Retrieval of glutamate from extracellular sites in the retina involves at least five excitatory amino acid transporters. Immunocytochemical analysis of the cat retina indicates that each of these transporters exhibits a selective distribution which may reflect its specific function. The uptake of glutamate into Müller cells or astrocytes appears to depend upon GLAST and EAAT4, respectively. Staining for EAAT4 was also seen in the pigment epithelium. The remaining transporters are neuronal with GLT-1α localized to a number of cone bipolar, amacrine, and ganglion cells and GLT-1v in cone photoreceptors and several populations of bipolar cells. The EAAC1 transporter was found in horizontal, amacrine, and ganglion cells. Staining for EAAT5 was seen in the axon terminals of both rod and cone photoreceptors as well as in numerous amacrine and ganglion cells. Although some of the glutamate transporter molecules are positioned for presynaptic or postsynaptic uptake at glutamatergic synapses, others with localizations more distant from such contacts may serve in modulatory roles or provide protection against excitoxic or oxidative damage.

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45–50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.

The visual responsiveness and spatial tuning of frontal eye field (FEF) neurons were determined using a delayed memory saccade task. Neurons with visual responses were then tested for direction selectivity using moving random dot patterns centered in the visual receptive field. The preferred axis of motion showed a significant tendency to be aligned with the receptive-field location so as to favor motion toward or away from the center of gaze. Centrifugal (outward) motion was preferred over centripetal motion. Motion-sensitive neurons in FEF thus appear to have a direction bias at the population level. This bias may facilitate the detection or discrimination of expanding optic flow patterns. The direction bias is similar to that seen in visual area MT and in posterior parietal cortex, from which FEF receives afferent projections. The outward motion bias may explain asymmetries in saccades made to moving targets. A representation of optic flow in FEF might be useful for planning eye movements during navigation.

Inside-out patches containing cGMP-gated channels were excised from catfish rod or cone outer segments and held under voltage clamp. The net cGMP-dependent currents elicited by saturating and subsaturating concentrations of cGMP at ±30 mV were measured and the dependence of current upon cGMP concentration was determined. The apparent affinity of the channel for its ligand was estimated by fitting these data with the Hill equation. The concentration of cGMP required to give half the maximum current (K1/2) in rod and cone channels at +30 mV was ~28 μM and ~37 μM, respectively, and was weakly voltage dependent. Thus, cone channels have an intrinsically higher K1/2 than rod channels. For both types of channel, the Hill coefficient was ~2.3. In the presence of calcium-calmodulin, the apparent affinity of the rod channel for cGMP decreased by about twofold, but the apparent affinity of the cone channels was unaffected. These results indicate that the open probability of the cone channel for its ligand cannot be modulated by calmodulin. This represents the first significant departure between rod and cone photoreceptors in mechanisms used by phototransduction and suggests that the β subunit of the cone channel must be different from that of the rod channel.

X- and Y-mediated input to areas 17 and 18 of the cat visual cortex was studied using current-source-density analysis of field potentials evoked by stimulation of the optic nerves. A cuff-shaped electrode was used for stimulation so that Y axons, by virtue of their larger diameters, would have lower electrical thresholds than X axons. The effect in each cortical area of activating Y axons alone could therefore be determined by low-&litude stimulation of the optic nerves. Current-source densities were calculated by two separate methods. (1) In five experiments, field potentials were measured sequentially at different cortical depths with a single tungsten electrode. Current densities were then calculated by computer. (2) In two experiments, current densities were derived in real time from field potentials recorded simultaneously from three sites with a multi-electrode probe. The calculation was performed by an analog circuit specially designed for this purpose. This method has several advantages over the standard, single-electrode method. At stimulus strengths sufficient to activate the majority of Y axons in the optic nerves, but subthreshold to most X axons, the field potentials evoked in area 17 changed little from layer to layer. When the current-source-density analysis was applied to these potentials, no significant sources or sinks were detectable. Only when the stimulus strength was raised to the point that both X and Y axons were activated by the stimulus were any current sources or sinks detected in area 17. The currents were similar in time course and laminar pattern to those recorded after stimulation of the optic chiasm. In area 18, large sources and sinks were evoked by stimulation of Y axons alone. These currents changed little when the stimulus strength was increased to activate X axons as well. Area 18, therefore, in contrast to area 17, seems to be dominated by Y input and receives little X input. These results support the conclusions of the accompanying paper in which synaptic potentials were recorded intracellularly from cortical neutrons. The intracellular experiments failed to show substantial Y input to area 17. The projections of X and Y axons may therefore be much more highly segregated into areas 17 and 18 than previously thought. Alternatively, the nature of the Y input to area 17 may be very different from that to area 18 in that it cannot be easily detected with intracellular or current-source-density techniques.

