent substitution paradigms. Temporal frequencies were 8 and 12 Hz, to reflect cone opponent activity, and 30, 36 and 48 Hz to reflect luminance activity. The responses were measured for full-field stimuli and for different circular and annular stimuli. The ERG data confirm the presence of two different mechanisms at intermediate and high temporal frequencies. The responses measured at high temporal frequencies strongly depended upon spatial stimulus configuration. In the full-field conditions, the L-cone driven responses were substantially larger than the full-field M-cone driven responses and also than the L-cone driven responses with smaller stimuli. The M-cone driven responses at full-field and with 70° diameter stimuli displayed similar amplitudes. The L- and M-cone driven responses measured at 8 and 12 Hz were of similar amplitude and approximately in counter-phase. The amplitudes were constant for most stimulus configurations. The results indicate that, when the ERG reflects luminance activity, it is positively correlated with stimulus size. Beyond 35° retinal eccentricity, the retina mainly contains L-cones. Small stimuli are sufficient to obtain maximal ERGs at low temporal frequencies where the ERGs are also sensitive to cone-opponent processing.

Horizontal cell morphology in nocturnal and diurnal primates: A comparison between owl-monkey ( Aotus ) and capuchin monkey ( Cebus )

Horizontal cell morphology was studied in the retina of the nocturnal owl-monkey, Aotus, and compared with that of its diurnal, close relative, the capuchin monkey, Cebus . Cells were initially labeled with DiI and the staining was later photoconverted in a stable precipitated using DAB as chromogen. The sizes of cell bodies, dendritic fields, and axon terminals, number of dendritic clusters, intercluster spacing, and intercone spacing were measured at increasing eccentricities. Two distinct morphological classes of horizontal cells were identified, which resembled those of H1 and H3 cells described in diurnal monkeys. A few examples of a third class, possibly corresponding to the H2 cells of diurnal monkeys, were labeled. Both H1 and H3 cells increased in size and had increasing numbers of dendritic clusters with eccentricity. H3 cells were larger and had a larger number of dendritic clusters than H1 cells. Owl-monkey H1 cells had larger dendritic fields than capuchin monkey H1 cells at all quadrants in the central and midperipheral retinal regions, but the difference disappeared in the far periphery. Owl-monkey and capuchin monkey H1 cells had about the same number of dendritic clusters across eccentricity. As owl-monkey H1 cells were larger than capuchin monkey H1 cells, the equal number of clusters in these two primates was due to the fact that they were more spaced in the owl-monkey cells. H1 intercluster distance closely matched intercone spacing for both the owl-monkey and capuchin monkey retinas. On the other hand, H3 intercluster distance was larger than intercone spacing in the retina of both primates. Owl-monkey H1 axon terminals had 2-3 times more knobs than capuchin monkey H1 axon terminals in spite of having about the same size and, consequently, knob density was 2-3 times higher for owl-monkey than capuchin monkey H1 axon terminals across all eccentricities. The differences observed between owl-monkey and capuchin monkey horizontal cells, regarding the morphology of their dendritic trees and axon terminals, may be related to the differences found in the cone-to-rod ratio in the retina of these two primates. They seem to represent retinal specializations to the nocturnal and diurnal life styles of the owl-monkey and capuchin monkey, respectively.

Alouatta Trichromatic Color Vision: Cone Spectra and Physiological Responses Studied with Microspectrophotometry and Single Unit Retinal Electrophysiology

The howler monkeys (Alouatta sp.) are the only New World primates to exhibit routine trichromacy. Both males and females have three cone photopigments. However, in contrast to Old World monkeys, Alouatta has a locus control region upstream of each opsin gene on the X-chromosome and this might influence the retinal organization underlying its color vision. Post-mortem microspectrophotometry (MSP) was performed on the retinae of two male Alouatta to obtain rod and cone spectral sensitivities. The MSP data were consistent with only a single opsin being expressed in each cone and electrophysiological data were consistent with this primate expressing full trichromacy. To study the physiological organization of the retina underlying Alouatta trichromacy, we recorded from retinal ganglion cells of the same animals used for MSP measurements with a variety of achromatic and chromatic stimulus protocols. We found MC cells and PC cells in the Alouatta retina with similar properties to those previously found in the retina of other trichromatic primates. MC cells showed strong phasic responses to luminance changes and little response to chromatic pulses. PC cells showed strong tonic response to chromatic changes and small tonic response to luminance changes. Responses to other stimulus protocols (flicker photometry; changing the relative phase of red and green modulated lights; temporal modulation transfer functions) were also similar to those recorded in other trichromatic primates. MC cells also showed a pronounced frequency double response to chromatic modulation, and with luminance modulation response saturation accompanied by a phase advance between 10–20 Hz, characteristic of a contrast gain mechanism. This indicates a very similar retinal organization to Old-World monkeys. Cone-specific opsin expression in the presence of a locus control region for each opsin may call into question the hypothesis that this region exclusively controls opsin expression.

Visual Dysfunction of Type I and VI Mucopolysaccharidosis Patients Evaluated with Visual Evoked Cortical Potential

Purpose: To evaluate the visual system of patients suffering from type I or VI mucopolysaccharidosis (MPS) by recording the visual evoked cortical potential (VECP). Methods: Two patients with MPS VI and 2 patients with MPS I were tested before and after enzyme replacement therapy (ERT). A control group of 20 subjects was tested for statistical comparison. VECP was elicited by monocular stimulation with 1-Hz phase-reversal checkerboard patterns at 0.5 and 2 cycles per degree and with 16° of visual field. In all patients, both eyes were tested. VECP amplitude and latency were measured and compared with tolerance limits obtained from controls. Results: MPS I and VI patients have a severe visual impairment that can be quantified by measuring VECPs. Even after several weeks of ERT, the visual impairment remained unaltered, indicating that the treatment had no significant influence on the visual conditions of MPS patients. Visual responses to high spatial frequencies were more deeply impaired than responses to low spatial frequencies. This can be explained by the kind of damage in the visual system that preferentially targets the eye optics. Conclusion: VECPs can be used to monitor the degree of visual impairment of MPS patients and to check ERT efficacy.