Amacrine cell contributions to red-green color opponency in central primate retina: a model study.

To investigate the contributions of amacrine cells to red-green opponency, a linear computational model of the central macaque retina was developed based on a published cone mosaic. In the model, amacrine cells of ON and OFF types received input from all neighboring midget bipolar cells of the same polarity, but OFF amacrine cells had a bias toward bipolar cells whose center responses were mediated by middle wavelength sensitive cones. This bias might arise due to activity dependent plasticity because there are midget bipolar cells driven by short wavelength sensitive cones in the OFF pathway. The model midget ganglion cells received inputs from neighboring amacrine cells of both types. As in physiological experiments, the model ganglion cells showed spatially opponent responses to achromatic stimuli, but they responded to cone isolating stimuli as though center and surround were each driven by a single cone type. Without amacrine cell input, long and middle wavelength sensitive cones contributed to both the centers and surrounds of model ganglion cell receptive fields. According to the model, the summed amacrine cell input was red-green opponent even though inputs to individual amacrine cells were unselective. A key prediction is that GABA and glycine depolarize two of the four types of central midget ganglion cells; this may reflect lower levels of the potassium chloride co-transporter in their dendrites.

A high frequency resonance in the responses of retinal ganglion cells to rapidly modulated stimuli: a computer model.

Brisk Y-type ganglion cells in the cat retina exhibit a high frequency resonance (HFR) in their responses to large, rapidly modulated stimuli. We used a computer model to test whether negative feedback mediated by axon-bearing amacrine cells onto ganglion cells could account for the experimentally observed properties of HFRs. Temporal modulation transfer functions (tMTFs) recorded from model ganglion cells exhibited HFR peaks whose amplitude, width, and locations were qualitatively consistent with experimental data. Moreover, the wide spatial distribution of axon-mediated feedback accounted for the observed increase in HFR amplitude with stimulus size. Model phase plots were qualitatively similar to those recorded from Y ganglion cells, including an anomalous phase advance that in our model coincided with the amplification of low-order harmonics that overlapped the HFR peak. When axon-mediated feedback in the model was directed primarily to bipolar cells, whose synaptic output was graded, or else when the model was replaced with a simple cascade of linear filters, it was possible to produce large HFR peaks but the region of anomalous phase advance was always eliminated, suggesting the critical involvement of strongly non-linear feedback loops. To investigate whether HFRs might contribute to visual processing, we simulated high frequency ocular tremor by rapidly modulating a naturalistic image. Visual signals riding on top of the imposed jitter conveyed an enhanced representation of large objects. We conclude that by amplifying responses to ocular tremor, HFRs may selectively enhance the processing of large image features.

Abnormal centrifugal axons in streptozotocin-diabetic rat retinas.

PURPOSE: To characterize the effects of diabetes on the expression of histidine decarboxylase mRNA and on the morphology of the histaminergic centrifugal axons in the rat retina. METHODS: Rats were made diabetic by streptozotocin. After 3 months, retinal histidine decarboxylase expression was analyzed by in situ hybridization in radial sections. Flatmount retinas from a second group of rats were labeled with an antiserum to histamine or an antibody to phosphorylated neurofilament protein. RESULTS: Histidine decarboxylase mRNA was expressed in cells in the inner and outer nuclear layers of diabetic retinas, but not in normal retinas. However, immunoreactive (IR) histamine was not localized to perikarya in either the normal or the diabetic retinas. Instead, a population of centrifugal axons was labeled. These axons emerged from the optic disc and had varicose terminal branches in the inner plexiform layer (IPL) of the peripheral retina. Some branches ended on large retinal blood vessels and others in dense clusters in the IPL. In rats with streptozotocin-induced diabetes, the centrifugal axon terminals developed many large swellings that contained neurofilament immunoreactivity; these swellings were rare in normal retinas. CONCLUSIONS: Diabetes perturbs the retinal histaminergic system, causing increases in histidine decarboxylase mRNA expression in neurons or glia and abnormal focal swellings on the centrifugal axons.