Lateral inhibition is one of the first and most important stages of visual processing. There are at least four theories related to information theory in the literature for the role of early retinal lateral inhibition. They are based on the spatial redundancy in natural images and the advantage of removing this redundancy from the visual code. Here, we contrast these theories with data from the retina's outer plexiform layer. The horizontal cells' lateral-inhibition extent displays a bell-shape behavior as function of background luminance, whereas all the theories show a fall as luminance increases. It is remarkable that different theories predict the same luminance behavior, explaining “half” of the biological data. We argue that the main reason is how these theories deal with photon-absorption noise. At dim light levels, for which this noise is relatively large, large receptive fields would increase the signal-to-noise ratio through averaging. Unfortunately, such an increase at low luminance levels may smooth out basic visual information of natural images. To explain the biological behavior, we describe an alternate hypothesis, which proposes that the role of early visual lateral inhibition is to deal with noise without missing relevant clues from the visual world, most prominently, the occlusion boundaries between objects.

A sinusoidal mask grating oriented orthogonally to and superimposed onto an optimally oriented base grating reduces a cortical neuron's response amplitude. The spatial selectivity of cross-orientation suppression (XOR) has been described, so for this paper we investigated the temporal properties of XOR. We recorded from single striate cortical neurons (n = 72) in anesthetized and paralyzed cats. After quantifying the spatial and temporal characteristics of each cell's excitatory response to a base grating, we measured the temporal-frequency tuning of XOR by systematically varying the temporal frequency of a mask grating placed at a null orientation outside of the cell's excitatory orientation domain. The average preferred temporal frequency of the excitatory response of the neurons in our sample was 3.8 (± 1.5 S.D.) Hz. The average cutoff frequency for the sample was 16.3 (± 1.7) Hz. The average preferred temporal frequency (7.0 ± 2.6 Hz) and cutoff frequency (20.4 ± 6.9 Hz) of the XOR were significantly higher. The differences averaged 1.1 (± 0.6) octaves for the peaks and 0.3 (± 0.4) octaves for the cutoffs. The XOR mechanism's preference for high temporal frequencies suggests a possible extrastriate origin for the effect and could help explain the low-pass temporal-frequency response profile displayed by most striate cortical neurons.

The diameter of the pupil is affected by changes in ambient illumination, color, spatial structure, movement, and mental effort. It has now been found that pupil diameter can be affected by cognitive processes. That is, it can be entrained by alternations between broadly spread and narrowly focused attention that are cued exogenously (attention is “summoned” by the cue) or endogenously (attention changes under the perceiver’s intentional control). Pupil diameter also is affected by post-eye-blink constrictions that occur most often when attention is narrowed, and possibly by changes evoked by the near reflex, although changes in attention state parsimoniously account for the entirety of the results. Changes in pupil diameter produce differences in spherical aberration that alternately blur (when the pupil dilates) and sharpen the retinal image (when the pupil constricts), affecting the relative sensitivity of large receptive fields that mediate broadly spread attention compared with smaller receptive fields that mediate more narrowly focused attention. Results for endogenously cued, intentional changes in attentional spread provide definitive behavioral evidence for cortical feedback to subcortical nuclei that control pupil diameter, either directly or through pupil-constricting eye blinks. Analyses of convergent and divergent changes in eye position indicate that the near reflex was activated long after the initiation of relatively gradual attentionally cued changes in pupil diameter, and further, that when it occurs, the near reflex facilitates ongoing changes in pupil diameter.

