The transcription factor Nr2e3 is an essential component for development and specification of rod and cone photoreceptors; however, the mechanism through which it acts is not well understood. In this study, we use Nr2e3rd7/rd7 mice that harbor a mutation in Nr2e3, to serve as a model for the human retinal disease Enhanced S Cone Syndrome. Our studies reveal that NR2E3 is expressed in late retinal progenitors and differentiating photoreceptors of the developing retina and localized to the cell bodies of mature rods and cones. In particular, we demonstrate that the abnormal increase in cone photoreceptors observed in Nr2e3rd7/rd7 mice arise from ectopic mitotic progenitor cells that are present in the outer nuclear layer of the mature Nr2e3rd7/rd7 retina. A prolonged phase of proliferation is observed followed by abnormal retinal lamination with fragmented and disorganized photoreceptor synapses that result in a progressive loss of rod and cone function. An extended and pronounced wave of apoptosis is also detected at P30 and temporally correlates with the phase of prolonged proliferation. Approximately twice as many apoptotic cells were detected compared to proliferating cells. This wave of apoptosis appears to affect both rod and cone cells and thus may account for the concurrent loss of rod and cone function. We further show that Nr2e3rd7/rd7 cones do not express rod specific genes and Nr2e3rd7/rd7 rods do not express cone specific genes. Our studies suggest that, based on its temporal and spatial expression, NR2E3 acts simultaneously in different cell types: in late mitotic progenitors, newly differentiating post mitotic cells, and mature rods and cones. In particular, this study reveals the function of NR2E3 in mitotic progenitors is to repress the cone generation program. NR2E3 is thus one of the few genes known to influence the competency of retinal progenitors while simultaneously directing the rod and cone differentiation.

During development, the projection from the lateral geniculate nucleus to striate cortex becomes segregated into monocular regions called ocular dominance columns. Prior studies in cats have suggested that experimental strabismus or alternating monocular occlusion increases the width and segregation of columns. In the squirrel monkey, strabismus has been reported to induce the formation of ocular dominance columns. However, these studies are difficult to interpret because no animal can serve as its own control and the degree of inter-individual variability among normal subjects is considerable. We have re-examined the effect of strabismus on ocular dominance columns in a large group of strabismic and normal squirrel monkeys. Five animals rendered strabismic at age one week had well-developed, widely spaced columns. Among 16 control animals, a wide spectrum of column morphology was encountered. Some control animals lacked ocular dominance columns, whereas others had columns similar to those observed in strabismic animals. Natural variation in column expression in normal squirrel monkeys, and potential uncontrolled genetic influences, made it impossible to determine if strabismus affects ocular dominance columns. It was evident however, that strabismus does not affect the binocular projection from the lateral geniculate nucleus to each CO patch in the upper layers. In strabismic monkeys, just as in normal animals, each patch received input from geniculate afferents serving both the left eye and the right eye. In addition, in strabismic monkeys, as in normal animals, patches were not aligned with ocular dominance columns.

In normal viewing, the visual system effortlessly assigns approximately constant attributes of color and shape to perceived objects. A fundamental component of this process is the compensation for illuminant variations and intervening media to recover reflectance properties of natural surfaces. We exploited the phenomenon of transparency perception to explore the cortical regions implicated in such processes, using fMRI. By manipulating the coherence of local color differences around a region in an image, we interfered with their global perceptual integration and thereby modified whether the region appeared transparent or not. We found the major cortical activation due to global integration of local color differences to be in the anterior part of the parahippocampal gyrus. Regions differentially activated by chromatic versus achromatic geometric patterns showed no significant differential response related to the coherence/incoherence of local color differences. The results link the integration of local color differences in the extraction of a transparent layer with sites activated by object-related properties of an image.