Diffuse bipolar cells provide input to OFF parasol ganglion cells in the macaque retina.

Parasol retinal ganglion cells are more sensitive to luminance contrast and respond more transiently at all levels of adaptation than midget ganglion cells. This may be due, in part, to differences between bipolar cells that provide their input, and the goal of these experiments was to study these differences. Midget bipolar cells are known to be presynaptic to midget ganglion cells. To identify the bipolar cells presynaptic to parasol cells, these ganglion cells were intracellularly injected with Neurobiotin, cone bipolar cells were immunolabeled, and the double-labeled material was analyzed. In the electron microscope, we found that DB3 diffuse bipolar cells labeled by using antiserum to calbindin D-28k were presynaptic to OFF parasol cells. In the confocal microscope, DB3 bipolars costratified with OFF parasol cell dendrites and made significantly more appositions with them than expected due to chance. Flat midget bipolar cells were labeled with antiserum to recoverin. Although they made a few appositions with parasol cells, the number was no greater than would be expected when two sets of processes have overlapping distributions in the inner plexiform layer. DB2 diffuse bipolar cells were labeled with antibodies to excitatory amino acid transporter 2, and they also made appositions with OFF parasol cells. These results suggest that DB2 bipolar cells are also presynaptic to OFF parasol ganglion cells, but midget bipolar cells are not. We estimate that midperipheral OFF parasol cells receive approximately 500 synapses from 50 DB3 bipolar cells that, in turn, receive input from 250 cones.

Synaptic connections of DB3 diffuse bipolar cell axons in macaque retina.

In primate retinas, the dendrites of DB3 diffuse bipolar cells are known to receive inputs from cones. The goal of this study was to describe the synaptic connections of DB3 bipolar cell axons in the inner plexiform layer. DB3 bipolar cells in midperipheral retina were labeled with antibodies to calbindin, and their axons were analyzed in serial, ultrathin sections by electron microscopy. Synapses were found almost exclusively at the axonal varicosities of DB3 axon terminals. There were 2.14 synaptic ribbons per varicosity. There were 33 varicosities per DB3 cell, giving an average of 71 ribbons per axon terminal. Because there were 1.5 postsynaptic ganglion cell dendrites per DB3 axonal varicosity, we estimate that there is at least 1 synapse per varicosity onto a parasol ganglion cell dendrite. There were 3.4 input synapses from amacrine cells per axonal varicosity. Among these were feedback synapses to the DB3 bipolar cell axon varicosities, which were made by 47% of the postsynaptic amacrine cell processes. Some of the feedback synapses could be from amacrine cells immunoreactive for cholecystokinin precursor or choline acetyltransferase, because both types of amacrine cells costratify with parasol cells and are known to be presynaptic to bipolar cells. AII amacrine cells were both presynaptic and postsynaptic to DB3 axons, a finding consistent with the large rod input to parasol ganglion cells reported in physiological experiments. DB3 bipolar cell axons also made frequent contacts with neighboring DB3 axons, and gap junctions were always found at these sites.

Histamine immunoreactive axons in the macaque retina.

PURPOSE: The goal of these experiments was to identify the neurotransmitter in centrifugal axons of the macaque retina. METHODS: Macaca mulatta retinas and optic nerves were fixed overnight in carbodiimide and labeled with an antiserum to histamine with the use of an immunofluorescence technique. RESULTS: Several large histamine-immunoreactive axons ran from the optic nerve head to the peripheral retina, where they branched extensively and terminated in the inner plexiform layer, occasionally alongside retinal blood vessels. Other axons that emerged from the optic nerve head ran in the optic fiber layer to the central retina, circled the fovea, and then returned to the optic disc. These may be the source of histamine-immunoreactive axons that have been observed in central visual areas. No labeled cell bodies were present in the retina. Because perikarya in the posterior hypothalamus are the only known source of histamine in the primate central nervous system and because neurons there can be retrogradely labeled from the cut optic nerve, the histamine-immunoreactive axons must have originated there. CONCLUSIONS: Centrifugal axons in the macaque retina are part of the system of axons containing histamine that originate in the hypothalamus and project throughout the brain. Because the activity of these neurons is highest during the morning, histamine might play a role in preparing the retina to operate in daylight. The contacts of histamine-immunoreactive axons with blood vessels suggest that histamine may also play a role in regulating the retinal microvasculature.

