Acetylcholine is present in and released from starburst amacrine cells in the inner plexiform layer (IPL), but its role in retinal function except, perhaps, in early development, is unclear. Nicotinic acetylcholine receptors are thought to be present on ganglion, amacrine, and bipolar cell processes in the IPL, and it is known that acetylcholine increases the spontaneous and light-evoked responses of retinal ganglion cells. The effects of acetylcholine on bipolar cells are not known, and here we report the effects of nicotine on the b-wave of the electroretinogram in larval zebrafish. The b-wave originates mainly from ON-bipolar cells, and the larval zebrafish retina is cone-dominated. Only small rod responses can be elicited with dim lights in wild-type larval zebrafish retinas, but rod responses can be recorded over a range of intensities in a mutant (no optokinetic response f) fish that has no cone function. We find that nicotine strongly enhances cone-driven b-wave response amplitudes but depresses rod driven b-wave response amplitudes without, however, affecting rod- or cone-driven b-wave light sensitivity.

Adult teleost retinas grow throughout life, in part by the addition of cells from an encircling, proliferative neuroepithelium. In some species, this proliferative growth is asymmetric around the retina. The present study evaluated two hypotheses regarding asymmetric proliferative growth in adult green sunfish retina: (1) the generation of rod photoreceptors in central retina from proliferative rod precursor cells is also asymmetric; and (2) asymmetric proliferative growth patterns are regulated by mechanisms that are organized retinotopically and are independent of body-axis coordinates. In all retinas examined, rod precursor distribution and rod production were asymmetric, and both were in coarse spatial register with the asymmetric pattern of cellular addition at the retinal margin. In adult eyes that were surgically rotated, the asymmetric patterns of proliferative growth were present and appropriate for the retina's prerotation orientation. The results suggest that proliferative growth at both marginal and central adult sunfish retina is asymmetric, and that these asymmetric growth patterns are regulated by a retinotopic mechanism that is independent of body-axis coordinates.

Dopaminergic interplexiform cells in the retina of the blue acara were investigated using an antiserum against tyrosine hydroxylase and PAP visualization. In whole-mount preparations, we observed a homogeneous distribution of cell bodies throughout the retina without any indication of regional specialization. At the fine and ultrastructural level, we studied the morphology of labeled telodendria within the outer plexiform layer. Apart from contacts with horizontal cell perikarya and bipolar cell dendrites, we observed direct contacts, mostly in the form of close appositions, with cone pedicles and rod spherules. Quantitative evaluation and reconstruction of serial sections showed that all cone pedicles and most rod terminals were approached in this way. The dopaminergic pathway terminating on horizontal cells and photoreceptors is discussed with respect to the localization of dopamine receptors in the outer retina, and the control of adaptive changes such as retinomotor movements, spinule formation, and horizontal cell coupling.

This study examines the spatiotemporal relationships between retinal pigment epithelium (RPE) and photoreceptors (PR) during development of Macaca nemestrina retina. Our aim was to learn more about the developmental dynamics of these two important cell populations, particularly whether developmental changes in RPE cell densities mimic those of PR at selected retinal points. Twelve eyes ranging in age from 100 fetal days (Fd) to adulthood were flatmounted; the retinal perimeters were traced; and then sample punches were taken of the RPE and neural retina at the fovea, optic disc, mid- and far-nasal periphery, and far temporal, inferior and superior periphery. The two tissues were gently separated and the RPE cells and photoreceptors from the same region of the punch were counted using Nomarski contrast interference optics. We found that the total number of cones remains stable around 4 million between Fd100 and adulthood, but RPE number increases from 1.6 million at Fd100 to 2.56 million in adulthood. At the fovea, the core:RPE ratio increases from 5.4:1 at Fd100 to 28:1 by adulthood. In the temporal periphery by contrast, the cone:RPE ratio declines from 2.2:1 at Fd100–110 to less than 1:1 in the adult. In the vicinity of the optic disc, the ratio of (cones + rods): RPE remains around 35:1 throughout development, but in the retinal periphery it decreases to the adult value of 22:1. These changing ratios indicate that photoreceptors and RPE cells are redistributed independently during development, and that these two cellular sheets slide over one another to achieve their final distribution. This situation suggests that the forces or factors causing foveation are intrinsic to the neural retina.

