We have used monolayer cultures prepared from early postnatal rabbit retinae (days 2–5) by the sandwich technique to study the capacity of immature neurons to express specific neuronal phenotypes in a homogeneous in vitro environment. Applying morphological, immunocytochemical, and autoradiographic criteria, we demonstrate that a variety of phenotypes could be distinguished after 7–14 days in vitro, and correlated with known retinal cell types. Bipolar cell-like neurons (approximately 4% of total cell number) were identified by cell type-specific monoclonal antibodies (115A10) and their characteristic bipolar morphology. Small subpopulations (about 1%) of GABA-immunoreactive neurons acquired elaborate morphologies strikingly similar to those of A- and B-type horizontal cells. Amongst putative amacrine cells several different subpopulations could be classified. GABA-immunoreactive amacrine-like neurons (6.5%), which also showed high affinity [3H]-GABA uptake, comprised cells of varying size and shape and could be subdivided into subpopulations with respect to their response to different glutamate receptor agonists (NMDA, kainic acid, quisqualic acid). In addition, a small percentage of [3H]-GABA accumulating cells with large dendritic fields showed tyrosine-hydroxylase immunoreactivity. Presumptive glycinergic amacrine cells (18.5%) were rather uniform in shape and had small dendritic fields. Release of [3H]-glycine from these neurons was evoked by kainic and quisqualic acid but not by NMDA. Small [3H]-glutamate accumulating neurons with few short processes were the most frequent cell type (73%). This cell type also exhibited opsin immunoreactivity and probably represented incompletely differentiated photoreceptor cells. Summing the numbers of characterized cells indicated that we were able to attribute a defined retinal phenotype to most, if not all of the cultured neurons. Thus, we have demonstrated that immature neuronal cells growing in monolayer cultures, in the absence of a structured environment, are capable of maintaining or producing specific morphological and functional properties corresponding to those expressed in vivo. These results stress the importance of intrinsic factors for the regulation of neuronal differentiation. On the other hand, morphological differentiation was far from perfect indicating the requirement for regulatory factors.

This study examined the consequences of visual system lesions on visual aftereffects produced by achromatic stimuli of various luminance contrasts and chromatic stimuli of various wavelength compositions. The effects of repeated exposure to such adapting stimuli were assessed using probes whose luminance contrast and wavelength composition were systematically varied using both detection and discrimination paradigms. Interocular tests revealed that both peripheral and central mechanisms contribute to the visual aftereffects produced by the adapting stimulus arrays used in this study. Contrary to the hypothesis according to which the midget system of the retina is the conveyor of visual afterimages, we found that blocking this system with lesions of parvocellular lateral geniculate nucleus, through which the midget cells make their way to the striate cortex in primates, did not eliminate the visual aftereffects. It appears therefore that the parasol system of the retina, which courses through the magnocellular layers of the lateral geniculate nucleus to cortex, can convey the necessary signals for the generation of visual aftereffects. Lesions of areas V4 and MT did not have significant effects on the visual aftereffects studied suggesting that the central factors that contribute to the visual aftereffects occur either already in area VI or are conveyed to higher centers through regions other than areas V4 and MT.

It has been assumed that the ‘ON’ and ‘OFF’ responses of the human electroretinogram (ERG) interact to produce a single waveform when brief flashes are used. To test this assumption, we examined the separate effects of stimulus intensity, the level of retinal illuminance and stimulus duration on the ERG. Both ‘ON’ and ‘OFF’ response amplitude decreased as stimulus intensity was decreased and as the level of retinal illumination was reduced. When stimulus duration was reduced, the amplitude of the ‘OFF’ response decreased; however, the amplitude of the ‘ON’ response increased. Summing of the ‘ON’ and ‘OFF’ response waveforms could not account for the increased amplitude in response to brief stimuli or for the changes in ERG wave shape. These results indicate that there is not a significant corneally recordable ‘OFF’ response elicited by brief stimuli, such as those commonly used to record the flash ERG.

