The superfused retinal slice preparation was used to examine the morphology and glutamate-activated whole-cell currents of rabbit bipolar cells. There were six morphologically distinct types of cone bipolar cells and a rod bipolar cell that had axon terminals stratifying in stratum 3 to 5 of sublamina-b. All of these bipolar cell types exhibited an outward current in response to the application of the metabotropic glutamate receptor, mGluR6, agonist AP-4 (APB), and had I/V curves indicative of membrane channel closure. Conversely, there were no currents activated during the application of kainate, the AMPA/kainate receptor agonist. These data demonstrate they were on-bipolar cells. In addition, there were six morphologically distinct cone bipolar cells that stratified in sublamina-a. Every cell with axonal arborizations in stratum 1 and 2 exhibited an inward current when the ionotropic glutamate receptor agonist kainate was applied. This current was blocked by application of the AMPA/kainate receptor antagonist CNQX. These cells also decreased their membrane resistance in response to kainate, a characteristic of the opening of channels within the plasma membrane. Without exception, no cells stratifying in sublamina-a responded to the mGluR6 agonist AP-4, further identifying them as off-bipolar cells.

We examined whether lateral spread of adaptation can be observed in the electroretinogram in humans. Specifically, we tested whether the luminance level of a surrounding, nonmodulated annulus affects the multifocal electroretinogram (ERG) response of a modulated central area. Multifocal electroretinograms were recorded in response to an array of 37 unscaled hexagons subtending a retinal area of 38 deg × 35 deg. Responses were recorded in six control subjects. In the first series of experiments, only the center hexagon was modulated, while the surrounding 36 hexagons were held constant at either 0.45, 172, or 340 cd/m2. In a subsequent series of control experiments, modulation depth of the center hexagon was varied and the proximity of the surrounding hexagon systematically altered. For the center-modulated condition, response amplitude and implicit time for the first-order kernel response significantly decreased as a function of increasing surround luminance. Control experiments demonstrated that the effect of the surround illumination was not due to scattered light but was influenced by the proximity of the surrounding annulus. These results demonstrate that lateral adaptation influences can be measured using the multifocal ERG.

In the retina of the African clawed frog (Xenopus laevis), melatonin is synthesized by the photoreceptors at night, and binds to receptors that likely mediate paracrine responses. Melatonin appears to alter the sensitivity of the retinal cells to light, and may play a key role in regulating important circadian events that occur in the eye. A polyclonal antibody was raised against a 13 amino acid peptide corresponding to a region of the third cytoplasmic loop of the Xenopus laevis Mel1c melatonin receptor. Western blot analysis revealed a major immunoreactive band of approximately 60 kD in neural retina and retinal pigment epithelium (RPE) membranes. Immunocytochemical labeling of sections of Xenopus eyes demonstrated intense melatonin receptor-like immunoreactivity in the inner plexiform layer (IPL). Immunolabeling with antibodies to glutamate decarboxylase (GAD) or tyrosine hydroxylase (TOH) appeared to co-localize with the melatonin receptor immunoreactivity in different sublaminas of the IPL. This suggests that both GABAergic and dopaminergic amacrine cells express melatonin receptor protein. There were also some melatonin receptor immunoreactive varicose fibers in the IPL that did not co-localize with either TOH or GAD, and may represent efferent fibers, since they could be followed into the optic nerve. Melatonin receptor immunoreactivity was also present on cell soma in the ganglion cell layer. Furthermore, a moderate level of melatonin receptor immunoreactivity was observed in the RPE and rod and cone photoreceptor cells. The presence of melatonin receptor immunoreactivity in these cells supports previous observations of melatonin receptor RNA expression in multiple cell types in the Xenopus retina. Expression of melatonin receptor protein in the photoreceptors suggests that melatonin may have a direct action on these cells.

Glutamate and kainate-induced currents of primate ganglion cells were studied using the whole-cell patch-clamp technique in a retinal slice preparation. Antagonists and allosteric modulators of desensitization selective for either α-amino-3-hydroxy-5-methyl-4-isoazoleprionic acid (AMPA)- or kainate-preferring receptors were used to determine the contributions of each type of receptor to excitatory responses. With synaptic transmission and NMDA receptors blocked, the AMPA-preferring receptor antagonist GYKI 52466 (30 μM–100 μM) reversibly blocked most of the glutamate-induced current in ganglion cells. GYKI 52466 also blocked the response in ganglion cells to focally applied kainate, suggesting that the current response to kainate arises from activation of AMPA-preferring receptors, and not kainate-preferring receptors. Both cyclothiazide (10 μM–100 μM) and the novel drug 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA, 20 μM–100 μM), which selectively enhance responses mediated by AMPA-preferring receptors, enhanced glutamate-induced responses of ganglion cells. Since these drugs preferentially inhibit desensitization of the flip and flop splice variants, respectively, of AMPA-preferring receptors, it is likely that both splice variants are present on these ganglion cells. Concanavalin A, which selectively suppresses the desensitization of kainate-preferring receptors, had no effect on the glutamate-induced responses of ganglion cells. We conclude that the non-NMDA component of the excitatory, glutamatergic input to primate ganglion cells is mediated largely by AMPA-preferring receptors, with little, if any, kainate-preferring receptor-mediated response.