We studied rod function in the chicken by recording corneal electroretinograms (ERGs). The following experiments were performed to demonstrate rod function during daytime: (1) determining the dark-adaptation function; (2) measuring the spectral sensitivity by a a–b-wave amplitude criterion in response to monochromatic flickering light of different frequencies ranging from 6.5–40.8 Hz (duty cycle 1: I); (3) analyzing the response vs. log stimulus intensity (V–log I) function in order to reveal a possible two phase process; and (4) determining the spectral sensitivity function either in a non-dark adapted state or after dark adaptation of the animals for I and 24 h. None of these experiments demonstrated clear evidence of rod function during daytime. On the other hand, we found rods histologically by light- and electron microscopy. Therefore, we repeated our ERG recordings during the night (between midnight and 3:00 A.M.). Without previous dark adaptation, rod function could be seen immediately in the same experiments described above. The result shows that, in the chicken, rods are turned on endogenously during the night but are scarcely functional during the day.

We tested the hypothesis that the dark-adapted sensitivity of the near central retina is lower than that of peripheral retina in 10–week-old infants. A spatial two alternative forced-choice psychophysical method was used to estimate the threshold for 2–deg spots presented 10 deg and 30 deg from the center of a screen. Each 10–week-old infant (n = 11) and adult (n = 5) subject was tested at both eccentricities. Every infant's threshold at 10 deg is higher than that at 30 deg. The median difference between the infants' thresholds at 10 deg and 30 deg is about 0.5 log unit. Whereas the scotopic thresholds of adults at 10 deg and 30 deg are virtually identical, the thresholds of infants' near central retina are higher than those in more peripheral retina. Delayed maturation of the near central rod outer segments with consequent lower quantum catch may be the primary determinant of this infantile sensitivity pattern.

The response to spatially focal flicker is enhanced by dim, spatially diffuse, rod-stimulating backgrounds. This effect is called suppressive rod-cone interaction (SRCI) as it reflects a tonic, suppressive influence of dark-adapted rods upon cone pathways which is removed by selective rod-light adaptation. SRCI is observed in amphibian retina with intracellular recordings from most cone-driven cells including the cones themselves, and is most obvious using stimuli flickering at frequencies too rapid for rods to follow. SRCI is blocked by glutamate analogs which selectively block the photic response of horizontal cells (HCs). In the presence of these agents, flicker responses from bipolar cells and cones are enhanced to levels normally seen only with selective rod-light adaptation. In the HCs themselves, SRCI is similarly blocked by lead chloride which blocks rod-, but not cone-related activity.

In amphibian and cat HCs and in human observers, SRCI is limited by a space constant of very similar value (between 100 and 150 μm). We suggest that SRCI in all three species is mediated by HCs: in amphibians, SRCI must at least partially reflect rod-modulation of HC feedback onto cones.

The habitat occupied by larval winter flounder (Pseudopleuronectes americanus) differs considerably in light regime from that of the adult. To understand how the visual system has adapted to such changes, photoreceptor spectral absorbance was measured microspectrophotometrically in premetamorphic and postmetamorphic specimens of winter flounder. Before metamorphosis, larval flounder retinas contain only one kind of photoreceptor which is morphologically cone-like with peak absorbance at 519 nm. After metamorphosis, the adult retina has three types of photoreceptors: single cones, double cones, and rods. The visual pigment in single cones has a peak absorbance at λmax = 457 nm, the double cones at λmax = 531 and 547 nm, and the rod photoreceptors at λmax = 506 nm. Double cones were morphologically identical, but the two members contained either different (531/547 nm) or identical pigments (531/531 nm). The latter type were found only in the dorsal retina. The measured spectral half-bandwidths (HBW) were typical of visual pigments with chromophores derived from vitamin A1 with the possible exception of the long-wavelength absorbing pigment in double cones which appeared slightly broader. Because the premetamorphic pigment absorbance has a different λmax than those of the postmetamorphic pigments, different opsin genes must be expressed before and after metamorphosis.