A postembedding immunogold procedure was used on thin sections of the dorsal lateral geniculate nucleus (LGN) and perigeniculate nucleus (PGN) of the cat to estimate qualitatively and quantitatively, at the electron-microscopic (EM) level, the intensity of glutamate or aspartate immunoreactivities on identifiable synaptic terminals and other profiles of the neuropil. On sections incubated with a glutamate antibody, terminals of retinal and cortical axons in the LGN, and of collaterals of geniculo-cortical axons in the PGN, contain significantly higher density of immunogold particles than GABAergic terminals, glial cells, dendrites, and cytoplasm of geniculate cells. By contrast, in sections incubated with an aspartate antibody, terminals of retino-geniculate, cortico-geniculate, and geniculo-cortical axons did not show a selective enrichment of immunoreactivity, but instead the density of immunogold particles was generally low in the different profiles of the neuropil, with the exception of nucleoli. These results suggest that glutamate, but not aspartate, is a neurotransmitter candidate in the retino-geniculo-cortical pathways.

The effect of unilateral depletion of retinal dopamine on goldfish visual behavior was studied using a behavioral reflex, the dorsal light reaction (DLR). Retinal dopamine was depleted by intraocular injections of 6–hydroxydopamine (6–OHDA) on two successive days. By 2 weeks postinjection, dopamine interplexiform cells (DA-IPC) were not detected using tyrosine-hydroxylase immunoreactivity (TH-IR). By 6 weeks postinjection, generation of DA-IPC was observed at the marginal zone and by 9 months postinjection, 2–3 rows of DA-IPC were present at the marginal zone. Neurites extended several hundred micrometers toward the central retina. By 2 weeks postinjection, all 6–OHDA lesioned fish tilted 7–15 deg toward the injected eye under uniform overhead illumination. The tilting did not occur under scotopic illumination and reappeared within 1 min of light adaptation. Quantitation of the DLR showed that 6–OHDA lesioned fish behaved as if light were 2.4 log units more intense to the injected eye. Partial recovery was observed by 9 months postinjection, paralleling the reappearance of DA-IPC at the marginal zone. Tilting also was induced by unilateral intraocular injection with Dl and D2 dopamine receptor antagonists, SCH 23390 and S(—)-sulpiride, respectively. Fish did not tilt if they were light adapted at the time of injection. Tilting was observed if the animals were dark-adapted for 3 h and left in the dark for 1 h postinjection. Fish tilted toward the drug-injected eye within 2 min of light adaptation and gradually returned to vertical within 2 h. The tilting response to S(—)-sulpiride was stronger (˜20 deg vs. ˜5 deg) and occurred at lower concentration (1 μM vs. 10 μM) compared to SCH 23390. We conclude that dopamine depletion mimics the dorsal light reaction by increasing the luminosity output of the eye and that dopamine is directly involved in photopic luminosity function.

Increment threshold functions of the electroretinogram (ERG) b–wave were obtained from goldfish using an in vivo preparation to study intraretinal mechanisms underlying the increase in perceived brightness induced by depletion of retinal dopamine by 6–hydroxydopamine (6–OHDA). Goldfish received unilateral intraocular injections of 6–OHDA plus pargyline on successive days. Depletion of retinal dopamine was confirmed by the absence of tyrosine-hydroxylase immunoreactivity at 2 to 3 weeks postinjection as compared to sham-injected eyes from the same fish. There was no difference among normal, sham-injected or 6–OHDA-injected eyes with regard to ERG waveform, intensity-response functions or increment threshold functions. Dopamine-depleted eyes showed a Purkinje shift, that is, a transition from rod-to-cone dominated vision with increasing levels of adaptation. We conclude (1) dopamine-depleted eyes are capable of photopic vision; and (2) the ERG b–wave is not diagnostic for luminosity coding at photopic backgrounds. We also predict that (1) dopamine is not required for the transition from scotopic to photopic vision in goldfish; (2) the ERG b–wave in goldfish is influenced by chromatic interactions; (3) horizontal cell spinules, though correlated with photopic mechanisms in the fish retina, are not necessary for the transition from scotopic to photopic vision; and (4) the OFF pathway, not the ON pathway, is involved in the action of dopamine on luminosity coding in the retina.