An investigation of retinal specializations was carried out in larval and juvenile dhufish, Glaucosoma hebraicum (Glaucosomidae, Teleostei). The development of photoreceptors and formation of the retinal mosaic was followed by light and electron microscopy. At hatching the eye was undifferentiated. Cone photoreceptors were present by day 3 posthatch (dph), when exogenous feeding began. Single and multiple cones were present in a row arrangement from 3 dph to 20 dph, when the first rod nuclei were observed. Between 20 dph and approximately 3 months posthatch (mph), the row arrangement was replaced by a square mosaic of four double cones surrounding a single cone, and the cones increased in size, with the outer segments reaching up to 30 μm in length. During the period of spatial rearrangement, triple cones were often observed. From their first appearance, rod photoreceptors were added rapidly. Investigation of ganglion cell topography in 3-mph fish that had attained the adult-like square photoreceptor mosaic was carried out using retinal wholemounts. The highest densities of neurones in the ganglion cell layer were in temporal retina but no well-defined area centralis was observed. Microspectrophotometric measurements of the visual pigments within the outer segments of the photoreceptors of 3-mph fish revealed double cones with identical absorption spectra in each member of the outer segment, and the wavelength of maximum absorption (λmax) located at 522 nm. Single cones were found to possess a visual pigment with λmax at 460 nm and rods with a λmax of 498 nm. The results imply that the larvae and juveniles are adapted for survival in coastal waters and may be active in relatively low light levels from early stages of development.

Anatomical findings are presented that identify cortical and subcortical sources of afferents to the nucleus reticularis tegmenti pontis (NRTP) and basal pontine nuclei. Projections from the middle temporal visual area (MT), medial superior temporal visual area (MST), lateral intraparietal area (LIP), and areas 7a and 7b to the basal pontine nuclei were studied using 3H-leucine autoradiography. The results complemented a parallel study of retrograde neuronal labeling attributable to injecting WGA-HRP into NRTP and neighboring pontine nuclei. Small 3H-leucine injections confined to MT, MST, LIP, area 7a, or area 7b, produced multiple patches of pontine terminal label distributed as follows: (1) An injection within MT produced terminal label limited to the dorsolateral and lateral pontine nuclei. (2) Injections restricted to MST or LIP showed patches of terminal label in the dorsal, dorsolateral, lateral, and peduncular pontine nuclei. (3) Area 7a targets the dorsal, dorsolateral, lateral, peduncular, and ventral pontine nuclei, whereas area 7b projects, additionally, to the dorsomedial and paramedian pontine nuclei. Notably, no projections were seen to NRTP from any of these cortical areas. In contrast, injections made by other investigators into cortical areas anterior to the central sulcus revealed cerebrocortical afferents to NRTP, in addition to nuclei of the basal pontine gray. With our pontine WGA-HRP injections, retrograde neuronal labeling was observed over a large extent of the frontal cortex continuing onto the medial surface which included the lining of the cingulate sulcus and cingulate gyrus. Significant subcortical sources for afferents to the NRTP and basal pontine nuclei were the zona incerta, ventral mesencephalic tegmentum, dorsomedial hypothalamic area, rostral interstitial nucleus of the medial longitudinal fasciculus, red nucleus, and subthalamic nucleus. The combined anterograde and retrograde labeling data indicated that visuo-motor cortico-pontine pathways arising from parietal cortices target only the basal pontine gray, whereas the NRTP, together with select pontine nuclei, is a recipient of afferents from frontal cortical areas. The present findings implicate the existence of parallel direct and indirect cortico-pontine pathways from frontal motor-related cortices to NRTP and neighboring pontine nuclei.

