Autoradiographic identification of acetylcholine in the rabbit retina.

Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells.

Visual motion perception: experimental modification.

If a human observer fixates a moving spiral pattern for 15 minutes, a negative aftereffect of motion is perceived when he inspects a stationary spiral 20 hours later. The illusory motion is seen only when the stationary test stimulus falls upon the portion of the retina which had been stimulated by real motion. Thus previous stimulation can cause a relatively long-term modification of vision.

Direct visualization of the dendritic and receptive fields of directionally selective retinal ganglion cells.

Optical methods were used to locate the cell bodies of directionally selective ganglion cells in isolated rabbit retinas. These neurons detect the direction in which images move across the retinal surface and transmit that information to the brain. The receptive field of each identified cell was determined, after which the cell was injected with Lucifer yellow. An image of the receptive field border was then projected onto the fluorescent image of the dendrites, allowing precise comparison between them. The size of the receptive field matched closely the size of the dendritic arbor of that cell. This result restricts the types of convergence that can be postulated in modeling the mechanism of retinal directional selectivity.

Monoclonal antibody to Thy-1 enhances regeneration of processes by rat retinal ganglion cells in culture.

Ganglion cells were dissociated from postnatal rat retinas, identified by specific fluorescent labels, and maintained in culture on a variety of substrates. Regeneration of processes by retinal ganglion cells was enhanced when the cells were plated on glass coated with a monoclonal antibody against the Thy-1 determinant. Plain glass and glass coated with polylysine, collagen, fibronectin, or other monoclonal antibodies supported the growth of neural processes, but were less effective than antibody to Thy-1.

Extreme diversity among amacrine cells: implications for function.

We report a quantitative survey of the population of amacrine cells present in the retina of the rabbit. The cells' dendritic shape and level of stratification were visualized by a photochemical method in which a fluorescent product was created within an individual cell by focal irradiation of that cell's nucleus. A systematically random sample of 261 amacrine cells was examined. Four previously known amacrine cells were revealed at their correct frequencies. Our central finding is that the heterogeneous collection of other amacrine cells is broadly distributed among at least 22 types: only one type of amacrine cell makes up more than 5% of the total amacrine cell population. With these results, the program of identification and classification of retinal neurons begun by Cajal is nearing completion. The complexity encountered has implications both for the retina and for the many regions of the central nervous system where less is known.

Receptive field microstructure and dendritic geometry of retinal ganglion cells.

We studied the fine spatial structure of the receptive fields of retinal ganglion cells and its relationship to the dendritic geometry of these cells. Cells from which recordings had been made were microinjected with Lucifer yellow, so that responses generated at precise locations within the receptive field center could be directly compared with that cell's dendritic structure. While many cells with small receptive fields had domeshaped sensitivity profiles, the majority of large receptive fields were composed of multiple regions of high sensitivity. The density of dendritic branches at any one location did not predict the regions of high sensitivity. Instead, the interactions between a ganglion cell's dendritic tree and the local mosaic of bipolar cell axons seem to define the fine structure of the receptive field center.

The fundamental plan of the retina.

The retina, like many other central nervous system structures, contains a huge diversity of neuronal types. Mammalian retinas contain approximately 55 distinct cell types, each with a different function. The census of cell types is nearing completion, as the development of quantitative methods makes it possible to be reasonably confident that few additional types exist. Although much remains to be learned, the fundamental structural principles are now becoming clear. They give a bottom-up view of the strategies used in the retina's processing of visual information and suggest new questions for physiological experiments and modeling.

Spatial scale and cellular substrate of contrast adaptation by retinal ganglion cells.

Human visual perception and many visual system neurons adapt to the luminance and contrast of the stimulus. Here we describe a form of contrast adaptation that occurs in the retina. This adaptation had a local scale smaller than the dendritic or receptive fields of single ganglion cells and was insensitive to pharmacological manipulation of amacrine cell function. These results implicate the bipolar cell pathway as a site of contrast adaptation. The time required for contrast adaptation varied with stimulus size, ranging from approximately 100 ms for the smallest stimuli, to seconds for stimuli the size of the receptive field. The differing scales and time courses of these effects suggest that multiple types of contrast adaptation are used in viewing natural scenes.