The two-dimensional organization of receptive fields (RFs) of 44 cells in the cat visual cortex and four cells from the cat LGN was measured by stimulation with either dots or bars of light. The light bars were presented in different positions and orientations centered on the RFs. The RFs found were arbitrarily divided into four general types: Punctate, resembling DOG filters (11%); those resembling Gabor filters (9%); elongate (36%); and multipeaked-type (44%). Elongate RFs, usually found in simple cells, could show more than one excitatory band or bifurcation of excitatory regions. Although regions inhibitory to a given stimulus transition (e.g. ON) often coincided with regions excitatory to the opposite transition (e.g. OFF), this was by no means the rule. Measurements were highly repeatable and stable over periods of at least 1 h. A comparison between measurements made with dots and with bars showed reasonable matches in about 40% of the cases. In general, bar-based measurements revealed larger RFs with more structure, especially with respect to inhibitory regions. Inactivation of lower cortical layers (V-VI) by local GABA injection was found to reduce sharpness of detail and to increase both receptive-field size and noise in upper layer cells, suggesting vertically organized RF mechanisms. Across the population, some cells bore close resemblance to theoretically proposed filters, while others had a complexity that was clearly not generalizable, to the extent that they seemed more suited to detection of specific structures. We would speculate that the broadly varying forms of cat cortical receptive fields result from developmental processes akin to those that form ocular-dominance columns, but on a smaller scale.

Immunohistochemistry and in situ hybridization were used to study the distribution of glycine receptor (GlyR) subunits and the GlyR-associated protein gephyrin in the rat retina. Monoclonal antibodies against the α and β subunits of the GlyR and gephyrin showed a strong punctate labeling pattern in the inner plexiform layer. Glycine receptor mRNAs were found in the inner nuclear layer and the ganglion cell layer. The α 1 subunit mRNA is predominantly present in the outer half of the INL and on some but not all ganglion cells. GlyR α2 subunit mRNA is predominantly present in the inner half of the INL and on nearly all cells in the ganglion cell layer. GlyR α3–, GlyR β-, and gephyrin-mRNAs are present in the entire INL and in cells in the ganglion cell layer. The differential expression of glycine receptor subunits indicates a functional diversity of glycine receptors in the retina.

The monoclonal antibody Cat-301 was used to stain neurons and neuropil in the visual thalamus and cortex of tree shrews —small, highly visual mammals that are closely related to primates. Previously, this antibody has been found to label neurons associated with the Y-cell stream of processing in cats and the magnocellular or M-cell stream in macaque monkeys. In tree shrews, the antibody selectively labeled layers 1, 2, 4, and 5 of the dorsal lateral geniculate nucleus, layers that are likely to contain neurons previously classified as Y-cells. Of the two layers that contain W-cells, layer 3 was unlabeled and layer 6 was lightly labeled. In area 17, layer 3c was densely stained, as in cats and macaque monkeys. The external half of layer 5 was also densely stained, in contrast to cats where the internal half of layer 5 is stained and macaques where layer 5 is sparsely stained. Area 18 was characterized by dense, uniform staining of inner layer 3 and outer layer 5, but no pattern of alternating light and dense bands crossed the width of area 18 as in macaques. Dense labeling of these same sublayers occurred in cortical areas TA and TD just lateral to area 18. Area TD may be the homologue of area MT of primates, which also stains densely with Cat-301 in macaques. These results indicate that Cat-301 differentially labels layers and areas in the visual system of tree shrews, and raise intriguing issues of comparison among tree shrews, primates, and cats.