Axon-bearing amacrine cells in mammalian retinae are encountered relatively infrequently during electrophysiological investigations, and thus very little is known about their physiological properties. Patch-clamp electrodes were used to record light responses from two axon-bearing amacrine cells in flat-mounted, dark-adapted rabbit retina. The recorded cells were stained, and the morphology visualized. Both cells were capable of generating action potentials. In one case, a linear relationship between mean depolarization and action-potential frequency was demonstrated. The cells had a proximal dendritic arbor and a morphologically distinct, much larger axon terminal system. The receptive field of the center response was coextensive with the dendritic arbor, and thus also much smaller than the axon terminal system. The center response was suppressed by activation of an inhibitory surround. Both cells responded to center illumination with an inward current which became more transient as the size of the illuminating spot was increased. It is suggested that axon-bearing amacrine cells receive input over a receptive field defined by the dendritic arbor, and distribute their output over a much more extensive axon terminal system, most probably via action potentials.

Voltage-gated potassium channels are differentially expressed in the brain, and recent studies have shown that K+ channels show subcellular localization. We characterized the distribution of five different K+ channels in the mouse retina. Each channel was distributed in a unique pattern in the retina and was localized to specific subcellular domains within a given retinal neuron. Kvl.4 and Kv4.2 were consistently found in axonal and somatodendritic portions, respectively, consistent with previous studies in brain. In contrast, Kvl.2, Kvl.3, and Kv2.1 showed variable subcellular distribution depending upon cellular context. These results suggest that no one K+ channel is distributed over the entire length of the neuron to provide a “housekeeping” level of membrane potential stabilization. Instead, we propose that each K+ channel is associated with a specific subcellular functional module, and each local K+ conductance responds uniquely to local voltage and second messenger signals.

The cortical contribution to the orientation and direction sensitivity of LGNd relay cells was investigated by recording the responses of relay cells to drifting sinusoidal gratings of varying spatial frequencies, moving bars, and moving spots in cats in which the visual cortex (areas 17, 18, 19, and LS) was ablated. For comparison, the spatial-frequency dependence of orientation and direction tuning of striate cortical cells was investigated employing the same quantitative techniques used to test LGNd cells. There are no significant differences in the orientation and direction tuning to relay cells in the LGNd of normal and decorticate cats. The orientation and direction sensitivities of cortical cells are dependent on stimulus parameters in a fashion qualitatively similar to that of LGNd cells. The differences in the spatial-frequency bandwidths of LGNd cells and cortical cells may explain many of their differences in orientation and direction tuning. Although factors beyond narrowness of spatial-frequency tuning must exist to account for the much stronger orientation and direction preferences of cells in area 17 when compared to LGNd cells, the evidence suggests that the orientation and direction biases present in the afferents to the visual cortex may contribute to the orientation and direction selectivities found in cortical cells.

