The mammalian neural retina contains single or multiple intrinsic circadian oscillators that can be directly entrained by light cycles. Dopaminergic amacrine (DA) cells represent an especially interesting candidate as a site of the retinal oscillator because of the crucial role of dopamine in light adaptation, and the widespread distribution of dopamine receptors in the retina. We hereby show by single-cell, end-point RT-PCR that retinal DA cells contain the transcripts for six core components of the circadian clock: Bmal1, Clock, Cry1, Cry2, Per1, and Per2. Rod photoreceptors represented a negative control, because they did not appear to contain clock transcripts. We finally confirmed that DA cells contain the protein encoded by the Bmal1 gene by comparing immunostaining of the nuclei of DA cells in the retinas of wildtype and Bmal1−/− mice. It is therefore likely that DA cells contain a circadian clock that anticipates predictable variations in retinal illumination.

In the fish retina, retinomotor movement, spinule formation, and alteration of connexon density within gap junctions occur in response to changes in ambient light conditions. All of these morphological parameters can also be influenced by the application of dopamine. This study examines whether the morphological alterations of these structures are correlated with the activity of endogenous dopamine during an entrained 12-h light/12-h dark cycle and after 1-h sort-term adaptation periods.

The two measured parameters of retinomotor movement, cone inner segment length and pigment dispersion, were well-correlated with endogenous cyclic dopamine activity. However, retinomotor movement was initiated already at the end of the entrained dark period, before the onset of light and before the onset of dopamine turnover. Furthermore, a 1-h dark-adaptation period in the middle of the light phase reduced dopamine activity but did not affect retinomotor movement. At the switch from light to dark and after a 1-h light period at midnight retinomotor movement correlated exactly with dopamine turnover and illumination conditions. The formation of spinules was correlated with dopaminergic activity during all phases of the light/dark cycle and during short-term adaptation periods. Spinules were expressed in the light when dopamine activity was high and they were retracted when dopamine activity was reduced during darkness. Connexon density of horizontal cell gap junctions showed a weaker correlation with the endogenous dopamine turnover. In this case, a high activity of endogenous dopamine was paralleled by a high density of connexons.

Our results suggest that endogenous dopamine is involved in the cyclic regulation of the observed morphological alterations and that dopamine is part of the light signal for these mechanisms.

Visually guided saccadic eye movements to singly presented stationary targets form a bimodal distribution. After superior colliculus lesions, the so called “express saccades” that form the first mode of the distribution are no longer obtained. The aim of this study was to determine what role several other neural systems play in the generation of express and regular saccades, with the latter being those that form the second mode in the bimodal distribution. Lesions were made in the parvocellular and magnocellular portions of the lateral geniculate nucleus to disrupt either the midget system or the parasol system that originates in the retina and areas V4 and MT. The effects of the lesions were examined on the accuracy and latency of saccadic eye movements made to stationary and to moving visual targets. Following magnocellular and MT lesions deficits were observed in smooth pursuit and in the amplitude of saccades made to moving targets. However, none of the lesions produced significant changes in the bimodal distribution of saccadic latencies to stationary targets. The results suggest that express saccades and regular saccades are not selectively mediated by either the midget or the parasol systems or by areas V4 and MT. Neither are the frontal eye fields involved as had previously been shown. We suggest that the superior colliculus plays a central role in producing both express and regular saccades by virtue of highly convergent input from numerous cortical structures.