Processing and encoding of visual information in the retina.

The retina is a remarkably sophisticated instrument and much of its internal circuitry is poorly characterized. A major problem for studies aimed at better understanding the retina is that the neurons in its middle layers are varied in type and relatively inaccessible. Two approaches that have facilitated progress towards elucidating retinal function include population-based studies of the anatomy of the retina and multi-electrode recordings from its output; in combination, they enable the neuronal system of the retina to be examined as a whole.

Neuronal diversity in the retina.

The listing of cell types present in the retina is nearing completion, the first time this can be said for any significantly complex sample of the central nervous system. Mammalian retinas contain approximately 55 separate neuronal types. The functions of 22 of them are known or can be strongly inferred. For these 22, in every instance, a cell defined as a 'type' by structural criteria carries out a distinct and individual physiological function. Electrophysiological experiments continue to reveal new features of the retina's handling of information, and there is every reason to believe that the remaining 33 types of cell will also have distinct physiological functions. Further subtleties clearly exist in both peripheral and central visual coding.

The nondiscriminating zone of directionally selective retinal ganglion cells: comparison with dendritic structure and implications for mechanism.

We have studied, at high resolution, the sizes and pattern of dendrites of directionally selective retinal ganglion cells in the rabbit. The dendrites had a distinctive pattern of branching. The major dendritic trunks were relatively thick, beginning at approximately 1 micrometer and tapering to approximately 0.5 micrometer in diameter. Higher order dendrites exiting from them generally stepped abruptly to a diameter of 0.4-0.6 micrometer, which they maintained throughout their length. Recording confirmed the existence of a zone within the receptive field, usually occupying 20-25% of its area, where direction of movement was only weakly discriminated. The dendritic arbors of cells, injected with Lucifer yellow after recording, revealed no difference in dendritic structure between the discriminating and nondiscriminating zones. The nondiscriminating zone was located on the preferred side of the receptive field (the side from which movement in the preferred direction originates). This is consistent with a mechanism of direction selectivity based on inhibition generated by movement in the null direction but not with feedforward excitation, as occurs in flies and is postulated in some models of mammalian direction selectivity.

Maturation of function in the developing rabbit retina.

Retinas isolated from rabbits aged less than eight hours to adult were maintained in a flowing physiological medium. The electroretinogram or activity of single ganglion cells were recorded, and receptive fields were studied using stimulation of the retina with focused light. Retinal activity was stable for at least eight hours of incubation. Retinal ganglion cells are electrophysiologically active on the first day of life. They generate spontaneous bursts of action potentials at rates of 10 to 30 spikes/sec, separated by silent intervals of one to six minutes. Maintained trains of action potentials follow elevation of the concentration of K+ in the incubating medium to 10 mM. Ganglion cells are also stimulated by acetylcholine, with apparent thresholds equal to or lower than those of ganglion cells in adult retinas. The first response of the retina to light is a small cornea-negative transretinal potential at day 6, presumably PIII of the electroretinogram. Responses of the ganglion cells are seen at eight days, but the responses are weak and adapt quickly to repeated stimulation. Many unresponsive cells are present. By ten days 60% of ganglion cells respond to light, and examples of mature receptive fields are present. Immature receptive fields at ten days fall into two rough classes, one characterized by a large responsive area with no antagonistic surround, and a second in which the surround can suppress the response to illumination of the center but can not itself cause a discharge. Immature fields are progressively replaced by mature ones, and by 20 days the qualitative organization of receptive fields is indistinguishable from adult.

A population of wide-field bipolar cells in the rabbit's retina.

