Anatomical and physiological approaches are beginning to reveal the synaptic origins of parallel ON- and OFF-pathway retinal circuits for the transmission of short (S-) wavelength sensitive cone signals in the primate retina. Anatomical data suggest that synaptic output from S-cones is largely segregated; central elements of synaptic triads arise almost exclusively from the “blue-cone” bipolar cell, a presumed ON bipolar, whereas triad-associated contacts derive primarily from the “flat” midget bipolar cell, a hyperpolarizing, OFF bipolar. Similarly, horizontal cell connectivity is also segregated, with only the H2 cell-type receiving numerous contacts from S-cones. Negative feedback from long (L-) and middle (M-) wavelength sensitive cones via the H2 horizontal cells elicits an antagonistic surround in S-cones demonstrating that S versus L + M or “blue-yellow” opponency is first established in the S-cone. However, the S-cone output utilizes distinct synaptic mechanisms to create color opponency at the ganglion cell level. The blue-cone bipolar cell is presynaptic to the small bistratified, “blue-ON” ganglion cell. S versus L + M cone opponency arises postsynaptically by converging S-ON and LM-OFF excitatory bipolar inputs to the ganglion cell’s bistratified dendritic tree. The common L + M cone surrounds of the parallel S-ON and LM-OFF cone bipolar inputs appear to cancel resulting in “blue-yellow” antagonism without center-surround spatial opponency. By contrast, in midget ganglion cells, opponency arises by the differential weighting of cone inputs to the receptive field center versus surround. In the macula, the “private-line” connection from a midget ganglion cell to a single cone predicts that S versus L + M opponency is transmitted from the S-cone to the S-OFF midget bipolar and ganglion cell. Beyond the macula, OFF-midget ganglion cell dendritic trees enlarge and collect additional input from multiple L and M cones. Thus S-OFF opponency via the midget pathway would be expected to become more complex in the near retinal periphery as L and/or M and S cone inputs sum to the receptive field center. An important goal for further investigation will be to explore the hypothesis that distinct bistratified S-ON versus midget S-OFF retinal circuits are the substrates for human psychophysical detection mechanisms attributed to S-ON versus S-OFF perceptual channels.

Using both NADPH diaphorase and anti-nNOS antibodies, we have identified—from retinal flatmounts—neuronal types in the inner retina of the chicken that are likely to be nitrergic. The two methods gave similar results and yielded a total of 15 types of neurons, comprising 9 amacrine cells, 5 ganglion cells, and 1 centrifugal midbrain neuron. Six of these 15 cell types are ubiquitously distributed, comprising 3 amacrine cells, 2 displaced ganglion cells, and a presumed orthotopic ganglion cell. The remaining nine cell types are regionally restricted within the retina. As previously reported, efferent fibers of midbrain neurons and their postsynaptic partners, the unusual axon-bearing target amacrine cells, are entirely confined to the ventral retina. Also confined to the ventral retina, though with somewhat different distributions, are the “bullwhip” amacrine cells thought to be involved in eye growth, an orthotopic ganglion cell, and two types of large axon-bearing amacrine cells whose dendrites and axons lie in stratum 1 of the inner plexiform layer (IPL). Intracellular fills of these two cell types showed that only a minority of otherwise morphologically indistinguishable neurons are nitrergic. Two amacrine cells that branch throughout the IPL are confined to an equatorial band, and one small-field orthotopic ganglion cell that branches in the proximal IPL is entirely dorsal. These findings suggest that the retina uses different processing on different regions of the visual image, though the benefit of this is presently obscure.

Retinal prosthetic devices are being developed to bypass degenerated retinal photoreceptors by directly activating retinal neurons with electrical stimulation. However, the retinal circuitry that is activated by epiretinal stimulation is not well characterized. Whole-cell patch clamp recordings were obtained from ganglion cells in normal and rd mice using flat-mount and retinal slice preparations. A stimulating electrode was positioned along the ganglion cell side of the preparation at different distances from the stimulated tissue. Pulses of cathodic current evoked action potentials in ganglion cells and less frequently evoked sustained inward currents that appeared synaptic in origin. Sustained currents reversed around ECl and were inhibited by blockade of α-amino-3-hydroxyl-5-methyl-4-isoxazole-proprionate (AMPA)-type glutamate receptors with 2,3-dihydroxy-6-nitro-sulfamoyl-benzo(f)-quinoxaline-2,3-dione (NBQX), γ aminobutyric acid a/c (GABAa/c) receptors with picrotoxinin, or glycine receptors with strychnine. This suggests that epiretinal stimulation activates glutamate release from bipolar cell terminals, which in turn evokes release of GABA and glycine from amacrine cells. Synaptic current thresholds were lower in ON ganglion cells than OFF cells, but the modest difference did not attain statistical significance. Synaptic currents were rarely observed in rd mice lacking photoreceptors compared to normal retina. In addition, confocal calcium imaging experiments in normal mice retina slices revealed that epiretinal stimulation evoked calcium increases in the outer plexiform layer. These results imply a contribution from photoreceptor inputs to the synaptic currents observed in ganglion cells. The paucity of synaptic responses in rd mice retina slices suggests that it is better to target retinal ganglion cells directly rather than to attempt to engage the inner retinal circuitry.