Gap junctions with amacrine cells provide a feedback pathway for ganglion cells within the retina.

In primates, one type of retinal ganglion cell, the parasol cell, makes gap junctions with amacrine cells, the inhibitory, local circuit neurons. To study the effects of these gap junctions, we developed a linear, mathematical model of the retinal circuitry providing input to parasol cells. Electrophysiological studies have indicated that gap junctions do not enlarge the receptive field centres of parasol cells, but our results suggest that they make other contributions to their light responses. According to our model, the coupled amacrine cells enhance the responses of parasol cells to luminance contrast by disinhibition. We also show how a mixed chemical and electrical synapse between two sets of amacrine cells presynaptic to the parasol cells might make the responses of parasol cells more transient and, therefore, more sensitive to motion. Finally, we show how coupling via amacrine cells can synchronize the firing of parasol cells. An action potential in a model parasol cell can excite neighbouring parasol cells, but only when the coupled amacrine cells also fire action potentials. Passive conduction was ineffective due to low-pass temporal filtering. Inhibition from the axons of the coupled amacrine cells also produced oscillations that might synchronize the firing of more distant ganglion cells.

Light-stimulated release of dopamine from the primate retina is blocked by 1-2-amino-4-phosphonobutyric acid (APB).

Macaca mulatta retinas were superfused, in vitro, to measure the efflux of dopamine. Steady light, in the low photopic range, stimulated dopamine release slightly. Flashing light (3 Hz) superimposed over the steady background increased dopamine efflux significantly. This increase was completely blocked by the addition of d,1-2-amino-4-phosphonobutyric acid (d,l-APB, 10-100 microM) to the superfusion medium, but not by the addition of the inactive enantiomer d-APB (10 microM). The results suggest that ON bipolar cells provide the excitatory drive to dopaminergic amacrine cells in primates, as in other species.

Morphology of P and M retinal ganglion cells of the bush baby.

P/midget ganglion cells mediate red-green color opponency in anthropoids. It has been proposed that these cells evolved as a specialization to subserve color vision in primates. If that is correct, they must have evolved about the same time as the long-wavelength ('red') and medium-wavelength ('green') pigment genes diverged, thirty million years ago. Strepsirhines are another group of primates that diverged from the ancestor of the anthropoids at least 55 million years ago. If P/midget ganglion cells evolved to subserve color vision, they should be absent in strepsirhines. We tested this hypothesis in a nocturnal strepsirhine, the greater bush baby Otolemur. The retinal ganglion cells were labeled with the lipophilic tracer Dil and the results show that bush babies have P/midget and M/parasol cells similar to those found in the peripheral retinas of anthropoids. A number of studies have shown that the P and M pathways of bush babies share many similarities with those of anthropoids, and our results show that the same is true for their retinal ganglion cells. These results support the hypothesis that the P system evolved prior to the emergence of red-green color opponency.

The topographical relationship between two neuronal mosaics in the short wavelength-sensitive system of the primate retina.

The short wavelength-sensitive (blue) cone bipolar cells was found to have a nonrandom distribution by analyzing the nearest neighbors and by calculating the density recovery profile (DRP). Blue cones had been shown previously to have a nonrandom distribution (Curcio et al., 1991). The relationship between the two arrays was then analyzed by calculating the cross-correlational density recovery profile (cDRP), which indicates the local density of blue cones around each blue cone bipolar cell. Although both cell types appeared to be distributed uniformly at the macroscopic level, the cDRP was 1.7 times higher within 15 microns of each bipolar cell perikaryon than in the surrounding area. The area of higher density was approximately the same as that in which the blue cone bipolar cells made synaptic contacts with blue ones. The finding that the blue cones and the blue cone bipolar cells were closer together than expected suggested that the positions of the perikarya of these neurons were influenced by their synaptic connections or other developmental interactions.