We have studied the effect of retinal illumination on the concentration of the extracellular space marker tetramethylammonium (TMA+) in the dark-adapted cat retina using double-barreled ion-selective microelectrodes. The retina was loaded with TMA+ by a single intravitreal injection. Retinal illumination produced a slow decrease in , which was maximal in amplitude in the most distal portion of the space surrounding photoreceptors, the subretinal space. The light-evoked decrease in was considerably slower and of a different overall time course than the light-evoked decrease in , also recorded in the subretinal space. decreased to a peak at 38 s after the onset of illumination, then slowly recovered towards the baseline, and transiently increased following the offset of illumination. It resembled the light-evoked decreases previously recorded in the in vitro preparations of frog (Huang & Karwoski, 1990, 1992) and chick (Li et al., 1992, 1994) but was considerably larger in amplitude, 22% compared with 7%. As in frog, where it was first recorded, the light-evoked decrease is considered to originate from a light-evoked increase in the volume of the subretinal space (or subretinal hydration). A mathematical model accounting for diffusion predicted that the volume increase underlying the response was 63% on average and could be as large as 95% and last for minutes. The estimated volume increase was then used to examine its effect on K+ concentration in the subretinal space. We conclude that a light-dependent hydration of the subretinal space represents a significant physiological event in the intact cat eye, which should affect the organization of the interphotoreceptor matrix, and the concentrations of all ions and metabolites located in the subretinal space.

We have studied light-evoked changes in extracellular Ca2+ concentration in the intact cat eye using ion-sensitive double-barreled microelectrodes. Two prominent changes in Ca2+ concentration were observed that differed in retinal location. There was a light-evoked increase in accompanied by brief ON and OFF transients, which was maximal in the inner plexiform layer and was not further studied. There was an unexpected sustained light-evoked decrease in of relatively rapid onset and offset, which was maximal in the distalmost region of the subretinal space (SRS). in the SRS was 1.0 mM higher than in the vitreous humor during dark adaptation and this transretinal gradient disappeared during rod-saturating illumination. After correcting for the light-evoked increase in the volume of the SRS, an increase in the total Ca2+ content of the SRS during illumination was revealed, which presumably represents the Ca2+ released by rods. To explain the light-evoked changes, we used the diffusion model described in the accompanying paper (Li et al., 1994b), with the addition of light-dependent sources of Ca2+ at the retina/retinal pigment epithelium (RPE) border and rod outer segments. We conclude that a drop in around photoreceptors, which persists during illumination and reduces a transretinal Ca2+ gradient, is the combined effect of the light-evoked SRS volume increase, Ca2+ release from photoreceptors, and an unidentified mechanism(s), which is presumably Ca2+ transport by the RPE. The relatively rapid onset and offset of the decrease remains unexplained. These steady-state shifts in should have significant effects on photoreceptor function, especially adaptation.

The hypothesis that a large, possibly toxic, increase in cellular calcium accompanies photoreceptor cell degeneration in several different Drosophila mutants was tested. The calcium content of wild type and mutant photoreceptors of Drosophila was measured using rapid freezing of the eyes and energy-dispersive x-ray analysis (e.d.x.) of cryosections and semithin sections of cryosubstituted material. Light- and dark-raised mutants of the following strains were studied: retinal degeneration B (rdgB); retinal degeneration C (rdgC); neither inactivation nor afterpotential C (ninaC), and no receptor potential A (norpA). These are light-dependent retinal degeneration mutants in which the affected gene products had been previously shown as myosin-kinase (ninaC), calcium-dependent phosphoprotein phosphatase (rdgC), phosphoinositide transfer protein (rdgB), and phospholipase C (norpA). In light-raised mutants, ommatidia of variable degrees of degeneration were observed. Mass-dense globular bodies of 200–500 nm diameter in relatively large quantities were found in the degenerating photoreceptor of all the mutants tested. These subcellular globules were found to have a very high calcium content, which was not found in wild type or in nondegenerating photoreceptors of the mutants. Nondegenerating photoreceptors were found not only in dark-raised mutants, but in smaller quantities also in light-raised mutants. Usually these globular structures contained high levels of phosphorus, indicating that at least part of the calcium in the mutant photoreceptors is precipitated as calcium phosphate. The results indicate that a large increase in cellular calcium accompanies light-induced photoreceptor degeneration in degenerating Drosophila mutants even when induced by very different mutations, suggesting that the calcium accumulation is a secondary rather than a primary effect in the degeneration process.