Vertebrate photoreceptors respond to light with changes in membrane conductance that reflect the activity of cyclic-nucleotide gated channels (CNG channels). The functional features of these channels differ in rods and cones; to understand the basis of these differences we cloned CNG channels from the retina of striped bass, a fish from which photoreceptors can be isolated and studied electrophysiologically. Through a combination of experimental approaches, we recovered and sequenced three full-length cDNA clones. We made unambiguous assignments of the cellular origin of the clones through single photoreceptor RT-PCR. Synthetic peptides derived from the sequence were used to generate monospecific antibodies which labeled intact, unfixed photoreceptors and confirmed the cellular assignment of the various clones. In rods, we identified the channel α subunit gene product as 2040 bp in length, transcribed into two mRNA 1.8 kb and 2.9 kb in length and translated into a single 96-kDa protein. In cones we identified both α (CNGA3) and β (CNGB3) channel subunits. For α, the gene product is 1956 bp long, the mRNA 3.4 kb, and the protein 74 kDa. For β, the gene product is 2265 bp long and the mRNA 3.3 kb. Based on deduced amino acid sequence, we developed a phylogenetic map of the evolution of vertebrate rod and cone CNG channels. Sequence comparison revealed channels in striped bass, unlike those in mammals, are likely not N-linked-glycosylated as they are transported within the photoreceptor. Also bass cone channels lack certain residues that, in mammals, can be phosphorylated and, thus, affect the cGMP sensitivity of gating. On the other hand, functionally critical residues, such as positively charged amino acids within the fourth transmembrane helix (S4) and the Ca2+-binding glutamate in the pore loop are absolutely the same in mammalian and nonmammalian species.

Amblyopia is usually considered to be a deficit in spatial vision. But there is evidence that amblyopes may also suffer specific deficits in motion sensitivity as opposed to losses that can be explained by the known deficits in spatial vision. We measured sensitivity to visual motion in random dot displays for strabismic and anisometropic amblyopic monkeys. We used a wide range of spatial and temporal offsets and compared the performance of the fellow and amblyopic eye for each monkey. The amblyopes were severely impaired at detecting motion at fine spatial and long temporal offsets, corresponding to fine spatial scale and slow speeds. This impairment was also evident for the untreated fellow eyes of strabismic but not anisometropic amblyopes. Motion sensitivity functions for amblyopic eyes were shifted toward large spatial scales for amblyopic compared to fellow eyes, to a degree that was correlated with the shift in scale of the spatial contrast sensitivity function. Amblyopic losses in motion sensitivity, however, were not correlated with losses in spatial contrast sensitivity. This, combined with the specific impairment for detecting long temporal offsets, reveals a deficit in spatiotemporal integration in amblyopia which cannot be explained by the lower spatial resolution of amblyopic vision.

Australian lungfish Neoceratodus forsteri may be the closest living relative to the first tetrapods and yet little is known about their retinal ganglion cells. This study reveals that lungfish possess a heterogeneous population of ganglion cells distributed in a horizontal streak across the retinal meridian, which is formed early in development and maintained through to adult stages. The number and complement of both ganglion cells and a population of putative amacrine cells within the ganglion cell layer are examined using retrograde labelling from the optic nerve and transmission electron-microscopic analysis of axons within the optic nerve. At least four types of retinal ganglion cells are present and lie predominantly within a thin ganglion cell layer, although two subpopulations are identified, one within the inner plexiform and the other within the inner nuclear layer. A subpopulation of retinal ganglion cells comprising up to 7% of the total population are significantly larger (>400 μm2) and are characterized as giant or alpha-like cells. Up to 44% of cells within the retinal ganglion cell layer represent a population of presumed amacrine cells. The optic nerve is heavily fasciculated and the proportion of myelinated axons increases with body length from 17% in subadults to 74% in adults. Spatial resolving power, based on ganglion cell spacing, is low (1.6–1.9 cycles deg−1, n = 2) and does not significantly increase with growth. This represents the first detailed study of retinal ganglion cells in sarcopterygian fish, and reveals that, despite variation amongst animal groups, trends in ganglion cell density distribution and characteristics of cell types were defined early in vertebrate evolution.

The ganglion cell layer of mammalian retina contains numerous amacrine cells. Many belong to one type, the cholinergic starburst cell, but the other types have not been systematically identified. Using a new method to target sparsely represented cell types, we filled about 200 amacrine neurons in the ganglion cell layer of the guinea pig visual streak and identified 11 types. Ten of these resemble types identified in other species with somas in the inner nuclear layer, but one type has not been previously reported. Most of the types and nearly all the injected cells (95%) arborized low in the synaptic layer where they would co-stratify with various classes of ON ganglion cell. The displaced somas (7% of all amacrine cells) thus represent a heterogeneous pool, which are relatively accessible for study of their interactions with ON ganglion cells.