Amino acid receptors of midget and parasol ganglion cells in primate retina.

Primate retinas contain two major ganglion cell types. Midget (or P type) cells have relatively sustained responses to light; the amplitude and polarity of these responses vary with stimulus wavelength. Parasol (or M type) cells are more sensitive to stimulus contrast and respond more transiently but are not selective for color. Both types can be further subdivided into a and b subtypes, according to the level of their dendritic stratification in the inner plexiform layer. To determine whether differences in receptors for amino acid transmitters are the basis for any differences in ganglion cell light responses, we made whole-cell, patch-clamp recordings from identified ganglion cells in slice preparations of macaque and baboon retinas. We found that midget and parasol cells of both a and b types had similar responses to excitatory amino acids, including kainate, alpha-amino-3-hydroxy-5-methylisoxalzole-4-propionic acid, and N-methyl-D-aspartate, with reversal potentials near the equilibrium potential for cations. Kainate responses were blocked by 6,7-dinitroquinoxaline, and N-methyl-D-aspartate responses were blocked by D-(-)-2-amino-7-phosphonoheptanoic acid. The four types of ganglion cells also had similar responses to bath-applied inhibitory amino acids. All cells had both gamma-aminobutyric acid and glycine receptors with reversal potentials near the equilibrium potential for Cl-, and the relative amplitudes of the responses to excitatory and inhibitory amino acids were similar among the various cell types. These results suggest that the differences in response properties of the different classes of ganglion cells in primate retina may be determined, to a significant degree, by the properties of the amacrine and bipolar cells that provide their input rather than by the nature of their postsynaptic receptors.

Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.

Spatial density and distribution of choline acetyltransferase immunoreactive cells in human, macaque, and baboon retinas.

Whole-mounted human, macaque, and baboon retinas were labelled with an antiserum to human choline acetyltransferase (ChAT), by the immunoperoxidase technique. Previous work in nonprimate species has shown that these cells correspond to the starburst amacrine cells. Labelled somata were disposed on either side of the inner plexiform layer, and their processes formed two narrow zones within it. In human retinas, the ratio of labelled somata in the ganglion cell layer (GCL) to those in the inner nuclear layer (nominal Sb/Sa ratio) was about 60/40 at all locations, similar to that found in nonprimate mammalian species. The density of labelled cells in the human GCL ranged from 1,000 to 1,150 mm-2 near the fovea to 300 to 400 mm-2 in the periphery. Labelling tended to be more erratic in macaque retinas. Nevertheless the Sb/Sa ratio was as high as 70/30 and spatial densities were similar to those of humans. The overlap factor in macaque retinas outside the nasal quadrant was about 10 at all retinal eccentricities, based upon dendritic-field sizes from a Golgi study. About each labelled soma there was a region 20 to 120 microns in diameter in which the probability of the occurrence of other labelled somata was lower than elsewhere. No such nonrandomness was found between labeled cells in the GCL and those in the amacrine cell layer. The packing factor was about 0.3 in well-labelled regions, independent of retinal position or spatial density. Published data on ChAT-labelled cells in rabbit and rat show a similar value. This invariance is consistent with the hypothesis that this nonrandomness is a residual consequence of somal contiguity at an early developmental stage.

Bipolar cells specific for blue cones in the macaque retina.

