Whole-cell recordings were obtained from retinal ganglion cells of the tiger salamander (Ambystoma tigrinum) in a superfused slice preparation to evaluate contributions of NMDA (N-methyl-D-aspartate) and KA/AMPA (kainate/α-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid) receptors to excitatory postsynaptic potentials (EPSPs) of retinal ganglion cells. Synaptic activation of retinal ganglion cells was achieved through the use of a brief pressure pulse of hyperosmotic Ringer (Ringer + sucrose) delivered through a microelectrode visually placed in the inner plexiform layer while whole-cell recordings were obtained from adjacent cells in the ganglion cell layer. Separation of NMDA and KA/AMPA excitatory postsynaptic currents (EPSCs) was achieved through the application of the antagonists NBQX and D-AP7, while inhibitory currents were blocked by strychnine and picrotoxin. Simple addition of the two independent EPSCs showed, most often, that the sum of the KA/AMPA and NMDA currents was less than the control response, but in some cases the sum of the two currents exceeded the magnitude of the control response. Neither result was consistent with expectations based on voltage-clamp principles and the assumption that the two currents were independent; for this reason, we considered the possibility of nonlinear interactions between KA/AMPA and NMDA receptors. Computer simulations were carried out to evaluate the summation experiments. We used both an equivalent cylinder model and a more realistic, compartmental model of a ganglion cell constrained by a passive leakage conductance, a linear KA/AMPA synaptic current, and a nonlinear NMDA current based on the well-known, voltage-sensitive Mg2+ block. Computer simulation studies suggest that the hypo- and hyper-summation of NMDA and KA/AMPA currents, observed physiologically, can be accounted for by a failure to adequately space clamp the neuron. Clamp failure leads to enhanced NMDA currents as the ion channels are relieved of the Mg2+ block; their contribution is thus exaggerated depending on the magnitude of the conductance change and the spatial location of the synaptic input.

In this study, the possible role of the retinal enkephalin system in form-deprivation myopia (FDM) in the chick eye was investigated. Daily intravitreal injection of the nonspecific opiate antagonist naloxone blocked development of FDM in a dose-dependent manner, while injection of the opiate agonist morphine had no effect at any dose tested. The ED50 for naloxone (calculated maximum concentration in the vitreous) was found to be in the low picomolar range. The results using receptor-subtype-specific drugs were contradictory. Drugs specific for μ and δ receptors had no effect on FDM. The κ-specific antagonist nor-binaltorphimine (nor-BNI) reduced FDM by about 50% at maximum daily retinal doses ranging between 4 X 10-10 and 4 X 10 -7 M, while the κ-specific agonist U50488 blocked FDM in a dose-dependent manner with an ED50 between 5 X 10-8 and 5 X 10-7 M. Met-enkephalin immunoreactivity (ME-IR) was localized immunocytochemically to a subset of amacrine cells (ENSLI cells) and their neurites in the inner plexiform layer (IPL). As reported previously, ENSLI cells from untreated chick retinas showed a cyclical pattern of immunoreactivity, with increased immunoreactivity in the light compared to the dark. Form-deprivation did not appear to change this pattern. Amounts of preproenkephalin mRNA from normal or form-deprived eyes were approximately the same under all conditions. Daily injection of naloxone, however, did increase ME-IR in the dark. These results suggest that naloxone may affect release of enkephalin from the ENSLI cells. The results as presented are inconclusive with regards to the role of the enkephalin system in FDM. While the κ receptor may participate, there is no conclusive evidence here for a direct effect of opiate receptors. The effect of naloxone on form-deprived eyes may be due to its effect on release of peptides from the ENSLI cells.