We have stained an unusual population of retinal bipolar cells. When the low molecular weight tracer biocytin was injected into the vitreous body of rabbits, it subsequently accumulated in the somata and processes of a population of wide-field bipolar cells. The cells have 2-4 primary dendrites. Their dendritic arbors span a field 50 to 200 microns in diameter. The axonal arbors are sparse and often highly asymmetric. The longest dimension of the axonal arbor ranges from 100 to 300 microns. The cells are moderately evenly spaced. They make up less than 1% of the total population of bipolar cells in the rabbit retina. With the whole population stained, regularities in the spatial arrangement of nearby cells can be recognized. Their dendrites often run to a common point, where they have the appearance of making contact with each other. A similar arrangement is seen for the cells' axonal arbors, so that the whole population is spatially linked in both the outer retina and the inner. The exact nature of the points of conjunction cannot be learned from light microscopy. One possibility is that the processes run together because they contact a common target. If so, the target structures (one in the outer retina and one in the inner) must be sparse. An alternative is that the points of conjunction represent synapses or gap junctions among wide-field bipolars of this type.

Costratification of a population of bipolar cells with the direction-selective circuitry of the rabbit retina.

We have stained a new population of bipolar cells in rabbit retina by using antibodies against the carbohydrate epitope, CD15. The CD15-positive bipolar cells comprise 6-8% of the total cone bipolar cells in peripheral retina. Their axonal and dendritic arbors are similar in size and range from 15 to 50 pm in diameter. The axonal arbors are narrowly stratified in sublamina b of the inner plexiform layer. Double label experiments using an antibody against the calcium binding protein, calbindin, or an antibody against protein kinase C, demonstrate that the CD15-positive bipolar cells are a separate population from the previously identified calbindin-positive cone bipolar cells and the rod bipolar cells. Labeling the processes of starburst amacrine cells with antibodies against choline acetyltransferase showed that the CD15-positive bipolar cells stratify within and slightly more distally to the processes of the ON-starburst amacrine cells. Confocal images of retinal wholemounts showed that the axons of the CD15-positive bipolar cells follow the pattern of the ON-starburst cells' processes. Axonal varicosities of the CD15-positive bipolar cells penetrate the bundles formed by the processes of the ON-starburst cells. This finding suggests that the CD15-positive bipolar cell provides input to the ON-starburst amacrine cells and/or the ON-plexus of the ON-OFF direction-selective ganglion cells.

Shape and distribution of an unusual retinal neuron.

Rabbit retinas were exposed to exogenous indoleamines and fixed with mixed aldehydes. The indoleamines were accumulated by two types of amacrine cell and by an unusual cell (type 3) that branches widely in both plexiform layers. The type 3 cells were studied after immunohistochemistry, photooxidation of the fluorescent label, or injection with Lucifer Yellow. Their cell bodies are located at the scleral margin of the inner nuclear layer. The cells' arbors in the outer plexiform layer range from 800 to 1,500 microns in diameter. A descending process crosses the inner nuclear layer and branches in layer 5 of the inner plexiform layer. The arbor in the inner retina can exceed 500 microns in diameter. The distribution of type 3 cells was mapped in a series of retinal whole mounts. The number of type 3 cells ranged from 58 to 270 in different retinas. In two retinas from a single animal, however, it was virtually identical. Type 3 cells are concentrated in the region ventral to the visual streak, so that large areas of the retina are not covered by any type 3 cell. Because of their incomplete retinal coverage and variable number from animal to animal, the type 3 cells appear to be developmental anomalies. Paradoxically, their generation must be precisely controlled because of the numerical symmetry between an individual animal's two eyes.

Shapes and distributions of the catecholamine-accumulating neurons in the rabbit retina.