A distinct subpopulation of bipolar cells in macaque monkey retina was labeled with antisera that recognize glycine-extended cholecystokinin precursors. The labeled bipolar cells were found throughout the retina and had dendrites contacting a subpopulation of cone pedicles and axons ramifying in the fifth stratum of the inner plexiform layer. Several lines of evidence indicate that the labeled bipolar cells are a single type despite some variations in their morphology. First, the density of perikarya and their diameters varied continuously as a function of eccentricity. Second, the positions of perikarya within the inner nuclear layer and the level at which the axons branched in the inner plexiform layer were constant at all eccentricities. Bipolar cells with similar morphology have been described previously as "blue cone bipolar cells" (Mariani, 1984b), but there was no direct evidence that this was the case. In this study, we show by light microscopy that labeled bipolar cells have dendrites ending exclusively upon presumptive blue cones labeled by Procion black dye. All blue cones were contacted by labeled bipolar cells, and virtually all bipolar cells contacted blue cones, the only exceptions being in regions where blue cones had been lost. Approximately 20% more labeled bipolar cells than blue cones were found at every eccentricity; thus, connections between blue cones and labeled bipolar cells were not strictly one to one. The mean number of cones presynaptic to each bipolar cell was 1.2, and the mean number of bipolar cells postsynaptic to each cone was 1.8. By an electron microscopic study of labeled bipolar cell dendrites, we determined that they became central elements of ribbon synapses in blue cones. Some of their ribbon synapses were unusual: in one type, a single, large labeled dendrite was postsynaptic to two or more ribbons, while in the other type, ribbons had two or more central elements. The presence of these invaginating contacts and the axonal terminals in the proximal inner plexiform layer suggest that the labeled bipolar cells depolarize to short-wavelength stimuli and function to relay information from blue cones to the inner plexiform layer. There were also other, unlabeled bipolar cell dendrites that received inputs from blue cones at basal junctions and triad-associated flat contacts, which suggests that there are additional types of bipolar cells conveying information from short-wavelength cones in the primate retina.

Peptidergic neurons of teleost retinas.

In retinas of teleost fish, neuropeptides typically have subtle, modulatory actions. The peptide effects typically have long latencies and durations, and, in some instances, they are known to be mediated by second messengers. Peptidergic neurons in teleost retinas have certain morphological features in common that are consistent with their function. Most peptidergic neurons are stratified amacrine cells with long, varicose processes; the processes of peptidergic centrifugal axons are also narrowly stratified and ramify extensively in the retina. The peptidergic amacrine cells are relatively infrequent, and, likewise, the centrifugal axons originate from a small number of perikarya in the brain. Cells that are so sparsely distributed and whose processes overlap so extensively are better-suited for modulation than for conveying detailed representations of visual space.

Serotoninergic varicosities make synaptic contacts with pleural sensory neurons of Aplysia.

Serotonin is a modulatory neurotransmitter that produces many of the cellular changes associated with sensitization of reflexes in Aplysia. These changes have been carefully documented in sensory neurons located in the abdominal ganglion that mediate the gill-siphon withdrawal reflex and in sensory neurons located in the pleural ganglion that mediate the tail-siphon withdrawal reflex. Although serotonin appears to be necessary for sensitization, there is no direct evidence that serotoninergic neurons make synaptic contacts with sensory neurons. In this study, the immunoperoxidase technique was used to label serotonin-immunoreactive neurites surrounding the cell bodies of sensory neurons in the pleural ganglion. Serotonin-immunoreactive neurites had varicosities whose mean short axis diameter was 1.1 +/- 0.6 microns (mean +/- S.D.). The shape of the size distribution was skewed toward larger sizes, however, suggesting that there were multiple subpopulations of varicosities. One subpopulation was that of varicosities located at branch points whose average short axis diameter was larger than normal (1.7 +/- 0.5 microns). Serotonin-immunoreactive varicosities were directly apposed to the sensory neurons without intervening glial cells. In most contacts, serotonin-immunoreactive neurites invaginated into the plasma membranes of the sensory neurons. There were also a few contacts onto spinelike processes, but these were flat rather than invaginated. Serotoninergic neurons whose activity produces changes in the electrophysiological properties of sensory neurons have been identified, but this study provides the first direct evidence for synaptic connections between serotoninergic neurons and sensory neurons in Aplysia.

Localization of immunoreactive cholecystokinin precursor to amacrine cells and bipolar cells of the macaque monkey retina.