Neurons sensitive to optic flow patterns have been recorded in the the olivo-vestibulocerebellar pathway and extrastriate visual cortical areas in vertebrates, and in the visual neuropile of invertebrates. The complex spike activity (CSA) of Purkinje cells in the vestibulocerebellum (VbC) responds best to patterns of optic flow that result from either self-rotation or self-translation. Previous studies have suggested that these neurons have a receptive-field (RF) structure that “approximates” the preferred optic flowfield with a “bipartite” organization. Contrasting this, studies in invertebrate species indicate that optic flow sensitive neurons are precisely tuned to their preferred flowfield, such that the local motion sensitivities and local preferred directions within their RFs precisely match the local motion in that region of the preferred flowfield. In this study, CSA in the VbC of pigeons was recorded in response to a set of complex computer-generated optic flow stimuli, similar to those used in previous studies of optic flow neurons in primate extrastriate visual cortex, to test whether the receptive field was of a precise or bipartite organization. We found that these RFs were not precisely tuned to optic flow patterns. Rather, we conclude that these neurons have a bipartite RF structure that approximates the preferred optic flowfield by pooling motion subunits of only a few different direction preferences.

Platyrrhine monkeys typically have only a single X-chromosome opsin gene. Alleles of this gene code for multiple versions of middle- to long-wavelength cone photopigments. X-chromosome inactivation provides heterozygous females with a retinal mosaic of cones containing either of two types of M and L pigment, thus establishing the photopigment basis for trichromatic color vision. This study examined the proportions of L and M cones created by this process. For that purpose, electroretinogram flicker photometry was used to obtain complete spectral sensitivity functions from 60 heterozygous female monkeys drawn from seven genera of platyrrhine monkeys. To obtain estimates of cone proportions, these functions were subsequently fit with linear combinations of L and M cone fundamentals that were derived from similar recordings made on conspecific animals having only one type of M/L pigment. Consistent with a random X-chromosome inactivation process, the average L:M cone weighting across the sample was close to unity. At the same time, there were significant individual variations in L:M cone proportions. The genesis of this variation and its implications for seeing are discussed.

Early in development, before the retina is responsive to light, neurons exhibit spontaneous activity. Recently it was demonstrated that starburst amacrine cells, a unique class of neurons that secretes both GABA and acetylcholine, spontaneously depolarize. Networks comprised of spontaneously active starburst cells initiate correlated bursts of action potentials that propagate across the developing retina with a periodicity on the order minutes. To determine whether other retinal interneurons have similar “pacemaking” properties, we have utilized cultures of dissociated neurons from the rat retina. In the presence of antagonists for fast neurotransmitter receptors, distinct populations of neurons exhibited spontaneous, uncorrelated increases in intracellular calcium concentration. These increases in intracellular calcium concentration were sensitive to tetrodotoxin, indicating they are mediated by spontaneous membrane depolarizations. By combining immunofluorescence and calcium imaging, we found that 44% of spontaneously active neurons were GABAergic and included starburst amacrine cells. Whole cell voltage clamp recordings in the absence of antagonists for fast neurotransmitters revealed that after 7 days in culture, individual retinal neurons receive bursts of GABA-A receptor mediated synaptic input with a periodicity similar to that measured in spontaneously active GABAergic neurons. Low concentrations of GABA-A receptor antagonists did not alter the inter-burst interval despite significant reduction of post-synaptic current amplitude, indicating that pacemaker activity of GABAergic neurons was not influenced by network interactions. Together, these findings indicate that spiking GABAergic interneurons can function as pacemakers in the developing retina.

To assess the effects of macular pigment optical density (MPOD) on isoluminant stimuli and to quantify MPOD electrophysiologically, MPOD distribution profiles were obtained in normal subjects using minimum motion and minimum flicker photometry. Isoluminance of VEP stimuli was determined using minimum flicker and tritan confusion lines were determined using a minimum distinct border criterion. Onset–offset and reversal VEPs to isoluminant red/green, blue/green, and subject-specific tritan gratings of different diameters were recorded from the same 14 subjects tested psychophysically. VEPs were additionally recorded to annular gratings. Chromatic VEP selectivity was assessed by Fourier analysis and as an index; onset negativity/(onset negativity + onset positivity). Peak MPOD varied between 0.2–0.8. Chromatic onset VEPs to all isoluminant 3-deg fields were predominantly negative. Larger blue/green and tritan stimuli elicited VEPs with additional positive, achromatic components; for 9-deg gratings, peak MPOD showed negative correlation with the power of the VEP fundamental (r = −0.70) and with the selectivity index (r = −0.83). Annular gratings elicited chromatic-specific B/G VEPs but only when isoluminance was determined for the annulus. Chromatic selectivity loss in VEPs to large B/G or Tritan gratings can be used to estimate subject-specific MPOD. An important implication is that isoluminant Tritan stimuli with short-wavelength components must be restricted in size in order to optimize koniocellular selectivity.