In birds, neurons of the isthmo-optic nucleus (ION), as well as “ectopic” neurons, send axons to the retina, where they synapse on cells in the inner nuclear layer (INL). Previous work has shown that centrifugal axons can be divided into two anatomically distinct types depending on their mode of termination: either “convergent” or “divergent” (Ramon y Cajal, 1889; Maturana & Frenk, 1965). We show that cytochrome-oxidase histochemistry specifically labels “convergent” centrifugal axons and target neurons which appear to be amacrine cells, as well as three “types” of ganglion cells: two types found in the INL (displaced ganglion cells) and one in the ganglion cell layer. Labeled target amacrine cells have distinct darkly labeled “nests” of boutons enveloping the somas, are associated with labeled centrifugal fibers, and are confined to central retina. Lesions of the isthmo-optic tract abolish the cytochrome-oxidase labeling in the centrifugal axons and in the target amacrine cells but not in the ganglion cells. Cytochrome-oxidase-labeled ganglion cells in the INL are large; one type is oval and similar to the classical displaced ganglion cells of Dogiel, which have been reported to receive centrifugal input; the other type is rounder. Rhodamine beads injected into the accessory optic system results in retrograde label in both types of cells, showing that two distinct types of displaced ganglion cells project to the accessory optic system in chickens. The ganglion cells in the ganglion cell layer that label for cytochrome oxidase also project to the accessory optic system. These have proximal dendrites that ramify in the outer inner plexiform layer. Neither the target amacrine cells nor either of the displaced ganglion cells are immunoreactive for the inhibitory transmitter gamma aminobutyric acid. At least some of the target amacrine cells may, however, be cholinoceptive: we found that the antibody to the alpha-7 subunit of the nicotinic ACh receptor labels a population of cells in the INL that are similar in location, size, and the presence of labeled bouton-like structures to those we find labeled with cytochrome oxidase. This antibody also labels neurons in the ION proper but not ectopic cells. In conclusion, it appears that cytochrome oxidase may be a marker for “convergent” centrifugal axons and at least one of their target cells in the INL.

The retina possesses subpopulations of amacrine cells, which utilize different transmitters, including acetylcholine (ACh), GABA, and dopamine. We have examined interactions between these neurones by studying the effects of nicotinic agonists on GABA and dopamine release. Isolated rabbit retinas were incubated with [3H]dopamine and then superfused. Fractions of the superfusate (2 min) were collected and the [3H]dopamine in each sample was measured. Endogenous GABA release was examined by incubating retinas in a small chamber. At 5-min intervals, the medium was changed and the GABA measured by high-pressure liquid chromatography (HPLC). Exposure of the retina to nicotine, epibatidine, and other nicotinic agonists increased the release of both GABA and dopamine. The effects of nicotine and epibatidine were blocked by mecamylamine, confirming an action on nicotinic receptors. The action of epibatidine on dopamine release was unaffected by glutamate antagonists but was blocked by picrotoxin and gabazine. These results suggested that nicotine might increase dopamine release indirectly by stimulating the release of GABA, which in turn inhibited the release of an inhibitory transmitter acting tonically on the dopaminergic amacrines. Exposure of the retina to GABA caused a small increase in dopamine release. This hypothetical inhibitory transmitter was not GABA, an opioid, adenosine, glycine, nociceptin, a cannabinoid, or nitric oxide because appropriate antagonists did not affect the resting release of dopamine. However, metergoline, a 5HT1/5HT2 receptor antagonist, and ketanserin, a 5HT2A receptor antagonist, but not the 5HT1A antagonist WAY100635, increased the resting release of dopamine and blocked the effects of nicotine. The 5HT1A/5HT7 agonist 8-hydroxy DPAT inhibited both the nicotine and GABA-evoked release of dopamine. We conclude that nicotinic agonists directly stimulate the release of GABA, but the evoked release of dopamine is indirect, and arises from GABA inhibiting the input of an inhibitory transmitter, which we tentatively identify as serotonin.

Cannabinoid CB1 receptor (via Gs) and dopamine D2 receptor (via Gi/o) antagonistically modulate goldfish cone membrane currents. As ON bipolar cells have CB1 and D1 receptors, but not D2 receptors, we focused on whether CB1 receptor agonist and dopamine interact to modulate voltage-dependent outward membrane K+ currents IK(V) of the ON mixed rod/cone (Mb) bipolar cells. Whole-cell currents were recorded from Mb bipolar cells in goldfish retinal slices. Mb bipolar cells were identified by intracellular filling with Lucifer yellow. The bath solution was calcium-free and contained 1 mM cobalt to block indirect calcium-dependent effects. Dopamine (10 μM) consistently increased IK(V) by a factor of 1.57 ± 0.12 (S.E.M., n = 15). A CB receptor agonist, WIN 55212-2 (0.25–1 μM), had no effect, but 4 μM WIN 55212-2 suppressed IK(V) by 60%. If IK(V) was first increased by 10 μM dopamine, application of WIN 55212-2 (0.25–1 μM) reversibly blocked the effect of dopamine even though these concentrations of WIN 55212-2 had no effect of their own. If WIN 55212-2 was applied first and dopamine (10 μM) was added to the WIN-containing solution, 0.1 μM WIN 55212-2 blocked the effect of dopamine. All effects of WIN 55212-2 were blocked by coapplication of SR 141716A (CB1 antagonist) and pretreatment with pertussis toxin (blocker of Gi/o) indicating action via CB1 receptor activation of G protein Gi/o. Coactivation of CB1 and D1 receptors on Mb bipolar cells produces reciprocal effects on IK(V). The CB1-evoked suppression of IK(V) is mediated by G protein Gi/o, whereas the D1-evoked enhancement is mediated by G protein Gs. As dopamine is a retinal “light” signal, these data support our notion that endocannabinoids function as a “dark” signal, interacting with dopamine to set retinal sensitivity.