We used antisera that recognized precursors of the neuropeptide cholecystokinin extended at the carboxyl terminus in an immunocytochemical study of the macaque retina. A subpopulation of bipolar cells with long, obliquely oriented dendrites was labeled. Their axons terminated exclusively in the fifth stratum of the inner plexiform layer, where they contacted processes of amacrine and ganglion cells. Based on their morphology, these cells appeared to be the type that contacts short-wavelength cones selectively. Two types of amacrine cells were also labeled, and processes from both types formed dense plexuses in the second and fourth strata of the inner plexiform layer. The majority of their synaptic connections were with other amacrine cells, but they had more contacts with bipolar cell axons and retinal ganglion cell dendrites than any other peptidergic cells in the macaque retina. We studied extracts of macaque retina with gel-filtration chromatography and radioimmunoassays to confirm our immunohistochemical results. We found cholecystokinin octapeptide and other immunoreactive forms that were amidated at their carboxyl termini and were therefore likely to be biologically active. Unlike most other regions of the CNS, however, the retina had relatively low concentrations of amidated forms, and forms with extended carboxyl termini that are presumably their precursors were far more abundant. These findings suggest that the rate of cholecystokinin synthesis in the retina is quite high, as we would expect if the peptide were found in tonically active neurons.

Peptidergic neurons of the macaque monkey retina.

Until recently, peptides were thought to act as neuromodulators in the retina, and the localizations of peptides in wide field amacrine cells, associational cells and interplexiform cells seemed to support this hypothesis. Anatomical studies in the macaque monkey retina, however, found that some types of peptidergic amacrine cells made extensive contacts with bipolar cell axons and retinal ganglion cell dendrites. The most striking exception to the earlier generalizations about retinal peptide function was the localization of immunoreactive cholecystokinin in bipolar cells that contacted short wavelength cones selectively. These results suggested that peptides were not only interacting with the most direct pathway for visual information; they also appeared to be used as transmitters by the neurons that comprise that pathway. Taken with the localizations of peptides in retinal ganglion cells and recent electrophysiological evidence, these findings suggest that peptides can also act more like conventional neurotransmitters.

A peptide histidine isoleucine/peptide histidine methionine-like peptide in the rabbit retina: colocalization with vasoactive intestinal peptide, synaptic relationships and activation of adenylate cyclase activity.

Antisera against peptide histidine isoleucine and peptide histidine methionine were found to label a subpopulation of amacrine and displaced amacrine cells in the rabbit retina with processes ramifying in sublaminas 1, 3 and 5 of the inner plexiform layer. Preadsorption controls demonstrated that this immunoreactivity was specific for a peptide histidine isoleucine- or peptide histidine methionine-like (peptide histidine isoleucine/peptide histidine methionine-like) peptide, and was not caused by cross-reactivity of the peptide histidine isoleucine or peptide histidine methionine antibodies with vasoactive intestinal peptide vasoactive intestinal peptide. In double-label studies, vasoactive intestinal peptide and peptide histidine isoleucine/peptide histidine methionine-like immunoreactivity were colocalized in the same population of retinal neurons. Electron microscopic analysis revealed that the peptide histidine isoleucine/peptide histidine methionine-labelled cells interacted with processes of bipolar cells, amacrine cells and ganglion cells. Peptide histidine methionine and peptide histidine isoleucine were slightly less potent than vasoactive intestinal peptide in stimulating adenylate cyclase activity in the rabbit retina, while the related peptides secretin, glucagon, and the C-terminal vasoactive intestinal peptide fragment, vasoactive intestinal peptide (10-28), showed little or no stimulatory activity. Stimulation of adenylate cyclase by high concentrations of vasoactive intestinal peptide and peptide histidine methionine were non-additive. These results suggest that a peptide histidine isoleucine/peptide histidine methionine-like peptide may function as a neuroactive peptide in the mammalian retina, and that this peptide appears to be cosynthesized and colocalized with vasoactive intestinal peptide and to mimic the activity of vasoactive intestinal peptide through interaction with vasoactive intestinal peptide receptor-adenylate cyclase complexes.