There are four types of horizontal cell in the goldfish retina, three cone- and one rod-type. The neurotransmitter of only one type, the H1 (cone) horizontal cell, has been identified as GABA. 3H-adenosine uptake was examined as a possible marker for the other classes of horizontal cell. Isolated goldfish retinae were incubated in 3H-adenosine (10–40 μCi) in HEPES-buffered saline for 30 min, then fixed, embedded in plastic, and processed for light-microscopic autoradiography (ARG). For double-label immuno/ARG studies, l-μm-thick sections were processed for GABA postembed immunocytochemistry, then for ARG. 3H-adenosine uptake was localized to cone photoreceptors, presumed precursor cells in the proximal outer nuclear layer, and to a single, continuous row of horizontal cell bodies in the inner nuclear layer. No uptake was localized to the region of horizontal cell axon terminals. 3H-adenosine uptake did not colocalize with GABA-IR in H1 horizontal cells, but it did colocalize with adenosine deaminase immunoreactivity. It is concluded that 3H-adenosine uptake selectively labels rod horizontal cells in the goldfish retina based on position and staining pattern, which are similar to rod horizontal cells stained by Golgi or HRP injection methods. The use of 3H-adenosine uptake may provide a useful tool to study other properties of rod horizontal cells (i.e. development) as well as provide clues as to the transmitter used by these interneurons.

Effects of ATP on the activity of cGMP-gated channels from carp cone photoreceptors were studied. In 29% of the patches examined (N = 45), ATP (1 mM) enhanced a current evoked by cGMP (20 μM, up to about 100%), in 33%, ATP suppressed it by up to about 90%, and in the remaining 38%, ATP had no effect. ATP showed similar effects on a current evoked by 8-bromoguanosine 3′,5′-cyclic monophosphate (2 μM, enhancing in 42% of the patches, suppressing in 25%, no effect in 33%, N = 12), suggesting that the effects were not through modulation of the phosphodiesterase. Both of the effects, enhancement and suppression, were produced by a change in apparent affinity for cGMP, since (1) the maximum current evoked by cGMP of the saturating concentration (≥1 mM) was not affected, and (2) the A1/2 value decreased by approximately 45% (N = 2) or increased by approximately 25% (N = 2). A lower pH (approximately 6) facilitated the enhancing effect. ATP-γ-S (1 mM) showed a suppressing effect in 80% of the patches and no effect in 20% of the patches (N = 10). However, ATP-γ-S did not show an enhancing effect. Thus, ATP had two opposite effects through different mechanisms on the apparent sensitivity of the channel to cGMP; increasing and decreasing.

Psychophysical research has documented the existence of three processes in light adaptation: a fast subtractive process, a divisive process that is fast at light onset and slower at light offset, and a very slow subtractive process (Hayhoe et al., 1987). In the neural model developed here, the fast subtractive process is identified with horizontal cell feedback onto cones and the divisive process with amacrine cell feedback onto bipolar cells. The very slow subtractive process is identified with the modulatory feedback circuit from amacrines via interplexiform cells to horizontal cells. A nonlinear dynamical model is developed incorporating these aspects of retinal circuitry along with both ON- and OFF-center M and P pathways. This model is shown to account for many aspects of foveal light adaptation, including negative afterimage formation, and to explain a number of the physiological differences between M and P ganglion cells, including their differing contrast-response functions.

Population-based studies of retinal neurons have helped to reveal their natural types in mammals and teleost fishes. In this, the first such study in a frog, labeled ganglion cells of the mesobatrachian Xenopus laevis were examined in flatmounts. Cells with large somata and thick dendrites could be divided into three mosaic-forming types, each with its own characteristic stratification pattern. These are named αa, αab, and αc, following a scheme recently used for teleosts. Cells of the αa mosaic (~0.4% of all ganglion cells) had very large somata and trees, arborizing diffusely within sublamina a (the most sclerad). Their distal dendrites were sparsely branched but achieved consistent coverage by intersecting those of their neighbors. Displaced and orthotopic cells belonged to the same mosaic, as did cells with symmetric and asymmetric trees. Cells of the αab mosaic (~1.2%) had large somata, somewhat smaller trees that appeared bistratified at low magnification, and dendrites that branched extensively. Their distal dendrites arborized throughout sublamina b and the vitread part of a, tessellating with their neighbors. All were orthotopic; most were symmetric. Cells of the αc mosaic (~0.5%) had large somata and very large, sparse, flat, overlapping trees, predominantly in sublamina c. All were orthotopic; some were asymmetric. Nearest-neighbor analyses and spatial correlograms confirmed that each mosaic was regular and independent, and that spacings were reduced in juvenile frogs. Densities, proportions, sizes, and mosaic statistics are tabulated for all three types, which are compared with types defined previously by size and symmetry in Xenopus and potentially homologous mosaic-forming types in teleosts. Our results reveal strong organizational similarities between the large ganglion cells of teleosts and frogs. They also demonstrate the value of introducing mosaic analysis at an early stage to help identify characters that are useful markers for natural types and that distinguish between within-type and between-type variation in neuronal populations.