Observation of the spread of biotinylated or fluorescent tracers following injection into a single cell has become one of the most common methods of demonstrating the presence of gap junctions. Nevertheless, many of the fundamental features of tracer movement through gap junctions are still poorly understood. These include the relative roles of diffusion and iontophoretic current, and under what conditions the size of the stained mosaic will increase, asymptote, or decline. Additionally, the effect of variations in amount of tracer introduced, as produced by variation in electrode resistance following cell penetration, is not obvious. To examine these questions, Neurobiotin was microinjected into the two types of horizontal cell of the rabbit retina and visualized with streptavidin-Cy3. Images were digitally captured using a confocal microscope. The spatial distribution of Neurobiotin across the patches of coupled cells was measured. Adequate fits to the data were obtained by fitting to a model with terms for diffusion and amount of tracer injected. Results indicated that passive diffusion is the major source of tracer movement through gap junctions, whereas iontophoretic current played no role over the range tested. Fluorescent visualization, although slightly less sensitive than peroxidase reactions, produced staining intensities with a more useful dynamic range. The rate constants for movement of Neurobiotin between A-type horizontal cells was about ten times greater than that for B-type horizontal cells. Although direct extrapolation to ion conductances cannot be assumed, tracer movement can be used to give an estimate of relative coupling rates across cell types, retinal location, or modulation conditions in intact tissue.

Visual information is processed in a series of subsequent steps. The performance of each of these steps depends not only on the computations it performs itself but also on the representation of the visual surround on which it operates. Here we investigate the consequences of signal preprocessing for the performance of the motion-detection system of the fly. In particular, we analyze whether the retinal input signals are rectified and segregate into separate ON and OFF channels, which then feed independent parallel motion-detection pathways. We recorded the activity of an identified directionally selective interneuron (HI-cell) in response to apparent motion stimuli, i.e. sequential brightness changes at two neighboring locations in the visual field, as well as to brightness changes at only a single location. For apparent motion stimuli, the motion-dependent response component was determined by subtracting from the overall response the responses to the individual stimulus components when presented alone. The following conclusions could be derived: (1) Apparent motion consisting of a sequence of increased or decreased brightness at two locations in the visual field have the same optimum interstimulus time interval (Fig. 3). (2) Sequences of brightness steps of like polarity (either increments or decrements) elicit positive and negative motion-dependent response components when mimicking motion in the cell's preferred and null direction, respectively. The motion-dependent response components are inverted in sign when the brightness steps of a stimulus sequence have a different polarity (Fig. 7). (3) The responses to the beginning and the end of a brightness pulse depend on the pulse duration. For pulse durations of less than 2 s, both events interact with each other (Fig. 9). All of these results do not provide any indication that the fly processes motion information in independent ON and OFF motion detectors. Brightness changes of both signs are rather represented at the input of the same movement detectors, and interactions between signals resulting from both brightness increments and decrements take their sign into account. This type of preprocessing of the retinal input is argued to render a motion-detection system particularly robust against noise.

Monkey electrophysiological and human neuroimaging studies indicate the existence of specialized neural systems for the perception and execution of actions. To date, the dynamics of these neural systems in humans have not been well studied. Here, we investigated the spatial and temporal behavior of human neural responses elicited to viewing motion of the face, hand, and body. Scalp event-related potentials (ERPs) were recorded in 20 participants viewing videotaped mouth (opening, closing), hand (closing, opening), and body stepping (forward, backward) movements. ERP peak differences within the movements of each body part were compared using topographical maps of voltage, voltage difference, and Student's t-test at ERP peak latencies. Predominantly temporoparietal negative ERPs occurred to motion of all body parts within 200 ms postmovement onset. Hand closure elicited a significantly greater negativity than opening, particularly in the left hemisphere. Vertex positive ERPs within 300 ms postmovement onset were elicited to hand and body motion. A significantly greater positivity occurred for the body stepping forward relative to stepping backward. The ERP topography was consistent with observed activation foci in human neuroimaging studies. Our data indicate that the neural activity of a system dedicated to the perception of high-level motion stimuli can rapidly differentiate between movements across and within body parts.

Studies of visual development show that basic metrics of visual development such as spatial resolution develop over the first 6–9 months in monkeys and over the first 6 or so years in humans. However, more complex visual functions may develop over different, or more protracted, time courses. To address the question of whether global perceptual processing is linked to or otherwise dependent on the development of basic spatial vision, we studied the development of contour integration, a global perceptual task, in comparison to that of grating acuity in macaque monkeys. We find that contour integration develops substantially later than acuity. Contour integration begins to develop at 5–6 months, near the time that acuity development is complete and continues to mature well into the second postnatal year. We discuss this later development in terms poor central efficiency and consider the relevant anatomy and physiology of the developing visual system. We conclude that contour integration is not likely to be limited by the same mechanisms that are permissive to acuity development, and may instead reflect the emergence of function central to V1.