A 6.4–kb sequence upstream of the mouse blue visual pigment gene has been assayed in transgenic mice for the ability to direct cell-type-specific expression of a linked β-galactosidase (lacZ) reporter. The construct is expressed specifically in cone photoreceptors in three independent lines. Transgene expression is found in the developing retina on the first postnatal day, increases rapidly in subsequent days, and persists through adulthood. A gradient of transgene expression is observed across the retina, with the transgene-expressing cones found almost exclusively in the lower retina and rarely in the upper retina, a pattern that parallels the distribution of blue cones in the mouse retina. Double-labeling with anti-cone pigment antibodies shows that transgene expression is confined to blue cones. These results imply that all of the sequence elements necessary for the control of blue cone-specific expression are encoded within the 6.4–kb DNA fragment tested.

Transmission through the lateral geniculate nucleus is facilitated following activation of the cholinergic input from the brain stem, which is thought to reflect activity patterns seen during arousal. One of the underlying mechanisms is the suppression of inhibitory circuits local to the lateral geniculate nucleus. However, evidence exists that some visually driven inhibitory inputs to geniculate relay cells may be preserved or even enhanced under conditions of arousal, and during electrical activation of the parabrachial region of the brain stem. We have therefore reexamined the effect of brain-stem activation on the visual responses of one group of local inhibitory inputs to geniculate relay cells, those emanating from the adjacent perigeniculate nucleus. We recorded single perigeniculate cells in anesthetized, paralyzed cats. Axons innervating the lateral geniculate and perigeniculate nuclei from the parabrachial region of the brain stem were electrically activated, and the effect of this activation was assessed on both spontaneous and visually evoked responses. Visual stimulation consisted of sinusoidally modulated sine–wave gratings of varying spatial and temporal frequency. For the great majority of perigeniculate cells (32 of 40), brain-stem activation inhibited spontaneous activity, while one cell was excited, three showed a mixed effect and four were unaffected. Nevertheless, the responses of most cells (30 of 40) were facilitated when brain-stem activation was paired with certain spatio-temporal patterns of visual stimulation. Spatial tuning curves were constructed for 17 cells and temporal tuning curves for 14, before and during parabrachial activation. The responses of any one cell could be facilitated, unchanged, or suppressed, depending on the visual stimulus used. In some cases, this substantially modified the cell’s spatial and temporal tuning properties. We conclude that activation of the brain stem disinhibits geniculate relay cells in the absence of visual stimulation, but it has the potential to enhance either the magnitude or specificity of visually driven inhibition arising from the perigeniculate nucleus.

Postembedding immunocytochemistry was used to determine the retinal distribution of the amino acid glutamine, and characterize amino acid signatures in the avian retinal ganglion cell layer. Glutamine is a potential precursor of glutamate and some glutamatergic neurons may use this amino acid to sustain production of glutamate for neurotransmission. Ganglion cells, cells in the inner nuclear layer, and some photoreceptors exhibited glutamine immunoreactivity of varying intensity. Ganglion cells demonstrated the highest level of immunoreactivity which indicates either slow glutamine turnover or active maintenance of a large standing glutamine pool relative to other glutamatergic neurons. Müller's cells in the avian retina are involved in glutamate uptake and carbon recycling by the rapid conversion of glutamate to glutamine, thus explaining the low glutamate and high glutamine immunoreactivity found throughout Müller's cells. Most chicken retinal ganglion cells are glutamate (E) and glutamine (Q) immunoreactive but display diverse signatures with presumed functional subsets of cells displaying admixtures of E and Q with GABA (7) and/or glycine (G). The four major ganglion cell signatures are (1) EQ; (2) EQγ; (3) EQG; and (4) EQγG.