Mutations in the human rod opsin gene have been shown to segregate with autosomal dominant retinitis pigmentosa (ADRP) and photoreceptor degeneration in transgenic mice. While these degenerations are characterized by the primary degeneration of rods, cones eventually die as well. To determine whether this subsequent cone degeneration is the result of expression of mutant rod opsin in the cones, the retinal cell-type specificity of a 221-bp fragment of the mouse rod opsin promoter was evaluated. Two transgenic mouse lines generated by injecting a fusion gene comprised of a 221-bp fragment of the mouse rod opsin promoter and the simian virus 40 large tumor antigen gene (Tag) were examined. The expression of Tag causes photoreceptor cell degeneration in members of both transgenic lines. However, the two lines differed with respect to the level of Tag expression and the rate and extent of photoreceptor cell degeneration. Immunocytochemical localization of opsin and Tag in surviving photoreceptor cells was determined and the results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Rod- and cone-mediated function was evaluated by electroretinography (ERG). In the higher Tag-expressing transgenic line only one row of nuclei remained in the outer nuclear layer at postnatal day (P) 150. While these nuclei showed no antigenicity for rod opsin or Tag, they did stain with an antibody that reacts with both rod and cone S-antigens (arrestins), indicating that these cells were surviving photoreceptor nuclei. Positive staining with peanut agglutinin, which uniquely decorates matrix domains surrounding cones in the normal retina, confirmed that the surviving photoreceptor nuclei were of cone origin. RT-PCR substantiated the results from immunostaining; amplification product was obtained using blue cone opsin transcripts but not from either Tag or rod opsin transcripts. The second transgenic mouse line exhibited a much slower photoreceptor cell death that was associated with low levels of Tag transgene transcript. At P120, ~50% of photoreceptors remained and an ~45% reduction in the rod ERG a-wave was observed. Cone-mediated ERGs, however, were normal. The results demonstrate the rod-specific expression of Tag as directed by the 221-bp fragment of the mouse rod opsin promoter and suggest that the cone degeneration in ADRP or transgenic mice associated with mutations in the rod opsin gene is a secondary effect of rod degeneration.

The expression of GABA in the human fetal (12–25 weeks of gestation), postnatal (five-month-old), and adult (35-year-old) retinas was investigated by immunohistochemistry. GABA expression was seen as early as 12 weeks in the undifferentiated cells of the inner neuroblast zone; a few optic nerve fiber layer axons were clearly labeled, suggesting that some of the stained cell bodies were prospective ganglion cells, others could be displaced amacrine cells. From 16–17 to 24–25 weeks, intense labeling was found in the amacrine, displaced amacrine, and some ganglion cells. During this time period, horizontal cells (identified by calbindin immunohistochemistry), undergoing migration (periphery) and differentiation (center), expressed GABA prominently. In the postnatal retina, some horizontal cells were moderately labeled, but very weakly in a few cells, in the adult. The Müller cells developed immunoreactivity first weakly at 12 weeks and then moderately from 16–17 weeks onward. The staining was also evident in the postnatal and adult retinas, showing labeled processes of these glial cells. Virtually no axons in the adult optic nerve and nerve fiber layer were stained; the staining was restricted to a few, large ganglion cells and displaced amacrine cells. Some amacrines were also labeled. The possibility that GABA might play a role in horizontal cell differentiation and maturation is highlighted. Other evidences suggest that GABA might play a role in metabolism during retinal development.

We have measured transient visual evoked potentials (VEPs) to low-contrast luminance stimuli favoring responses of magnocellular pathway cells and to low-contrast red-green stimuli favoring parvocellular cells. Stimuli were square-wave alternating, 3-deg homogeneous disks. Low-contrast stimuli modulated in luminance elicited relatively simple responses. For some observers, a negativity was present that saturated at low contrast. This may be the signature of inputs from magnocellular channels to the visual cortex. The slope of the contrast—response curve for low-contrast stimuli was about the same for all subjects. For medium contrasts, these contrast—response curves displayed an abrupt increase of slope. The shallower slope may reflect the responsivity of magnocellular-pathway inputs to the cortex, whereas the steeper slope may be caused by additional parvocellular activation.