The key steps in the evolution of full trichromatic color vision in primates include duplication of the ancestral pigment gene to form the L and M pigment gene array on the X chromosome, mutually exclusive expression of the L and M pigment genes in cone photoreceptors, and formation of a retinal mosaic with randomly distributed L and M cones. Previous work using transgenic mice has indicated that a locus control region adjacent to this array of genes plays an important role in their mutually exclusive expression in respective cone cells (Smallwood et al., 2002). However, the mechanism by which this is accomplished is unknown. We searched for a cellular model system to investigate the mechanism of this mutually exclusive expression. We previously showed that the undifferentiated human retinoblastoma cell line WERI expresses L and M cone opsin but not rod opsin genes. We now show that WERI cells express the L and M pigment genes in a mutually exclusive manner, in that either L or M pigment mRNA is expressed in a single cell. Importantly, clonal analysis showed that single WERI cells that express either L or M generate, upon cell division produce, a mixed population of L- or M-expressing cells. These results indicate, first, that cell division resets L or M pigment gene expression, most likely due to disassembly and reassembly of LCR-promoter DNA-protein complexes during cell division. Second, a retinal mosaic with near-random distribution of L and M cones may have been generated automatically after duplication of the ancestral gene to form the L and M pigment genes. Third, determination of L and M cone identity may not require external molecular cues during differentiation, and is consistent with the idea that L and M cones are not intrinsically different.

We measured the responses of single neurons in marmoset visual cortex (V1, V2, and the third visual complex) to moving first-order stimuli and to combined first- and second-order stimuli in order to determine whether first-order motion processing was influenced by second-order motion. Beat stimuli were made by summing two gratings of similar spatial frequency, one of which was static and the other was moving. The beat is the product of a moving sinusoidal carrier (first-order motion) and a moving low-frequency contrast envelope (second-order motion). We compared responses to moving first-order gratings alone with responses to beat patterns with first-order and second-order motion in the same direction as each other, or in opposite directions to each other in order to distinguish first-order and second-order direction-selective responses. In the majority (72%, 67/93) of cells (V1 73%, 45/62; V2 70%, 16/23; third visual complex 75%, 6/8), responses to first-order motion were significantly influenced by the addition of a second-order signal. The second-order envelope was more influential when moving in the opposite direction to the first-order stimulus, reducing first-order direction sensitivity in V1, V2, and the third visual complex. We interpret these results as showing that first-order motion processing through early visual cortex is not separate from second-order motion processing; suggesting that both motion signals are processed by the same system.

Light decrements are mediated by two distinct groups of rod pathways in the dark-adapted retina that can be differentiated on the basis of their sensitivity to the glutamate agonist DL-2-amino-phosphonobutyric (APB). By means of the APB sensitive pathway, rods transmit light decrements via rod bipolar cells to AII amacrine cells, then to Off cone bipolar cells, which in turn innervate the dendrites of Off ganglion cells. APB hyperpolarizes rod bipolar cells, thus blocking this rod pathway. With APB insensitive pathways, rods either directly synapse onto Off cone bipolar cells, or rods pass light decrement signal to cones by gap junctions. In the present study, whole-cell patch-clamp recordings were made from ganglion cells in the dark-adapted mouse retina to investigate the functional properties of APB sensitive and insensitive rod pathways. The results revealed several clear-cut differences between the APB sensitive and APB insensitive rod pathways. The latency of Off responses to a flashing spot of light was significantly shorter for the APB insensitive pathways than those for the APB sensitive pathway. Moreover, Off responses of the APB insensitive pathways were found to be capable of following substantially higher stimulus frequencies. Nitric oxide was found to selectively block Off responses in the APB sensitive rod pathway. Collectively, these results provide evidence that the APB sensitive and insensitive rod pathways can convey different types of information signaling light decrements in the dark-adapted retina.

This article appeared in Volume 23, Number 5, 2006, pages 765–778.