Photoreceptors in the ferret's retina have been shown to project transiently to the inner plexiform layer (IPL) prior to their differentiation of an outer segment. On postnatal day 15 (P-15), when this projection achieves maximal density, the photoreceptors projecting into the IPL extend primarily to one of two depths, coincident with the processes of cholinergic amacrine cells. The present study has used an excitotoxic approach employing subcutaneous injections of l-glutamate to ablate these cholinergic amacrine cells on P-7, in order to see whether their elimination alters this targeting of photoreceptor terminals within the IPL. The near-complete elimination of cholinergic amacrine cells at P-15 was confirmed, although the population of retinal ganglion cells was also affected, being depleted by roughly 50%. The rod opsin-immunopositive terminals in such treated ferrets no longer showed a stratified distribution, being found throughout the depth of the IPL, as well as extending into the ganglion cell layer. This effect should not be due to the partial loss of retinal ganglion cells, however, since optic nerve transection at P-2, which eliminates the ganglion cells entirely while leaving the cholinergic amacrine cell population intact, was shown not to affect the stratification pattern of the photoreceptors within the IPL. These results strongly suggest that the targeting of the photoreceptor terminals to discrete strata within the IPL is dependent upon the cholinergic amacrine cell processes.

Absorbance spectra of cone outer segments and oil droplets were recorded microspectrophotometrically in the retina of the red-eared turtle, Trachemys scripta elegans. There are four cone visual pigments, with λmax = 617 nm (red sensitive), 515 nm (green sensitive), 458 nm (blue sensitive), and 372 nm (UV-sensitive). The red-sensitive pigment resides in single cones with red or orange oil droplets, and in both members of double cones. The principal member of the double cone contains an orange oil droplet, and the accessory member is droplet free. The green-sensitive pigment is situated in single cones with orange/dark yellow droplets. The blue-sensitive pigment is combined with the UV-absorbing oil droplet in single cones. The UV-sensitive pigment resides in single cones with clear oil droplets that exhibited virtually no absorbance down to 325 nm. Thus, seven types of cones can be identified based on their morphology, oil droplet color, and the visual pigment absorbance. At the moment, this is the most complex cone system described for vertebrates.

Recent physiological experiments support behavioral and morphological evidence for a fourth type of cone in the turtle retina, maximally sensitive in the ultraviolet (UV). This cone type has not yet been included in the models proposed for connectivity between cones and horizontal cells. In this study, we examined the inputs of UV, S, M, and L cones to horizontal cells. We used the high-resolution Dynamic Constant Response Method to measure the spectral sensitivity of horizontal cells without background light and after adaptation to UV, blue (B), green (G), and red (R) light. We concluded the following: (1) Tetrachromatic input to a Y/B horizontal cell was identified. The spectral-sensitivity curves of the cell in three of the adaptation conditions were well represented by L-, M-, and S-cone functions. Adaptation to blue light revealed a peak at 372 nm, the same wavelength location as that determined behaviorally in the turtle. A porphyropsin template could be closely fitted to the sensitivity band in that region, strong evidence for input from a UV cone. (2) The spectral-sensitivity functions of R/G horizontal cells were well represented by the L- and M-cone functions. There was no indication of UV- or S-cone inputs into these cells. (3) The spectral sensitivities of the monophasic horizontal cells were dominated by the L cone. However, the shape of the spectral-sensitivity function depended on the background wavelength, indicating secondary M-cone input. Connectivity models of the outer retina that predict input from all cone types are supported by the finding of tetrachromatic input into Y/B horizontal cells. In contrast, we did not find tetrachromatic input to R/G and monophasic horizontal cells. Chromatic adaptation revealed the spectral-sensitivity function of the turtle UV cone peaking at 372 nm.

Most molecular studies on the visual system in fish have been performed on freshwater teleosts such as goldfish and zebrafish where cones and rods appear simultaneously. Many marine fishes have long larval phase in the upper pelagic zone before transformation into a juvenile and a benthic life style. The retina at the larval stages consists of only single cone cells; later during metamorphosis double cones and rods develop. The flatfish Atlantic halibut (Hippoglossus hippoglossus) is a typical example of a marine species with such a two-step retina development. In this study, we have cloned five different opsins from Atlantic halibut larvae and juvenile retinas. Sequence comparisons with other opsins and phylogenetic analysis show that the five genes belong to the opsins of long-wavelength sensitive (L); middle-wavelength sensitive, MCone and MRod; and short-wavelength sensitive, SBlue and SUltraviolet, respectively. In situ hybridization analysis reveals expression in double cone (L and MCone), single cone (SBlue and SUltraviolet), and rod (MRod) types of photoreceptor cells in juvenile halibut retina. The visual system in Atlantic halibut seems therefore to have all four types of cone photoreceptors in addition to rod photoreceptors. This work shows for the first time molecular isolation of a complete set of retinal visual pigment genes from a marine teleost and describes the first cloning of an ultraviolet-sensitive opsin type from a marine teleost.