Contrast-response curves for the most sensitive waveforms of the isoluminant green—red modulation also showed two branches, although not as clearly as for luminance. This may indicate parvocellular-mediated activity for small chromatic differences, and a combination of parvocellular and magnocellular inputs for larger contrasts. Curves of time-to-peak response as a function of contrast often changed their monotonous behavior near the kink of the corresponding contrast—response curve, thus supporting the notion of a contribution from several mechanisms to the main waveforms.

We have used Sutter's (1987) spatiotemporal m-sequence method to map the receptive fields of neurons in the visual system of the cat. The stimulus consisted of a grid of 16 X 16 square regions, each of which was modulated in time by a pseudorandom binary signal, known as an m-sequence. Several strategies for displaying the m-sequence stimulus are presented. The results of the method are illustrated with two examples. For both geniculate neurons and cortical simple cells, the measurement of first-order response properties with the m-sequence method provided a detailed characterization of classical receptive-field structures. First, we measured a spatiotemporal map of both the center and surround of a Y-cell in the lateral geniculate nucleus (LGN). The time courses of the center responses was biphasic: OFF at short latencies, ON at longer latencies. The surround was also biphasic—ON then OFF—but somewhat slower. Second, we mapped the response properties of an area 17 directional simple cell. The response dynamics of the ON and OFF subregions varied considerably; the time to peak ranged over more than a factor of two. This spatiotemporal inseparability is related to the cell's directional selectivity (Reid et al., 1987, 1991; McLean & Palmer, 1989; McLean et al., 1994). The detail with which the time course of response can be measured at many different positions is one of the strengths of the m-sequence method.

Brain-stem afferents to the n. isthmo-opticus (NIO) and ectopic neurons (EN) of the centrifugal visual system (CVS) were determined in the pigeon using the retrograde transneuronal transport of the fluorescent dye Rhodamine β-isothiocyanate (RITC) after its intraocular injection. In other experiments, either RITC was injected into various periocular tissues (controls) or the retrograde tracer Fluoro-gold (FG) was injected stereotaxically in the NIO. Following intraocular injections, the RITC retrograde labeling of cell bodies was observed contralaterally in the NIO and EN and transneuronally in layers 9/10 of the optic tectum, area ventralis-Tsai, zona peri-NIII, mesencephalic and pontine reticular formation (PRF), n. linearis caudalis-raphe, and bilaterally within a region referred to as zona peri-n.NVI (Zp-n.NVI) immediately underlying the abducens nerve nucleus. None of the above structures were labeled after RITC periocular injections. The FG labeling revealed that the tectal efferent neurons were mainly medium-sized, multipolar cells whose dendrites extended superficially to retino-recipient tectal layers 6 and 5. Quantitative measurements of the distribution of layers 9/10 RITC-labeled neurons indicated the highest densities to be localized within the ventral tectum corresponding to the representation of the dorsal retina and inferior visual field. We suggest that visual and nonvisual brain-stem afferents upon NIO and EN may play a role in the proposed mechanism of the avian CVS in attention, ground-feeding behavior, and modulation of retinal sensitivity.

AMPA and NMDA type glutamate receptors were studied in isolated catfish cone horizontal cells using the whole-cell and outside-out patch-recording techniques. In whole-cell recordings, cyclothiazide (CTZ) enhanced the peak current in response to glutamate (in the presence of NMDA receptor antagonists). In patch recordings, currents evoked by rapid and maintained applications of glutamate desensitized with a time constant of one millisecond. CTZ blocked this rapid desensitization. Recovery from desensitization of the AMPA receptors was rapid, having a time constant of 8.65 ms. In contrast, the whole-cell and patch responses to applications of NMDA were much smaller than the AMPA receptor responses and did not desensitize. The relative contribution of these two receptor subtypes depends critically on the condition of the synapse: if glutamate levels are tonically present, the NMDA receptors contribute significantly to the postsynaptic response. If glutamate levels fall rapidly following the release of a single quantum of glutamate, then AMPA receptor currents will dominate the postsynaptic response.