Single isolated photoreceptors can be taken through a visual cycle of light adaptation by bleaching visual pigment, followed by dark adaptation when supplied with 11–cis retinal. Light adaptation after bleaching is manifested by faster response kinetics and a permanent reduction in sensitivity to light flashes, presumed to be due to the presence of bleached visual pigment. The recovery of flash sensitivity during dark adaptation is assumed to be due to regeneration of visual pigment to pre-bleach levels. In previous work, the outer segments of bleached, light-adapted cells were exposed to 11–cis retinal. In the present work, the cell bodies of bleached photoreceptors were exposed. We report a marked difference between rods and cones. Bleached cones recover sensitivity when their cell bodies are exposed to 11–cis retinal. Bleached rods do not. These results imply that retinal can move freely along the cone photoreceptor, but retinal either is not taken up by the rod cell body or retinal cannot move from the rod cell body to the rod outer segment. The free transfer of retinal along cone but not along rod photoreceptors could explain why, during dark adaptation in the retina, cones have access to a store of 11–cis retinal which is not available to rods. Additional experiments investigated the movement of retinal along bleached rod outer segments. The results indicate that retinal can move along the rod outer segment, but that this movement is slow, occurring at about the same rate as the regeneration of visual pigment.

The Syngnathidae are specialized diurnal feeders that are known to possess a retinal fovea and use independent eye movements to locate, track, and strike individual planktonic prey items. In this study, we have investigated the spectral sensitivities of three syngnathid species: a pipefish and two seahorses. We used spectrophotometry to measure the spectral transmission properties of ocular lenses and microspectrophotometry to measure the spectral absorption characteristics of visual pigments in the retinal photoreceptors. The pipefish, Stigmatopora argus, together with the seahorse Hippocampus subelongatus, is found in “green-water” temperate coastal seagrass habitats, whereas the second seahorse, H. barbouri, originates from a “blue-water” tropical coral reef habitat. All species were found to possess short wavelength absorbing pigment(s) in their lenses, with the 50% cut-off point of S. argus and H. subelongatus at 429 and 425 nm respectively, whereas that of H. barbouri was located at 409 nm. Microspectrophotometry of the photoreceptors revealed that the rods of all three species contained visual pigment with the wavelength of maximum absorption (λmax) at approximately 500 nm. The visual pigment complement of the cones varied between the species: all possessed single cones with a λmax close to 460 nm but H. barbouri also possessed an additional class of single cone with λmax at 430 nm. Three classes of visual pigment were found in the double cones, the λmax being approximately 520, 537, and 560 nm in the two seahorses and 520, 537, and 580 nm in the pipefish. The spectral sensitivities of the syngnathids investigated here do not appear to conform to generally accepted trends for fishes inhabiting different spectral environments. The influence of the specialized feeding regime of the syngnathids is discussed in relation to our findings that ultra-violet sensitivity is apparently not necessary for zooplanktivory in certain habitats.