When a bar of light (215 x 5000 μm) illuminates the receptive field of an ON-β ganglion cell of cat retina, the cell depolarizes. Intracellular recording from the cat eyecup preparation shows that this depolarization is due to an increase in conductance (2.4 ± 0.6 nS). Different phases of this depolarization have different reversal potentials, but all of these reversal potentials are more positive than the cell’s resting potential in the dark. When the light is turned on, there is an initial transient depolarization; the reversal potential measured for this transient is positive (23 ± 11 mV). As the light is left on, the cell partially repolarizes to a sustained depolarization; the reversal potential measured for this sustained depolarization is close to zero (−1 ± 5 mV). When the light is turned off, the cell repolarizes further; the reversal potential measured for this repolarization is negative (−18 ± 7 mV), but still above the resting potential in the dark (−50 mV). To explain this variety of reversal potentials, at least two different synaptic conductances are required: one to ions which have a positive reversal potential and another to ions which have a negative reversal potential. Comparing the responses to broad and narrow bars suggests that these two conductances are associated with the center and surround, respectively. Finally, since an ON-β cell in the area centralis receives about 200 synapses, these results indicate that a single synapse produces an average conductance increase of about 15 pS during a near-maximal depolarization.

Computer simulations were carried out to evaluate the influence of varying the membrane resistance (Rm) on the dendritic integration capacity of three classes of ganglion cells in the mudpuppy (Necturus maculosus) retina. Three broadly different morphological classes of ganglion cells were selected for this study and represent the range of dendritic tree sizes found in the ganglion cell population of this species. Simulations were conducted on anatomical data obtained from cells stained with horseradish peroxidase; each cell was traced, using a computer as an entry device and later converted to a compartmental (electrical) representation of the cell. Computer-simulation analysis used a time-variant conductance change which was similar in waveform to light-activated bipolar cell input. The simulated membrane resistance for each cell varied between 5000 and 100,000 Ω cm2, and conductance changes were introduced into different regions of the soma-dendritic tree to evaluate dendritic integration efficiency. When higher values of Rm are used, even the largest cells become electrotonically compact and attenuation of voltage responses is minimized from distal to soma regions. Responses were less attenuated from proximal to distal regions of the cell because of the favorable impedance matching, and because less current is required to polarize small “sealed” dendritic terminations. Steady-state responses integrate more effectively than transient responses, particularly when Rm is high, since transient responses were more attenuated by the membrane capacitance. The possibility that Rm is a dynamic property of retinal ganglion cells is discussed in view of the functional organization of dendritic integration efficiency as Rm fluctuates from low to high values.

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

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

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

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

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

Ganglion cell activity in response to drifting or alternating stimulus patterns was recorded in an eyecup preparation of the turtle. The stimuli were sinusoidal gratings with various spatial frequencies and spatial phases. Spike histograms of the activity were Fourier-analyzed. The results indicated that the retinal circuitry always functioned in a nonlinear manner. The average, first, and second harmonics of the activity were all a function of stimulus conditions, including spatial phase. Similar outcomes were obtained with full-field (retina-wide) and a small-field (1 mm diameter) stimulation. In the middle of the spatial-frequency range, the ratio of second to first harmonic activity was higher in response to static, contrast-reversing gratings than for drifting, constant-contrast ones. The findings suggest that, as distinct from the usual X, Y, and W segregation of ganglion cell properties in other retinas, there is a single class of cells whose responses seem to have attributes of one or another of the usual categories depending on stimulus characteristics. This may be the result of a degree of stimulus-dependent functional modification in the retinal input circuitry to the turtle ganglion cells.