The objective of this research was to determine the sources and sinks of current underlying the slow PIII component of the electroretinogram. Current source density analysis of the ERG evoked by diffuse light flashes was performed in eyecup and isolated retinas of frog. Blockade of synaptic transmission with aminophosphonobutyric + kynurenic acids simplified the CSD profiles through the retina. In addition to the photoreceptor source/sink pair, there was evidence for a major slow PIII source near the outer limiting membrane, a major sink near the inner limiting membrane, and a small source near the inner plexiform layer. Addition of Ba2+ abolished the slow PIII source/sinks, and it left only the photoreceptor source and sink. The results support the idea that slow PIII originates through K+ spatial buffering by Müller cells. Specifically, the light-evoked decrease in [K+]0 in the subretinal space causes a primary K+ efflux from Müller cells (current source) and a primary K+ influx at the Müller cell endfeet (current sink). A decrease in [K+]0 in the proximal retina, caused by diffusion of K+ to the subretinal space, results in K+ efflux (the current source) at the inner plexiform layer.

Several retinal photoreceptor proteins involved in phototransduction have also been found in the mammalian pineal. This study demonstrates that rat and human pineals express protein kinases that are identical to the corresponding rod photoreceptor rhodopsin kinases. The deduced amino acid sequence of rat and human rhodopsin kinases have 84% sequence similarity to the earlier reported sequence of the bovine retinal enzyme, with complete conservation of the topological regions containing the position of the catalytic domain and sites of posttranslational modifications. Rat pineal also expresses rod opsin and putative blue cone opsin. Using immunocytochemistry, rod opsin and rhodopsin kinase were found to be co-localized in pinealocytes in the human tissue. These data demonstrate that the mammalian pineal contains light-sensitive opsins and a kinase involved in their inactivation. These findings correlate with an earlier report that neonatal rats show extraretinal light sensitivity, and suggest that a functional photoreceptive system may be present in the adult mammalian pineal.

The organization of the primate nerve fiber layer and optic nerve head with respect to the positioning of central and peripheral axons remains controversial. Data were obtained from 32 human fetal retinae aged between 15 and 21 weeks of gestation. Crystals of the carbocyanine dyes, DiI or DiA, and fluorescence microscopy were used to identify axonal populations from peripheral retinal ganglion cells. Peripheral ganglion cell axons were scattered throughout the vitreal-scleral depth of the nerve fiber layer. Such a scattered distribution was maintained as the fibers passed through the optic nerve head and along the optic nerve. There was a rough topographic representation within the optic nerve head according to retinal quadrant such that both peripheral and central fibers were mixed within a wedge extending from the periphery to the center of the nerve. There was no indication that the fibers were reorganized in any way as they passed through the optic disc and into the nerve. The present results suggest that any degree of order present within the fiber layer and optic nerve is not an active process but a passive consequence of combining the fascicles of the retinal nerve fiber layer. Optic axons are not instructed to establish a retinotopic order and the effect of guidance cues in reordering fibers, particularly evident prechiasmatically and postchiasmatically, does not appear to be present within the nerve fiber layer or optic nerve head in humans.

Visual function was evaluated in transgenic mice expressing the simian virus 40 early region under the control of the promoter for phenylethanolamine-N-methyltransferase. These transgenic mice undergo a degeneration of the retinal horizontal cells and the outer plexiform layer. Electroretinograms (ERGs) were recorded under stimulus conditions chosen to elicit both receptoral and postreceptoral responses. The dark-adapted a-waves obtained from transgenic mice were not different from control recordings, indicating that the degenerative process does not interfere with function of the rod photoreceptors. In comparison, the ERG b-wave was markedly reduced in transgenic mice under both dark- and light-adapted conditions. Reproducible visual evoked potentials (VEPs) were recorded from transgenic mice in response to both low luminance stimuli that isolate rod function, and to higher luminance stimuli, indicating that retinal activity is transmitted centrally to the visual cortex. However, VEPs were delayed at all stimulus luminances compared to controls. Analysis of luminance-response functions suggests that the VEP delays could reflect the combination of a decrease in synaptic efficacy and an overall loss in visual sensitivity. These functional abnormalities correlate well with the anatomical abnormalities that have been previously observed in the transgenic retina (Hammang et al., 1993), namely a reduced number of synapses between photoreceptors and second-order neurons.