The integrative properties of starburst amacrine cells in the rabbit retina were studied with compartmental models and computer-simulation techniques. The anatomical basis for these simulations was provided by computer reconstructions of intracellularly stained starburst amacrine cells and published data on dendritic diameter and biophysical properties. Passive and active membrane properties were included to simulate spiking and nonspiking behavior. Simulated synaptic inputs into one or more compartments consisted of a bipolar-like conductance change with peak and steady-state components provided by the sum of two Gaussian responses. Simulated impulse generation was achieved by using a model of impulse generation that included five nonlinear channels (INa, ICa, Ia,. Ik. Ik.Ca). The magnitude of the sodium channel conductance change was altered to meet several different types of impulse generation and propagation behaviors. We studied a range of model constraints which included variations in membrane resistance (Rm) from 4,000 Ω.cm2 to 100,000 Ω.cm2, and dendritic diameter from 0.1 to 0.3 μm. In a separate series of simulations, we studied the feasibility of voltage-clamping starburst amacrine cells using a soma-applied, single-electrode voltage clamp, based on models with and without dendritic and somatic spiking behavior. Our simulation studies suggest that single dendrites of starburst amacrine cells can behave as independent functional subunits when the Rm is high, provided that one or a small number of dendrites is synaptically co-activated. However, as the number of co-activated dendrites increases, the starburst cell behavior becomes more uniform and independent dendritic function is less prevalent. The presence of impulse activity in the dendrites raises new questions about dendritic function. However, dendritic impulses do not necessarily eliminate independent dendritic function, because dendritic impulses commonly fail as they propagate toward the soma, where they contribute EPSP-like responses which summate with conventional synaptic currents.

The presence of melatonin in retina has been widely reported for over two decades although studies of its functional importance within the retina have only recently been emphasized. We have analyzed the biochemical characteristics of melatonin synthesis and release, focusing on rapid changes in response to light/dark conditions. Our major findings are consistent with the following conclusions: (1) melatonin synthesis is stimulated within minutes after exposure to darkness, and may reflect an increase in N-acetyl transferase activity; (2) melatonin is not stored, but rather it diffuses freely throughout the retina immediately after it is synthesized; and (3) the dark-induced increase in retinal melatonin release is a synthesis-coupled response and does not involve separate secretion mechanisms. The characteristics of melatonin synthesis and release described herein would be consistent with the proposed role of melatonin as a local paracrine effector of dark-adaptive responses in retina.

An important mechanism for the discrimination of direction of motion in the retina is a spatially asymmetric inhibition. This inhibition has been postulated to operate either as a subtraction, like in difference-of-Gaussians' models, or as a division, like in shunting-inhibition models of directional selectivity. The latter, but not the former, is nonlinear. This raises the question of whether the inhibitory mechanism involved in directional selectivity is nonlinear. To investigate this issue, we studied the linearity of the contrast dependence of the extracellularly recorded responses to apparent motions in ON-OFF directionally selective ganglion cells of the rabbit retina. The results show that the inhibition underlying directional selectivity is nonlinear and fits shunting-inhibition models well. Other biophysical mechanisms that might account for the type of nonlinearity observed in the data are also considered.

We have studied the glial investment of ganglion cells of the cat's retina, orienting the sections taken for electron microscopy so that the investment could be traced from the soma along the axon. The soma of each ganglion cell is covered by a close-fitting, continuous sheath formed by Müller cells. The axon hillock and the first part of the initial segment are invested by an extension of the somal sheath, and are thus enclosed in the same glial compartment as the soma. The initial segment extends a few microns past the Müller cell sheath; this last length of the initial segment is contacted by numerous processes of astrocytes, which converge on it in a pattern found also on nodes of the same axons, in the optic nerve. Beyond the initial segment, the intraretinal lengths of the axons are invested by both Müller cells and astrocytes, but the investment is strikingly incomplete. Large areas of axonal membrane have no glial cover, and lie close to other axonal membranes. The sequential arrangement of these distinct forms of glial wrapping of the soma, initial segment, and axon is described here for the first time. It is suggested that this pattern of glial investment controls the flow of current between dendrite and initial segment of the ganglion cell, defines the site of initiation of action spikes, and controls the formation of synapses on the soma and initial segment.