The spectral sensitivities of rods and cones in macaque and human retinas were determined by recording the membrane current from single outer segments. In the macaque retina, the wavelengths of maximum sensitivity were at about 430, 530, and 561 nm for the blue, green, and red cones, respectively, and at 491 nm for the rods. The shapes of the spectra of the three cones were similar when plotted on a log wavenumber scale; the rod spectrum was slightly broader. Spectral sensitivities of the red and green cones from a human retina were virtually identical to those of macaque cones. For comparison with human psychophysical measurements, the rod and cone spectra were adjusted to give the sensitivities expected for light incident on the cornea of the human eye. These functions satisfactorily predicted the scotopic and photopic luminosity functions as well as results from human color-matching experiments. The adjusted spectra of the red and green cones also agreed well with the π-mechanisms of Stiles (1953, 1959).

Winter flounder (Pseudopleuronectes americanus) are hatched as bilaterally symmetric larvae which live near the ocean surface. At metamorphosis, they become laterally compressed, one eye migrates to the opposite side of the head, and they live the remainder of their lives lying on their blind side on the ocean floor. The present study characterizes and quantifies retinal cell distribution throughout the larval period and contrasts it with the adult retina. Based on light- and electron-microscopic analyses, retinas of larval flounder contain only a single cone-like photoreceptor type, arranged in a hexagonal array. In contrast, after metamorphosis, the adult retina has three types of photoreceptors: rods, single cones, and double cones. Rod photoreceptors are numerous in the ventral retina and decrease in density dorsad. The cone photoreceptor density, in contrast to rods, is higher in the dorsal retina decreasing ventrad. Adult cone photoreceptors are arranged in a square mosaic with four double cones surrounding one single cone. The differences in larval and adult retinal morphology reflect the distinctly different habitat each occupies.

In previous experiments we established that a light flash reduced cGMP levels of toad rod outer segments within the transduction time interval, but that recovery of the dark level of cGMP occurred more slowly than reported electrophysiological recovery of membrane potential. We now report that a second light flash accelerates the recovery rate of total cGMP following an initial flash, but that this acceleration is blocked in a medium which is both sodium and calcium deficient. We also noted that calcium deficiency only elevated cGMP levels when sodium was present. For other experiments, we recorded ERG or aspartate isolated PIII responses from eyecups or retinas mounted on our quick-freeze apparatus, the light stimuli originating from the double light-bench of the latter. Whereas background illumination depressed cGMP, no detectable further cGMP loss accompanied the electrical response to a flash superimposed on the background.

Intracellular recordings were made from rods in the superfused retina of the marine toad (Bufo marinus). It was found that injection of a brief depolarizing current pulse (0.04–1 nA) evoked a distinctive, long-lasting response, here called “the prolonged depolarization.” The response appears to be regenerative, has a stereotypical waveform, is typically about 6 mV in &litude and 3 s in duration, and has a relatively long recovery period (10–60 s). As a rule, the response cannot be directly evoked by light but the current-evoked response is significantly enhanced in the presence of steady illumination. The light-evoked hyperpolarization and the depolarizing spikes of the rod are both attenuated in the presence of the prolonged depolarization. The prolonged depolarization is not an altered manifestation of the depolarizing spikes of toad rods since both can be recorded simultaneously and steady illumination suppresses the spikes while enhancing the prolonged depolarization. The response is enhanced in chloride-free superfusate and also appears to be enhanced by the use of electrodes containing chloride. The response is markedly shortened in superfusates that lack calcium or contain 1–5 mM cobalt. On this and other evidence, it is suggested that the response may be generated by the sequential action of calcium channels and calcium-activated chloride channels. Although rarely evoked by light, the prolonged depolarization of toad rods is otherwise remarkably similar to the prolonged depolarization of turtle cones. It is proposed that the prolonged depolarization, in contrast to the feedback depolarization of cones, arises from mechanisms common to both rods and cones.