Evidence suggests that patients with schizophrenia show impaired performances on tests assessing the magnocellular (M) visual pathway. The aim of this study was to investigate M pathway functioning in persons at high-risk of psychosis. Sixteen high-risk persons at the prodromal phase of psychosis and 20 healthy controls participated. Two types of contrast sensitivity measurements were used, during which participants were asked to detect a briefly presented target Gabor patch. In the pulsed-pedestal paradigm, the luminance of the background field was decreased to saturate M pathways and to bias information processing to parvocellular (P) pathways. In the steady-pedestal paradigm, the luminance of the background field was constant and briefly presented targets were processed by the M pathway. Anomalous perceptual experiences were assessed using the Structured Interview for Assessing Perceptual Anomalies (SIAPA). Results revealed that the high-risk persons showed elevated contrast sensitivity during the M pathway test, and normal sensitivity during the P pathway test. The visual SIAPA scores showed significant positive correlations with the M pathway sensitivity values. These results suggest that the high-risk mental state is associated with hyper-reactive M pathways, which may be responsible for some anomalous perceptual experiences, including abnormal intensity of environmental stimuli, feelings of being flooded and inundated, and inability to focus attention to relevant details.

The short wavelength-sensitive (blue) cone bipolar cells was found to have a nonrandom distribution by analyzing the nearest neighbors and by calculating the density recovery profile (DRP). Blue cones had been shown previously to have a nonrandom distribution (Curcio et al., 1991). The relationship between the two arrays was then analyzed by calculating the cross-correlational density recovery profile (cDRP), which indicates the local density of blue cones around each blue cone bipolar cell. Although both cell types appeared to be distributed uniformly at the macroscopic level, the cDRP was 1.7 times higher within 15 μm of each bipolar cell perikaryon than in the surrounding area. The area of higher density was approximately the same as that in which the blue cone bipolar cells made synaptic contacts with blue cones. The finding that the blue cones and the blue cone bipolar cells were closer together than expected suggests that the positions of the perikarya of these neurons were influenced by their synaptic connections or other developmental interactions.

Using patch-clamp recording in combination with reverse transcriptase-polymerase chain reaction (RT-PCR), we show in individual bipolar cells acutely dissociated from the adult rat retina a correlation between the expression of the GABAA receptor ρ1 subunit mRNA and a bicuculline-resistant, diazepam-insensitive component of the GABA-activated whole-cell current response. This “GABAC-like” response, contributing to approximately 42% of the GABA-activated whole-cell current and displaying variable sensitivity to picrotoxin, was found in bipolar cells but not in any of the ganglion cells examined. Expression profiling of GABAA receptor subunit mRNAs in individual electrophysiologically tested retinal neurons revealed that, while both bipolar cells and ganglion cells may express numerous GABAA receptor subunit isoforms, including that of ρ2, the expression of the ρ1 subunit was strictly limited to bipolar cells. We propose a possible link between the presence of a receptor with GABAC-like pharmacological profile and the expression of the retina-specific ρ1 subunit isoform. The results presented in this study constitute the first direct demonstration of such a correlation at the single-cell level.

We previously showed that immunoreactivity to N-Methyl-D-aspartate (NMDA) receptors in primary visual cortex of Callithrix jacchus is regulated by visual activity during the second and third postnatal months (Fonta et al., 1997). The purpose of the present study was to show that the columnar pattern of high and low NMDAR1 immunoreactivity observed in monocularly deprived animals corresponds to ocular dominance columns linked to the nondeprived and deprived eye, respectively. We compared cortical distribution of NMDAR1 receptors and the projection zones of thalamic afferents, revealed by transneuronal transport of tritiated proline, in 2-month-old, either monocularly deprived or control, marmosets. The data show that ocular dominance columns exist in 2-month-old marmosets and that a 2-week monocular deprivation by means of eyelid suture leads to a modification of the thalamo-cortical afferents organization. Experiments of neuronal tracing and immunohistochemistry performed on the same animals demonstrated that cortical domains with decreased NMDAR1 level correspond to the deprived eye columns. These investigations, coupled to the previous results, strongly suggest that the NMDA receptors, regulated by visual activity, are involved in the refining of ocular dominance columns in the primary visual cortex of juvenile marmoset.