Previous lesion studies of color-reversal learning in pigeons show that an impairment results when (1) the tectofugal visual pathway is damaged at either the thalamic level (nucleus rotundus) or the telencephalic level (ectostriatum), or (2) the thalamofugal visual pathway is damaged at the telencephalic level (the visual Wulst). An impairment does not result, however, when the thalamic source of thalamofugal input (n. opticus principalis thalami or OPT) to the visual Wulst is damaged. These results suggest that the visual Wulst plays a role in color-reversal learning as a consequence of visual information routed from the tectofugal pathway via other visual areas in the telencephalon. One such area is the hyperstriatum ventrale (HV). In the present study, after ablation of the medial and lateral regions of HV, pigeons were trained postoperatively to discriminate between two colors presented simultaneously. After reaching criterion, the pigeons were required to perform a series of discrimination reversals in which the positive and negative stimuli were interchanged. Lesions of medial HV resulted in impaired performance of a color-discrimination task (i.e. original learning), but did not affect discrimination reversal. An impairment in color-reversal learning resulted from combined damage to lateral HV and the fronto-thalamic tract (FT), which carries ascending visual input from OPT to the visual Wulst. No deficits were observed when either lateral HV or FT were damaged alone. These findings suggest that both the thalamofugal and tectofugal pathways provide the visual Wulst with visual input relevant to color-reversal learning.

The retinal pigment epithelium (RPE) of the quokka wallaby, Setonix brachyurus, grows and changes throughout life. To investigate factors that determine changes in the quokka RPE, we have examined topography of this tissue in experimentally enlarged eyes. Unilateral eyelid suture was conducted at the time of normal eye opening, postnatal day (P) 110, and animals were examined at 1 or 1½ years of age. The numbers and densities of RPE cells and the extent of multinucleation were compared with those in normal animals. Eyelid suture resulted in a 9.8% and 17.4% increase in retinal area at 1 and 1½ years, respectively; a significant degree of myopia was associated with this enlargement. Cell density topography in experimental eyes was not the same as in controls. Cells from central retina were disproportionately larger in the experimental than control eyes. However, the RPE cell topography in sutured eyes was not the same as that of aged retinae of a similar size. Notably, in sutured eyes there was no development of the high or highest cell densities seen in equatorial and temporal central RPE in aged retinae, respectively. Furthermore, the degree of cell enlargement in peripheral regions was slight compared with that observed in similar-sized, aged retinae. There was no increase in RPE cell number; rather, average cell area increased accompanied by no change or a slight decrease in RPE thickness. Consequently, overall volume of cells did not change significantly. The large number of multinucleate cells normally seen in aged animals was not observed in experimentally enlarged eyes, implying that an increase in cell volume may be the trigger for multinucleation.

Architectonic subdivisions of the inferior pulvinar (PI) complex were delineated in New World owl and squirrel monkeys and Old World macaque monkeys. Brain sections were processed for Nissl substance, myelin, cytochrome oxidase (CO), acetylcholinesterase (AChE), calbindin-D28K (Cb), or with the monoclonal antibody Cat-301. In all three primates, we identified the posterior nucleus (PIp) and the medial nucleus (PIm) of previous reports, and divided the previously recognized central nucleus (PIc) into two subdivisions, medial (PIcm) and lateral (PIcl). Each nucleus had several features that allowed it to be readily distinguished. (1) PIp was dark in Cb, and moderately dark in AChE and CO preparations. (2) PIm was Cb light, and AChE and CO dark. (3) PIcm was Cb dark, and AChE and CO light. (4) PIcl was Cb moderate with a scattering of dark neurons, and moderately dark for AChE and CO. (5) In sections processed for Cat-301, PIm in macaque monkeys and PIcm and PIp in squirrel monkeys stained darkly, while little staining was apparent in owl monkeys. The results allowed subdivisions of the inferior pulvinar to be more clearly defined, homologized, and compared across taxa. All monkeys appear to have the same four subdivisions of the PI, although properties vary.