In the human rod visual system, self-cancellation of flicker signals is observed at high rod intensity levels near 15 Hz, both perceptually and in the electroretinogram (ERG). This and other evidence suggests that two rod signals are transmitted through the human retina with different speeds of transmission. Here we report a series of flicker ERG recordings from a normal observer and an observer who lacks cone vision. From these results, we propose a quantitative model of the two rod signals, which assumes (1) that the amplitude of the slow signal grows linearly with log intensity but then saturates at ~1 scot, td; (2) that the amplitude of the fast signal grows linearly with intensity; (3) that there is a difference in time delay of ~33 ms between two rod signals of the same polarity (or of ~67 ms if the signals are of inverted polarity); and (4) that the time delay of both signals declines linearly with log intensity (by ~10 ms per log scot. td). These simple assumptions provide a remarkably good account of the experimental data. Our results and model are relevant to current anatomical theories of the mammalian rod visual system. We speculate that the slower signal in the human ERG may reflect the transmission of the rod response via the rod bipolars and the An amacrine cells, while the faster signal may reflect its transmission via the rod-cone gap junctions and the cone bipolars. There are, however, several objections to this simple correspondence.

Dark-adapted adults' electroretinographic b−wave thresholds are —2 log units below a−wave thresholds and ~3 log units above the perceptual threshold, and their perceptual and the visually evoked cortical potential (VEP) thresholds are similar. Dark-adapted infants' scotopic a− and b−wave thresholds for full-field stimuli are both about 0.5 log units above those of adults, but their scotopic VEP thresholds for such stimuli have not been studied. We obtained scotopic VEP thresholds for brief, full-field stimuli from dark-adapted, infants and adults to consider the relationships of the cortical responses to the responses of more distal structures, namely the rod photoreceptors (scotopic a−wave) and ON-bipolars (scotopic b−wave). The median VEP threshold of infants is 0.5 log unit above that of adults. Thus, the relationships of a− and b−wave and VEP thresholds in infants are similar to those in adults. These results are consistent with rod cell immaturities being the primary determinant of the difference between infants' and adults' thresholds.

To identify ultraviolet (UV) and middle- (M) wavelength-sensitive cone and rod signals in murine retinal ganglion cells, single ganglion cell responses were studied in anesthetized, light-adapted C57/BL6 mice with tungsten microelectrodes driven through the sclera and vitreous to the neural retina. One hundred fifty-four ganglion cells were examined in 43 retinas of 34 mice. The retina was stimulated with diffuse flashes and/or pulses of ultraviolet (360 nm) or green (520 nm) light in the presence and absence of a strong steady orange adapting light. Twelve ganglion cells were studied in the dark-adapted retina in order to identify the signals of rods. Three functionally different types of ganglion cells were found: (1) phasic responding cells (31%) with no spontaneous activity and large impulse amplitudes; (2) tonic responding cells (60%) with irregular, low frequency (5–10 Hz) spontaneous activity and smaller impulse amplitudes; and (3) metronome-like cells (9%) with regular, relatively high-frequency (20–40 Hz) spontaneous activity. A few cells (1%) had habituating responses. Every cell encountered was affected by diffuse stimulation. The more common two types were excited at either the ON or OFF or at both the ON and OFF phases of stimulation. Type III cells had weaker responses, sometimes only inhibited by turning off a light. In the light-adapted state, most cells received signals of the same polarity from UV- and M-cones but UV-cone inputs were usually more dominant, especially in ventral retina. A fraction of cells received signals from only UV- (18%) or only M- (3%) cones. In rare cases (2%) these cone inputs had an opposite polarity on the same cell. In the dark-adapted state, all cells were at least four or five logarithmic units more sensitive and more to green than ultraviolet light. The results indicate that co-expression of both UV-and M-cone opsins cannot be ubiquitous in murine retina. Some cones, especially UV cones, exist without the presence of any functional M-cone opsin. This must be the case to explain the presence of ganglion cells that receive inputs only from UV-cones and others that receive inputs of opposite polarity from UV- and M-cones. The results support the hypothesis that murine retina has the physiological capacity to relay signals to the brain that allow the sensing of chromatic contrast and color vision.

We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12–14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A nonrecombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.