Goldfish cones contain CB1 receptors at the synaptic terminal, selectively accumulate 3H-anandamide, and contain fatty acid amide hydrolase-immunoreactivity, and voltage-gated calcium and potassium currents are modulated by CB1 ligands (Yazulla et al., 2000; Fan & Yazulla, 2003; Glaser et al., 2005). These data suggest that a retinal mechanism may account for some of the psychophysical effects of cannabis. Here, we studied the effect of a cannabinoid agonist on cone light responses. Whole-cell patch-clamp recordings were made from cones in the isolated goldfish retina. Cones were stimulated with a spot of light of variable wavelength and intensities in combination with voltage-and current-clamp protocols. Pharmacological manipulation was performed using the cannabinoid agonist WIN 55212-2 (10 μM). WIN had no effect on the absolute sensitivity of the cones or on the kinetics of the onset response. However, the light-offset response became faster, and the depolarizing overshoot was enhanced. Time constant of the offset response was reduced from 292 ± 28 ms to 180 ± 11 ms (n = 6) (P < 0.01) in the presence of WIN. Acceleration of the offset response was not affected by flash length from 200 ms to 10 s. This was found under current-clamp as well as under voltage-clamp conditions, indicating that the effect of WIN was mediated directly or indirectly by modulation of the cGMP-gated channels in the outer segment of the cones. The effects of WIN were not blocked by the CB1 antagonist SR141716A. With a train of “dark” flashes from a steady background, the photocurrent recovered toward baseline more quickly with WIN than in Control. In summary, cannabinoids speed up the dynamics of the phototransduction deactivation cascade in the cone outer segments. The functional consequence of this effect is to shorten the recovery time to the offset of bright flashes, perhaps resulting in an increase in contrast sensitivity.

Extracellular Ca2+ and Zn2+ influence many aspects of retinal function. Here, we examined the effect of external Ca2+ and Zn2+ on potassium channels of retinal horizontal cells. When extracellular Ca2+ was lowered from 3 mM to 0.3 mM, horizontal cell transient outward currents elicited by voltage steps from resting membrane potential (−70 mV) were decreased by approximately 50%, whereas the sustained currents remained unchanged. This effect was due to a hyperpolarizing shift in the steady-state inactivation curve of A-type K+ currents when extracellular Ca2+ concentration was lowered. The mean half inactivation potential of the steady-state inactivation curves was hyperpolarized from −56.3 ± 4.7 mV in 3 mM Ca2+ to −76.4 ± 3.9 mV in 0.3 mM Ca2+. Neither the state-steady activation curve nor the kinetics of inactivation was significantly changed in low extracellular Ca2+. The addition of 30 μM Zn2+ restored peak outward currents in 0.3 mM Ca2+. The half inactivation voltages were depolarized from −70 ± 2.8 mV in 0.3 mM Ca2+ to −56 ± 2.6 mV in 0.3 mM Ca2+ plus 30 μM Zn2+. Taken together, the results indicate that external Ca2+ and Zn2+ maintain the activity of A-type potassium channels in retinal horizontal cells by influencing the voltage dependence of steady-state inactivation.

Glaucoma is a major cause of blindness and is characterized by death of retinal ganglion cells. In a rat model of glaucoma in which intraocular pressure is raised by cautery of episcleral veins, the somata and dendritic arbors of surviving retinal ganglion cells expand. To assess physiological consequences of this change, we have measured visual receptive-field size in a primary retinal target, the superior colliculus. Using multiunit recording, receptive-field sizes were measured for glaucomatous eyes and compared to both those measured for contralateral control eyes and to homolateral eyes of unoperated animals. Episcleral vein occlusion increased intraocular pressure. This was accompanied by a significant increase in receptive-field size across the superior colliculus. The expansion of receptive fields was proportional to both degree and duration of the increase of intraocular pressure. We suggest that this increase in the size of receptive fields of glaucomatous eyes may be related to the increase in the size of dendritic arbors of the surviving ganglion cells in retina.

The topographical distributions of the relative ratio of long- (L) and middle- (M) wavelength sensitive cone opsin messenger RNA (mRNA) in human and baboon retinas were mapped using real-time polymerase chain reaction. The L:M mRNA ratio increased in a central-to-peripheral gradient in both species, being quite pronounced for humans.

A model of the development of L/M cone ratios in the Old World primate retina is presented. It is supposed that during gestation, the cone cycles randomly between states in which it transcribes either L or M opsin. The current state determines and increases the probability that it will transcribe the same opsin in future cycles. These assumptions are sufficient to formalize the process as a Markov chain that can be modeled as an urn containing two types of balls, L and M. Drawing one ball results in the increase of its species and the decrease of the other. Over the long run, the urn will become populated with a single type of ball. This state corresponds to the photoreceptor adopting a fixed identity for its lifetime. We investigate the effect of the number of states and the rule that regulates the advantage of transition toward one cone type or another on the relation between fetal and adult L/M cone ratios. In the range of 100 to 1000 states, small variations of the initial L/M ratio or the transition advantage can each generate large changes in the final L/M ratio, in qualitative accord with the variation seen in human adult retinas. The time course to attain stable L/M ratios also varies with these parameters. If it is believed that the cycling follows a circadian rhythm, then final L/M cone ratios would be expected to stabilize shortly after birth in the human being and the macaque.