White noise stimuli were used to estimate second-order kernels for complex cells in cortical area VI of the macaque monkey, and drifting grating stimuli were presented to the same sample of neurons to obtain orientation and spatial-frequency tuning curves. Using these data, we quantified how well second-order kernels predict the normalized tuning of the average response of complex cells to drifting gratings.

The estimated second-order kernel of each complex cell was transformed into an interaction function defined over all spatial and temporal lags without regard to absolute position or delay. The Fourier transform of each interaction function was then computed to obtain an interaction spectrum. For a cell that is well modeled by a second-order system, the cell’s interaction spectrum is proportional to the tuning of its average spike rate to drifting gratings. This result was used to obtain spatial-frequency and orientation tuning predictions for each cell based on its second-order kernel. From the spatial-frequency and orientation tuning curves, we computed peaks and bandwidths, and an index for directional selectivity.

We found that the predictions derived from second-order kernels provide an accurate description of the change in the average spike rate of complex cells to single drifting sine–wave gratings. These findings are consistent with a model for complex cells that has a quadratic spectral energy operator at its core but are inconsistent with a spectral amplitude model.

Previous research suggests binocular rivalry disrupts extrastriate, but not striate processes, although the locus along the visual pathway at which such disruption first occurs is uncertain. It has been argued that subjective contours arise via a two-stage process in which end-stopped cells feed into orientation-sensitive neurones in V2, and that orientation aftereffects induced with subjective contours are the product of mechanisms similar to those giving rise to real contour aftereffects. If binocular rivalry disrupts the acquisition of subjective contour aftereffects, then it follows from this model that rivalry disrupts processing in V2. Experiments reported here confirm this and provide evidence which suggests binocular rivalry arises through interactions between binocular neurones, rather than via some type of specialized binocular rivalry mechanism.

The rhabdomeral microvilli in the eyes of invertebrates have a cytoskeletal core (Saibil, 1982; Blest et al., 1982a, b, 1983) of actin (de Couet et al., 1984; Arikawa et al., 1990; Williams, 1991; Caiman & Chamberlain, 1992) which might be involved in phototransduction. Tsukita et al. (1988) suggested that light stimulation triggers the breakdown of the microvillar actin filament complex and that this may play a role in phototransduction. To test their suggestion, we pressure-injected phalloidin into Limulus ventral photoreceptors to prevent actin depolymerization (Cooper, J.A., 1987) and tested to see if this blocked phototransduction. We have previously shown that injected rho-damine-phalloidin brightly labels the microvillar actin filaments in living Limulus ventral photoreceptors for several hours (Feng et al., 1994). In the experiments reported here, phalloidin unconjugated with fluorophore was used, and the final concentration in the cell after many small injections from a pipette containing 10 mg/ml phalloidin was estimated to be 1 mg/ml (1.2 mM). This concentration is 100 times higher than that used in an extracted cellular preparation to stabilize actin (Biegel & Pachter, 1992). Fig. 1 shows that the responses to steps of light at different intensities are normal 1 h after phalloidin injection. Similar results were obtained in two other cells.

Ocular following responses induced by brief movements of the visual scene were examined in monkeys for their dependence on disparity in the peripheral field. A random dot pattern was projected onto a tangent screen and partitioned into central and peripheral regions. Test stimuli were velocity steps applied in the central region, while stimuli in the periphery were stationary. The visual images in the central region were seen always in the plane of the screen, while stimuli in the periphery could be seen in front, behind, or in the plane of the screen (achieved by a system of polarizing filters). Initial ocular following responses were larger when the peripheral stimuli were presented with an uncrossed disparity than without disparity. On the other hand, responses were smaller when the peripheral stimuli were presented with crossed disparity (<5.0 deg) than without disparity. The result is consistent with the idea that ocular following responses are dependent on the perceived viewing distance.

On page 397 of Visual Neuroscience 11(2), the paragraph at the top of the right column erroneously has a line of copy repeated and one deleted. The paragraph below has the correct copy, which actually ran from the bottom of the left column to the top of the right column on page 397.