Synaptic contacts onto fibers and somata in the nerve fiber layer (NFL) and ganglion cell layer (GCL) of macaque and human retina were demonstrated at the electron microscopical (EM) level. Many presynaptic processes in monkey NFL are gamma aminobutyric acid (GABA) immunoreactive, using anti-GABA antiserum with an EM immunogold procedure. Immunocytochemistry at the light microscopic level revealed that many GABA-reactive cells in the GCL send branching processes into the NFL, forming a sparse synaptic plexus. The presence of long, unbranched GABA-reactive fibers running horizontally in the NFL and entering the optic nerve suggests that some ganglion cells may be GABAergic. GABA-reactive cells contributing to the plexus appear to be a new class of displaced amacrines that arborize in the NFL.

The effects of vasoactive intestinal peptide (VIP) on the extracellularly recorded activity of ganglion cells were studied in superfused rabbit retinas. VIP, applied to the bathing solution, significantly increased the maintained activity of both OFF-center and ON-center brisk ganglion cells. The transient, excitatory responses of these cells to flashes of light (spots or annuli centered over the receptive field) were either unaffected or moderately reduced. VIP did not affect the responses of most ON/OFF directionally selective ganglion cells to a moving light stimulus. Furthermore, maintained activity of most of these cells remained absent.

Effects of VIP on the light responses of ganglion cells were pronounced in retinas that were bathed with the dopamine D1 antagonist (+)-SCH 23390. For OFF-center brisk ganglion cells, VIP brought out both the center and surround excitatory responses that were selectively reduced by (+)-SCH 23390. Similarly, VIP brought out the leading edge responses that were reduced by (+)-SCH 23390 in ON/OFF directionally selective ganglion cells to a moving light stimulus. VIP did not however reverse the effects of (+)-SCH 23390 on ON-center brisk ganglion cells. It is argued that VIP counteracts the effects of (+)-SCH 23390 on OFF-center brisk ganglion cells and ON/OFF directionally selective ganglion cells by stimulating adenylate cyclase activity in dopamine-receptive cells of the retina.

Extracellular recordings were made from the isolated goldfish retina during superfusion with various serotonergic agonists and antagonists to determine the effects of these drugs on the maintained activity and response properties of the ganglion cells. Superfusion of the retina with serotonin (25–500 μM) increased the maintained activity of OFF-center ganglion cells and decreased the maintained activity of ON-center ganglion cells. In addition, serotonin also attenuated the excitatory responses to annular stimuli, suggesting a decrease in the strength of surround input to the ganglion cells. The effects of serotonin on OFF-center ganglion cells were mimicked by the nonselective 5-HT1, agonist 5-MeOT and the 5-HT1 receptor agonist 8-OH-DPAT, while only 5-MeOT mimicked the action of serotonin on ON-center ganglion cells. The effects of exogenously applied serotonin on the ganglion cells could be blocked by the mixed 5-HT1/5-HT2 receptor antagonist methysergide but not by the 5-HT2 receptor antagonist mianserin or the dopamine receptor antagonist haloperidol.

These results support previous anatomical and biochemical evidence that serotonin functions in a neurotransmitter or neuromodulatory role in the teleost retina and suggest that serotonin may be involved in modulating the maintained activity and surround input to the ganglion cells. The results also indicate that two different types of receptors may mediate the actions of serotonin in the ON and OFF pathways, respectively.

Putative GABAergic mechanisms were studied in perfused cat retina by means of intracellular recording and application of GABA and the GABAA agonists δ-amino valeric acid (dAVA), muscimol, and THIP. In contrast to results reported previously for cold-blooded vertebrates, introduction of 20 mM GABA into the superfusate had no influence upon the response properties of cat retinal horizontal cells (HCs). In common with results reported in cold-blooded vertebrates, introduction of the GABAA agonists dAVA (2–12 mM) and THIP or muscimol (0.2–1 mM) had four consistent reversible influences upon the response properties of cat retinal HCs: (1) they reduced photic-response amplitude, (2) slowed response onset, (3) slowed response offset, and (4) depolarized the dark membrane potential. Both rod and cone signal components were affected. GABAA agonists had similar influences upon both the time course and amplitude of responses recorded from amacrine and ganglion cells. In all cell types examined, the influence upon response kinetics was made particularly apparent with rapidly flickering stimuli. Flicker responses were reduced in amplitude much more than sustained responses. These results suggest that, in addition to other influences, GABAergic action serves to modify the time course of photic responses in both the inner and outer plexiform layer of mammalian retina making responses slower and less phasic.