To determine how a progressive loss of photoreceptor cells and the concomitant loss of glutamatergic input to second-order neurons can affect inner-retinal signaling, glutamate receptor expression was analyzed in the Royal College of Surgeons (RCS) rat, an animal model of retinitis pigmentosa. Immunohistochemistry was performed on retinal sections of RCS rats and congenic controls between postnatal (P) day 3 and the aged adult (up to P350) using specific antibodies against N-methyl-D-aspartate (NMDA) subunits. All NMDA subunits (NR1, NR2A–2D) were expressed in control and dystrophic retinas at all ages, and distinct patterns of labeling were found in horizontal cells, subpopulations of amacrine cells and ganglion cells, as well as in the outer and inner plexiform layer (IPL). NR1 immunoreactivity in the inner plexiform layer of adult control retinas was concentrated in two distinct bands, indicating a synaptic localization of NMDA receptors in the OFF and ON signal pathways. In the RCS retina, these bands of NR1 immunoreactivity in the IPL were much weaker in animals older than P40. In parallel, NR2B immunoreactivity in the outer plexiform layer (OPL) of RCS rats was always reduced compared to controls and vanished between P40 and P120. The most striking alteration observed in the degenerating retina, however, was a strong expression of NR1 immunoreactivity in Müller cell processes in the inner retina which was not observed in control animals and which was present prior to any visible sign of photoreceptor degeneration. The results suggest functional changes in glutamatergic receptor signaling in the dystrophic retina and a possible involvement of Müller cells in early processes of this disease.

Previous work suggests that photoreceptor synaptic ribbon length and absolute dark-adapted threshold may vary during a 24-h diurnal cycle. To test this hypothesis, we examined the length of photoreceptor synaptic ribbons and the dark-adapted threshold in black (+/+) and albino (c2J/c2J) C57BL/6J mice at six times over a 24-h period. Testing began 2 h after light onset (ZT 2:00) and continued at successive 4-h intervals (12 h:12 h light:dark). We determined the length of the synaptic ribbons in frozen sections by labeling them with an antibody specific for synaptic ribbons. Synaptic ribbons vary in length at different points in the diurnal cycle in both types of mice, but the synaptic ribbons in black mice are longer than those in albino mice by an average of 0.33 μm. The synaptic ribbons of black mice also have a larger response to changes in the light cycle. Ribbon length in black mice ranges from 1.66 μm to 1.4 μm, whereas ribbon length in albino mice ranges from 1.32 μm to 1.25 μm. The shortest ribbons are evident 6 h after light onset in both types of mice, whereas the longest ribbons appear within 2 h after light onset. These changes in synaptic ribbon length support the idea that photoreceptor synaptic ribbons are dynamic structures whose length changes over a 24-h diurnal cycle. Examining black and albino mice with a water-maze behavioral assay showed that visual thresholds in black and albino mice vary over the 24-h diurnal cycle. The visual thresholds of albino mice are elevated compared with black mice at all times tested. This is consistent with previous findings of visual thresholds in hypopigmented mice. The lowest threshold (greatest sensitivity) is present 2 h after light onset (ZT 2:00) and corresponds to the time when synaptic ribbons are the longest. The highest threshold is observed 6 h after light onset, the time when synaptic ribbons are shortest. These results show that synaptic ribbon length and visual sensitivity vary together in relation to the time.

The voltage-gated calcium and sodium currents of starburst amacrine cells were examined in slices of the adult rabbit retina. ON-center starburst amacrine cells were targeted for whole-cell recording by prelabeling the retina with the nuclear dye 4′-6-diamidino-2-phenylindole hydrochloride (DAPI). Calcium currents were isolated using an external Ringer that contained tetrodotoxin to block sodium currents and barium to block potassium channels. When starburst amacrine cells were stepped to holding potentials positive to −50 mV, a series of voltage-dependent calcium currents were activated. The calcium current peaked at −10 mV. The calcium currents kinetics were mainly sustained in nature, showing only a small amount of slow inactivation. Nickel (100 μM), a T-type channel blocker, had no effect on the calcium current. Application of the L-type channel agonist BAY K8644 (1–2.5 μM) had small variable effects on the calcium current while the L-type channel antagonist nifedipine (10 μM) had no effect. However, addition of a reported N-type calcium channel antagonist, omega-conotoxin G6A (1 μM), blocked a large portion of the calcium current, as did a more nonselective antagonist, omega-conotoxin M7C (200 nM). Agatoxin 4A (500 nM) reduced a smaller sustained calcium current component, implying a P/Q-type calcium channel was present on these neurons. In addition to the calcium currents, a fast voltage-gated sodium current was observed in many starburst cells. This current could be blocked by tetrodotoxin (200–500 nM). The differing kinetics and durations of the sodium and calcium currents could play important roles in the regulation of synaptic release and in the coordination of spiking by starburst amacrine cell dendrites during retinal development and in the encoding of motion across the retinal surface.