Simultaneous extracellular ERG and intracellular recordings from horizontal and ON-bipolar cells were obtained from the dark-adapted retina of the dogfish. The light intensity–peak response relation (IR) and time course of on-bipolar cell responses closely resembled that of the ERG b-wave, but only at low light intensities [<10 rhodopsin molecules bleached per rod (Rh*)]. Block of on-bipolar cell responses with 50 μM 2-amino-4-phosphonobutyrate (APB) abolished the b-wave and unmasked a vitreal-negative wave. Subtraction from the control ERG resulted in the isolation of a vitreal-positive ERG with an IR which matched that of on-bipolar cells over the full range of light intensities. The D.C. component of the ERG arises as a result of sustained depolarization of on-bipolar cells in response to long (>0.5 s) dim light stimuli, or following bright light flashes. The IR of horizontal cells and the vitreal-negative wave unmasked by APB could be matched by scaling at low light intensities (<5 Rh*). However, horizontal cell responses saturated at about 30 Rh*, while the vitreal-negative wave continued to increase in amplitude. The time course of horizontal cell membrane current with dim flashes could be matched to the rising phase of the vitreal-negative wave, assuming that the delay in generating the voltage response in horizontal cells is due to their long (100 ms) membrane time constant. Blocking post-photoreceptor activity resulted in a much smaller vitreal-negative wave than that unmasked by APB alone. We conclude that the b-wave arises from on-bipolar cell depolarization, while the leading edge of the a-wave is a composite of the change in extracellular voltage drop across the rod layer and a component (proximal PIII) reflecting a decrease in extracellular K+ as horizontal cell synaptic channels close with light.

The effect of light stimuli and prolonged darkness on the release of endogenous dopamine was measured in the white perch and hybrid bass retinas. Isolated retinas were superfused and released dopamine was measured using extraction and high-pressure liquid chromatography separation techniques. Potassium-induced release did not depend on the background illumination nor on the period of previous darkness. Steady white light did not affect release, but flickering light of 2 Hz increased release about two-fold. During prolonged darkness, the release of dopamine increased steadily over the test period of 2 h, but only if the experiments were performed at night. During the day such an increase was not observed. The increased release during prolonged darkness at night was turned off by a short period of steady white light. The release patterns obtained from the white perch and the hybrid bass were similar. However, the hybrid bass retina showed much lower levels of dopamine than did the white perch retina.

Melatonin is a ubiquitous molecule and widely distributed in nature, with functional activity occurring in unicellular organisms, plants, fungi, and animals. Several studies have indicated that melatonin synthesis occurs in the retina of most vertebrates, including mammals. The retinal biosynthesis of melatonin and the mechanisms involved in the regulation of this process have been extensively studied. Circadian clocks located in the photoreceptors and retinal neurons regulate melatonin synthesis in the eye. Photoreceptors, dopaminergic amacrine neurons, and horizontal cells of the retina, corneal epithelium, stroma endothelium, and the sclera all have melatonin receptors, indicating a widespread ocular function for melatonin. In addition, melatonin is an effective antioxidant which scavenges free radicals and up-regulates several antioxidant enzymes. It also has a strong antiapoptotic signaling function, an effect that it exerts even during ischemia. Melatonin cytoprotective properties may have practical implications in the treatment of ocular diseases, like glaucoma and age-related macular degeneration.