The cellular connectivity of the lacertilian parietal eye is not well understood. Because the intercellular connections establish the foundation for information processing, we have investigated cellular connectivity of one cell type in this simple vertebrate retina. We also developed an in vitro preparation to study the anatomy of the parietal eye visual system. Horseradish peroxidase transport in the in vitro preparation revealed a class of displaced ganglion cells occupying positions among the photoreceptors, in a location where the presence of interneurons had been suggested. Three-dimensional reconstruction at the electron-microscopic level showed that the morphology and synaptic input of these displaced ganglion cells is different from that of the previously known ganglion cells. The displaced ganglion cells receive an average of about 13 ribbon synapses from photoreceptors. The ribbon input is equally distributed between the soma and dendritic arbor. Junctional membrane measurement and ethnolic phosphotungstic acid-staining provided evidence for the existence of non-ribbon synaptic contacts (synaptoid junctions). Displaced ganglion cells make about 20 synaptoid junctions, 65% of which are on the dendritic arbor. The morphology of the displaced ganglion cell is such that a significant measure of synaptic input to the dendritic arbor will be transmitted to the soma.

The distribution of ganglion cells in the retina of the silver lamprey, Ichthyomyzon unicuspis, was revealed by retrograde labeling from the optic nerve with horseradish peroxidase (HRP) and fluorescent-labeled dextrans in live animals and with the fluorescent dye DiI in aldehyde-fixed tissue. The majority of ganglion cells (74%) termed the “outer ganglion cells,” are multipolar and are located at the vitread boundary of the inner nuclear layer. The remaining ganglion cells (26%), termed the “inner ganglion cells” are bipolar and are distributed in a sublamina within the inner plexiform layer. The dense, dendritic meshwork of the outer ganglion cells is largely restricted to the sclerad half of the inner plexiform layer with some cells possessing dendrites which pass through the inner nuclear layer to terminate within the outer plexiform layer. The dendrites of the inner ganglion cells form a thin, dendritic network apposing the inner limiting membrane. Axons from both populations of ganglion cells originate from dendrites or the soma and form fascicles lying adjacent to the outer ganglion cell somata.

Retinopetal fibers, originating from bilaterally distributed neurons of the tegmental midbrain, were thin and varicose and ran parallel to the ganglion cell axons to terminate either with a varicose enlargement or a few short sidebranches in the sclerad third of the inner plexiform layer. The unusual organization of the lamprey retina and outgroup comparison with hagfish suggests that agnathans share a presumably primitive type of retinal ganglion cell organization compared to that of gnathostomes.

We have examined the retinotopy in the superior colliculus of pigmented and albino ferrets using both anatomical and electrophysiological methods. While the distribution of contralaterally projecting retinotectal ganglion cells is characterized by the presence of an area centralis superimposed on a visual streak in both strains, the ipsilateral projection from temporal hemiretina is strongly reduced in albinos. In spite of the significantly altered retinotectal projection pattern, the collicular visual field map in the albino ferret reveals the same characteristics as in the pigmented animal with a strongly enlarged representation of the center of visual space. An areal comparison between retinotectal ganglion cell distribution and collicular areal magnification shows that the increase in areal magnification factor between the periphery and the representation of the central visual hemifield exceeds the corresponding increase in retinal ganglion cell density between peripheral retina and area centralis by a factor of three in pigmented and a factor of four in albino ferrets. The areal magnification factor of the representation of the retinal visual streak does not exceed the increase in retinotectal ganglion cell density. Thus, our results suggest that the representation of visual space in the superior colliculus of albino and pigmented ferrets does not simply follow the retinotectal ganglion cell density, but that there is an enhanced representation of the frontal central visual field. The possibility is discussed that the collicular visual field map may be determined either by both retinotectal and corticotectal projections or by the colliculus' intrinsic structure.

Neurons likely to utilize glycine (GLY) as a neurotransmitter were identified immunocytochemically in the “all-cone” lizard retina and the basic anatomical organization of the retinal GLY and gamma-aminobutyric acid (GABA) systems was compared. Four types of GLY-immunoreactive (GLY-IR) neurons were identified. Most GLY-IR cells were amacrine cells, which comprised at least two types. GLY-IR interplexiform cells and ganglion cells also were identified. The first GLY-IR amacrine cell type was characterized by a small pyriform soma, located distal to the border of the inner plexiform layer (IPL), and fine dendrites. Most GLY-IR amacrine cells were of this type and several subtypes may exist within this group. The second amacrine cell type was characterized by a large, distally located soma and a large descending process. This amacrine cell type showed colocalization of GLY-IR and GABA-IR and comprised about 4% of the total GLY-IR amacrine cell population.

Comparison of GLY-IR and GABA-IR on serial sections showed that GLY and GABA were present in largely separate neuronal populations. Generally, GLY-IR amacrine cells were smaller, more distally located in the inner nuclear layer and had finer dendrites than GABA-IR amacrine cells. Distribution of GLY-IR and GABA-IR in the outer plexiform layer and the inner plexiform layer differed considerably.