We compared the effect of 2 days of monocular vision on the ocular dominance of cells in the visual cortex of light-reared kittens with the effect in dark-reared kittens at 6, 9, and 14 weeks of age, and analyzed the results by layer. The size of the ocular-dominance shift declined with age in all layers in light-reared animals. There was not a large change in the ocular-dominance shift with age in dark-reared animals in any layer, suggesting that dark rearing largely keeps the cortex in the immature 6-week state until 14 weeks or longer, although there was a slight decrease in layers II, III, and IV, and a slight increase in layers V and VI. At 14 weeks, the difference between light- and dark-reared animals was smallest in layer IV, larger in layers II/III, and largest in layers V/VI, suggesting that dark rearing has a large effect on intracortical synapses and a small effect on geniculocortical synapses. There was a significant ocular-dominance shift in layer IV at 14 weeks of age in both light- animals and dark-reared animals, showing that the critical period for ocular-dominance plasticity is not ended at this age. While the ocular-dominance shift after 26 h of monocular deprivation in 6-week animals was similar in light- and dark-reared animals, after 14 h it was smaller in dark-reared animals, showing that ocular-dominance changes occur more slowly in dark-reared animals at this age, in agreement with Mower (1991). Increases in selectivity for axis of movement after 26 h of monocular vision were seen in dark-reared animals at 6 weeks of age, but not at 9 or 14 weeks of age, showing that the critical period for axial selectivity ends earlier than the critical period for ocular dominance in dark-reared animals, as it does in light-reared animals.

Mature visual cortex shows a single, binocularly matched orientation map. This matching develops without visual experience. It persists despite early monocular deprivation that largely eliminates one eye's map, followed by reverse suture (deprivation of the previously open eye and opening of the previously deprived eye), even though the two eyes lack common visual experience in this case. These results have been interpreted to suggest that the structure of orientation maps either is innately predetermined or, if it arises through self-organization, is determined by external cues such as boundary conditions or a “scaffolding” of horizontal connections. We show, to the contrary, that these results are the expected outcomes if orientation maps develop through activity-instructed, correlation-based development of the geniculocortical connections without additional cues. A weak, binocularly correlated orientation map is known to exist before deprivation onset; we previously showed how this can arise through activity-instructed development. Now we show that this initial correlation between the two eyes' maps can persist or increase despite deprivation sufficient to cause massive loss of the deprived eye's geniculocortical synaptic strength, followed by reverse suture. Given sufficient early correlated map development, each map's fate is “dynamically committed”: the two eyes' maps will converge upon a common outcome, even if developing independently. This dynamic fate commitment is retained even after severe deprivation.

Lateral voltage spread in electrically coupled retinal horizontal cell networks is the substrate of center-surround antagonism in bipolar and ganglion cells. We studied its spatial and temporal properties in more detail in turtle L1 horizontal cells by using a contrast border as light stimulus. Experimental data were contrasted with expectations from a linear continuum model to specify the impact of nonlinearities. The assumptions for the diffusion term of the continuum model were justified by neurobiotin labeling. Measured voltage spread revealed two different length constants Λ+ and Λ0, under illuminated and nonilluminated regions of the retina, respectively, as predicted by the linear model. Length constants in the illuminated region showed strong temporal dynamics. For the initial phase of the horizontal cell responses Λ+ was larger than Λo. This was also in accordance with the model. Right at the peak of the response, however, Λ+ dropped below Λo and did not change any more. It is this temporal reversal of asymmetry in voltage spread and not the decrease of Λ+ itself that is lacked by the linear model. The observed independence of the mean ratio Λ+/Λo from light intensity in both the peak and the plateau phases of horizontal cell responses contradicts the linear assumption, too. These two effects have to be addressed to local nonlinearities in the horizontal cell network like a negative feedback loop from photoreceptors and/or voltage-dependent conductances. Due to the failure of the linear model, firm conclusions about the membrane resistance and the coupling resistance of the horizontal cell network cannot be drawn from length constant measurements.