This report describes a second-generation photostimulator with four primary lights that allows independent control of the stimulation of the four receptor types in the human eye. The new design uses LEDs (with light levels controlled by eight drivers that include voltage-to-frequency converters that provide 1-μs pulses at frequencies up to 250 kHz), with four center channels being combined by use of a fiber optic assembly, and likewise for four surround channels. Four fiber optic bundles are merged into a single bundle whose output is fed into a spatial homogenizer terminated by a diffuser. An interference filter is sandwiched between each LED and the fiber optic bundle. Two camera lenses collimate light from the diffusers, one for center and one for surround. The center-surround field configuration is formed by a photometric cube with a mirrored ellipse on the hypotenuse. A field lens places images of the diffusers in the plane of an artificial pupil. The fields are highly uniform. Following alignment and calibration, the center and surround fields are indistinguishable. An observer calibration procedure, designed to compensate for prereceptoral filtering, is shown by calculation to correct also for normal observer receptoral spectral sensitivity variation. With the instrument calibrated for the individual observer, a peripherally fixated 200-ms 40% contrast rod center field pulse, highly conspicuous under dark adaptation, is invisible following light adaptation.

To determine whether the photoreceptors change structurally and functionally during aging, and to analyze whether pigmentation in the retinal pigment epithelium might be a contributing factor. Young, adult, and aged C57BL/6 and Balb/c mice (1, 4, and 17 months of age) were housed under a 12-h light/12-h dark cycle, with an ambient light intensity at the eye level of the mice of 85 ± 18 lux. Scotopic single-flash and photopic-flicker electroretinograms (ERGs) after complete dark adaptation were used to assess rod and cone function, respectively. Numbers of rod photoreceptors were counted in plastic sections, and rhodopsin levels were measured using absorption difference spectrophotometry. Numbers and types of cones were determined using lectin staining in retinal flatmounts and cone-specific antibodies in radial frozen sections. Young pigmented C57BL/6 and nonpigmented Balb/c mice had similar numbers of rods. In both mouse strains, there was an overall decline in rod photoreceptor number during aging, which was more pronounced in albino mice. Rod cell numbers correlated with a drop in the overall amount of rhodopsin and a reduction in the maximum a-wave of the rod ERG. The number of short-wavelength cones was unaffected by age and pigmentation, whereas an age-related decline was observed in mid-wavelength (MWL) cones in albino, but not in pigmented mice. In contrast, MWL cone function was reduced during aging in both strains.

The cellular responses of the cone-dominant ground squirrel retina to retinal detachment were examined and compared to those in rod-dominant species. Retinal detachments were made in California ground squirrels. The retinas were prepared for light, electron, and confocal microscopy. Tissue sections were labeled with antibodies to cone opsins, rod opsin, glial fibrillary acidic protein (GFAP), vimentin, synaptophysin, cytochrome oxidase, and calbindin D 28K. Wax sections were probed with the MIB-1 antibody to detect proliferating cells. By 10 h postdetachment many photoreceptor cells in the ground squirrel already show structural signs of apoptosis. At 1 day many photoreceptors have collapsed inner segments (IS), yet others still have short stacks of outer segment discs. At 3 days there is a marked increase in the number of dying photoreceptors. Rod and medium-/long-wavelength opsins are redistributed in the cell membrane to their synaptic terminals. At 7 days photoreceptor cell death has slowed. Some regions of the outer nuclear layer (ONL) have few photoreceptor somata. IS remnants are rare on surviving photoreceptors. At 28 days these trends are even more dramatic. Retinal pigmented epithelium (RPE) cells do not expand into the subretinal space. The outer limiting membrane (OLM) appears flat and uninterrupted. Müller cells remain remarkably unreactive; they show essentially no proliferation, only negligible hypertrophy, and there is no increase in their expression of GFAP or vimentin. Horizontal cells show no dendritic sprouting in response to detachment. The speed and extent of photoreceptor degeneration in response to detachment is greater in ground squirrel than in cat retina—only a small number of rods and cones survive at 28 days of detachment. Moreover, the almost total lack of Müller cell and RPE reactivity in the ground squirrel retina is a significant difference from results in other species.