Based on the segregated distribution of GLY-IR and GABA-IR in the synaptic layers of the lizard retina, GLY and GABA may have fundamentally different roles in retinal processing.

Biological visual systems can detect positional changes that are finer than these systems' acuity to sine-wave gratings, a property known as hyperacuity. Some systems can even detect changes finer that the photoreceptor spacing. We report here that rabbit's directionally selective ganglion cells not only detect positional changes in the hyperacuity range, but also discriminate the direction of their motion. Our experiments show that directional selectivity occurs for edges of light moving as little as 1.1 μm (26” of visual angle) across the retina. This distance corresponds to a hyperacuity, since the acuity to sine-wave gratings of rabbit's On-Off DS ganglion cells is about 125 μm (50′). In addition, this distance is smaller than the minimal spacing between rabbit photoreceptors (1.9 μm or 46”), as estimated from cell-density studies (Young & Vaney, 1991). Such a hyperacuity suggests low-noise high-gain signal transmission from photoreceptors to ganglion cells and that directional selectivity can arise in small portions of retinal dendritic processes.

Putative GABAergic mechanisms were studied in the cat retina by exogenous application of the GABAA antagonists picrotoxin (PTX), native bicuculline (BCC), and bicuculline methyl bromide (BCC MeBr). When recording intracellular responses from horizontal cells (HCs) and amacrine cells as well as electroretinograms (ERGs), drugs were added to the perfusate used to maintain the isolated eyecup; when recording extracellular spikes from ganglion cells of anesthetized cats, drugs were introduced by iontophoretic injection. Both PTX and BCC MeBr had relatively little influence upon the response properties of HCs. In contrast, native BCC tended to decrease the amplitude of and to slow the photic response to light onset and both to quicken and to increase the amplitude of response to light offset; in the presence of native BCC, HC responses were dominated by a prominent spike-like “Off-overshoot.” The influence of GABAA agonists upon HC responses was not blocked by GABAA antagonists. ERG b−wave amplitude was reduced both by PTX and by native BCC, but was not influenced by BCC MeBr. Latency (time to half-peak) was increased by low doses of native BCC, and to a lesser extent PTX but not BCC MeBr. Rod-amacrine On-transient responses were increased in amplitude by PTX. Extracellular recordings from On- and Off- X and Y ganglion cell types became considerably more transient with application of either PTX, native BCC, or BCC MeBr; this tendency was greater in Off-type ganglion cells. Collectively, these results strengthen conclusions from the previous paper suggesting that GABA serves to slow onset and offset kinetics of retinal neurons, making them more sustained and less phasic. They also suggest that in mammalian retina heterogeneous types of GABAA receptors exist, segregated into different zones: a distal zone, sensitive only to native BCC, a central zone sensitive to both native BCC and PTX, and a proximal zone sensitive to native BCC, BCC methyl halides (BCC MeH), and PTX. Only the proximal zone obeys conventional GABAA pharmacology.

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

In order to elucidate the role of putative indoleaminergic amacrine cells in visual processing, we have employed pharmacological agents specific for the two classes of serotonin receptor, 5-HT2 and 5-HT1, which have been identified in both the retina and brain. Perfusion of the rabbit retina with 5-HT2 selective antagonists decreases the ON-excitation of all classes of ganglion cell as well as the spontaneous activity of these cells. The effect on OFF-responses depends on the cell type: OFF-excitation of center-surround brisk and sluggish cells is increased or not affected by these drugs, but OFF excitation of ON/OFF direction selective cells is reduced. No disruption of the trigger features of direction selective or orientation selective cells was discovered, suggesting that indoleaminergic amacrine cells do not play a role in the generation of the complex properties of these cells.

5-HT1 receptors are heterogeneous and classified as a, b, or c subtypes. Since no selective antagonists are available for these sites, we have employed specific agonists. The most specific of these are for the 5-HT1A receptor. Perfusion with these agonists had physiological effects similar to those with perfusion of 5-HT2 antagonists. Thus, we have suggested that these two classes of serotonin receptors mediate opponent processes in the neural pathway. Since indoleaminergic cells make reciprocal synaptic connections with rod bipolar cell terminals, which are depolarizing in the rabbit retina, we hypothesize that 5-HT2 receptors facilitate the synaptic transmission from the depolarizing rod bipolar cell thus facilitating ON-excitation in the retinal network while 5-HT1A receptors mediate an inhibitory process. Similar self-opponent processing appears to take place in the hypothalamic and hippocampal serotonergic systems as well as in the dopaminergic retinal system. Thus, it is likely that this organization is a general feature of monoamine signal transmission in the central nervous system.