Mammalian homologues of the Drosophila canonical Transient Receptor Potential (TRPC) protein have been proposed to encode the store-operated Ca2+ influx (SOC) channel(s). This study examines the role of TRPC1 in the SOC mechanism of retinal cells. htrpc1 transcript was detected in bovine retinal and in human adult retinal pigment epithelial (ARPE) cells. Western blot analysis also confirmed the expression of TRPC1 protein in neuronal cells including retina and ARPE cells. To determine the role of TRPC1 protein in retinal cells, TRPC1 was recombinantly expressed in ARPE cells and changes in intracellular Ca2+ were analyzed. ARPE cells stably transfected with htrp1 cDNA displayed 2-fold higher Ca2+ influx with no significant increase in the basal influx. Consistent with this the overexpressed TRPC1 protein was localized in the plasma membrane region of ARPE cells. Interestingly, both bovine retinal tissues and ARPE cells showed that TRPC1 protein co-localizes and could be co-immunoprecipitated with β-tubulin. Disruption of tubulin by colchicine significantly decreased both plasma membrane staining of the TRPC1 protein and Ca2+ influx in ARPE cells. These results suggest that TRPC1 channel protein is expressed in retinal cells, further, targeting/retention of the TRPC1 protein to the plasma membrane in retinal cells is mediated via its interaction with β-tubulin.

Monoclonal antibodies to the three isozymes of protein kinase C (PKC) (α, β, and γ) were applied to postmortem human retina. Immunostaining was done on wholemount, or cryostat-sectioned retina, and visualized after ABC/DAB procedures by light (LM) and electron (EM) microscopy.

The PCK-α antibody stained rod bipolar cells throughout the retina. EM analysis confirmed they were PKC-α-immunoreactive (IR) on their characteristic dendritic and axonal synaptology. Putative blue cone bipolar cells with wide-field axon terminals, stratifying in s5 of the inner plexiform layer (IPL), were also PKC-α-IR, and EM showed them to engage in narrow-cleft ribbon junctions in blue cone pedicles.

The PKC-β antibody stained cone bipolar cells, many amacrine cells, and most ganglion cells. Cone bipolar cells were stained all the way into the foveal center: both midget and diffuse varieties were included. The IPL was densely PKC-IR and individual neurons could not be identified on stratification patterns. EM of the outer plexiform layer (OPL) revealed that both flat and invaginating cone bipolar types were IR and that IR axon terminals were presynaptic in all strata of the IPL. The occurrence of PKC-β-IR bipolar axons in stratum 2 of the IPL suggests that OFF-center as well as ON-center types were included.

The PKC-γ antibody gave inferior staining compared with results from the other two antibodies; however, two varieties of wide-field monostratified amacrine cell and a large-bodied ganglion cell type were discernible.

PKC in one form or another appears to be a second messenger used in neurotransmission by both rod and cone systems and ON- and OFF-center systems in the human retina.

The role of cortical area V4 in complex shape discriminations was studied by testing the effects of V4 lesions in macaques on the ability to visually discriminate between images of three-dimensional (3D) objects from different viewpoints. Stimuli were presented in pairs in the lower left or lower right visual field quadrants about 4 deg from the fovea, and the monkeys judged on each trial whether the two views were of the same or of different objects. Object similarity was varied to determine a threshold shape difference. V4 lesions caused profound, retinotopic, and apparently permanent disruptions of discrimination, regardless of whether the images represented single or multiple viewpoints. In V4 lesioned portions of the visual field, monkeys could discriminate objects only when they differed much more grossly in shape than was true in control locations. These effects of the lesion were virtually identical for discriminations that had been learned before lesions were placed and for those learned afterwards. As in previous studies, V4 lesions elevated contrast thresholds by approximately a factor of two, but control observations showed that this was not the basis of the disruption of shape discrimination. Manipulation of cues to shape showed that in control locations, monkeys maintained excellent shape discrimination despite a variety of stimulus alterations, whereas in V4 lesioned areas their performance was easily disrupted. This finding suggests that V4 may support visual shape discriminations by facilitating the use of multiple visual cues. However, the fact that single-viewpoint and multiple-viewpoint discriminations were similarly affected indicates that the disruption was not specific to 3D shape discrimination, but may apply to a variety of subtle discriminations.

Retinal horizontal cells display large receptive fields as a result of extensive electrical coupling via gap junctions. There is abundant evidence that these gap junctions are dynamically regulated by changes in the adaptational state of the retina. The neuromodulator dopamine appears to play a major role in regulating gap junctional conductances of horizontal cells. Emerging evidence indicates that nitric oxide (NO) also acts as a neuromodulator in the retina and, more specifically, regulates the coupling between horizontal cells. In the present study, we examined the effects of a nitric oxide, and its secondary messenger cGMP, on electrical and tracer coupling between A-type and between B-type horizontal cells in the rabbit retina. Application of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) significantly reduced the coupling between horizontal cells as evidenced by a decrease in their space constants, annulus-to-small spot response ratios, and the extent of tracer coupling following injection with Neurobiotin. Further, application of SNP eliminated the increase in coupling of horizontal cells normally seen with exposure to dim background illumination. Application of 8-bromo-cGMP produced effects similar to those of the NO donors, consistent with the idea that the uncoupling actions of NO were mediated via a cGMP cascade.

We examined contrast, direction of motion, and concentration dependencies of the effects of GABAergic and cholinergic antagonists, and anticholinesterases on responses to movement of On—Off directionally selective (DS) ganglion cells of the rabbit's retina. The drugs tested were curare and hexamethonium bromide (cholinergic antagonists), physostigmine (anticholinesterase), and picrotoxin (GABAergic antagonist). They all reduced the cells' directional selectivity, while maintaining their preferred-null axis. However, cholinergic antagonists did not block directional selectivity completely even at saturating concentrations. The failure to eliminate directional selectivity was probably not due to an incomplete blockade of cholinergic receptors. In a extension of a Masland and Ames (1976) experiment, saturating concentrations of antagonists blocked the effects of exogenous acetylcholine or nicotine applied during synaptic blockade. Consequently, a noncholinergic pathway may be sufficient to account for at least some directional selectivity. This putative pathway interacts with the cholinergic pathway before spike generation, since physostigmine eliminated directional selectivity at contrasts lower than those saturating responses. This elimination apparently resulted from cholinergic-induced saturation, since reduction of contrast restored directional selectivity. Under picrotoxin, directional selectivity was lost in 33% of the cells regardless of contrast. However, 47% maintained their preferred direction despite saturating concentrations of picrotoxin, and 20% reversed the preferred and null directions. Therefore, models based solely on a GABAergic implementation of Barlow and Levick's asymmetric-inhibition model or solely on a cholinergic implementation of asymmetric-excitation models are not complete models of directional selectivity in the rabbit. We propose an alternate model for this retinal property.

Mammalian retinae express multiple connexins that mediate the metabolic and electrical coupling of various cell types. In retinal neurons, only connexin36, connexin45, connexin50, and connexin57 have been described so far. Here, we present an analysis of a novel retinal connexin, connexin30.2 (Cx30.2), and its regulation in the mouse retina. To analyze the expression of Cx30.2, we used a transgenic mouse line in which the coding region of Cx30.2 was replaced by lacZ reporter DNA. We detected the lacZ signal in the nuclei of neurons located in the inner nuclear layer and the ganglion cell layer (GCL). In this study, we focused on the GCL and characterized the morphology of the Cx30.2-expressing cells. Using immunocytochemistry and intracellular dye injections, we found six different types of Cx30.2-expressing ganglion cells: one type of ON-OFF, three types of OFF, and two types of ON ganglion cells; among the latter was the RGA1 type. We show that RGA1 cells were heterologously coupled to numerous displaced amacrine cells. Our results suggest that these gap junction channels may be heterotypic, involving Cx30.2 and a connexin yet unidentified in the mouse retina. Gap junction coupling can be modulated by protein kinases, a process that plays a major role in retinal adaptation. Therefore, we studied the protein kinase–induced modulation of coupling between RGA1 and displaced amacrine cells. Our data provide evidence that coupling of RGA1 cells to displaced amacrine cells is mediated by Cx30.2 and that the extent of this coupling is modulated by protein kinase C.