Rabbit retinas were fixed with mixed aldehydes and examined for the fluorescence of catecholamines. Labeled cell bodies were present in the layer of the amacrine cells. A band of fluorescent processes was present in layer 1 of the inner plexiform layer. Weaker labeling was present in two deeper strata, one near the middle of the inner plexiform layer (presumably layer 3) and one at the junction of layers 4 and 5. Immunohistochemistry showed tyrosine hydroxylase (TH) to be present in the same cells and the same strata of the inner plexiform layer as the endogenous catecholamines. Exposing the retina to exogenous dopamine or norepinephrine resulted in stronger labeling in the middle and deep levels of the inner plexiform layer. At the same time a second population of amacrine cell bodies became visible. Catecholamine fluorescence contained in the amacrine cell bodies was used as a guide to their injection with Lucifer yellow CH. The filled dendritic arbors revealed two main types of cells. The type 1 cells are monostratified at the most distal level of the inner plexiform layer. They have relatively uncomplicated, radially branching dendritic trees. They are the cells densely stained by immunohistochemistry with antibodies against TH. The type 2 cells are tristratified, with minor branching in layer 1 of the inner plexiform layer and major branching in the two deeper sublayers. The descending dendrites follow a complicated course, and it is not uncommon for intermediate dendrites to cross between strata more than once. The relationship of the cells to their dendritic plexuses was further studied in retinas in which the aldehyde-induced fluorescence of catecholamines was photoconverted to a diaminobenzidine product. The type 1 cells were found to dominate the plexus of dendrites in layer 1 of the inner plexiform layer. The catecholaminergic plexuses in the middle and deep levels of the inner plexiform layer are formed by dendrites of the type 2 cells. The position of every type 1 cell was mapped in retinas stained with antibodies directed against TH. In one retina we counted 5,613 type 1 cells, distributed evenly across the retina. In another retina, all of the catecholamine-accumulating cells were counted. There were 9,058 with a distribution that peaks in the visual streak. The type 1 cells appear to be the dopaminergic cells previously studied by others and thought to regulate the flow of information from rod bipolar cells to ganglion cells. The low density and wide spread of type 2 cells suggests that they, too, perform a generalized control function, presumably a novel one that dictates their intricate, tristratified shape.

Development of outer segments and synapses in the rabbit retina.

The peripheral retina of rabbits aged 0 to 60 days was studied by electron microscopy. Ribbon and conventional synaptogenesis was studied with serial sections, and the density of synapses of the inner plexiform layer was measured on large (1,500 micrometer 2) montages. Photoreceptor and bipolar ribbon synapses seem to develop similarly in that processes of the prospective dyad or triad contact the presynaptic ribbon-containing terminal one at a time. No statistically significant difference in the lengths of ribbon lamellae was found at 11 and 30 days. Conventional synapses appear to result from the aggregation of synaptic vesicles on one side of junctions that first existed as symmetrical membrane densities without vesicles. The length of the synaptic membrane specialization constant between 0 and 30 days. The density of inner plexiform layer conventional synapses remains at a low and roughly constant level from 0 to 9 days, after which there is an abrupt increase to a plateau at about 20 days. After nine days the density of ribbon synapses also increases, with an initially steep time course similar to that of conventional synapses. All subcategories of synapse studied (amacrine-to-amacrine, amacrine-to-bipolar, serial, and reciprocal) participate in the general increase between 9 and 20 days. Functional circuits of the inner plexiform layer thus seem to be assembled primarily during the second and third weeks of life.

Connections of indoleamine-accumulating cells in the rabbit retina.

To study the connections of the neurons of the rabbit retina that accumulate indoleamines, we injected 5,7-dihydroxytryptamine into the vitreous body. It accumulated within a subset of amacrine cells and could be visualized there by aldehyde-induced fluorescence. The fluorescent labeling was photo-converted to an insoluble, osmiophilic product by irradiation in the presence of diaminobenzidine, and the tissue was examined by electron microscopy. Preservation of the structure of the tissue after photoconversion was satisfactory and the dendrites of the indoleamine-accumulating cells could easily be identified. They form a dense plexus near the junction of the inner plexiform and ganglion cell layers, where they exhibit large synaptic endings that occupy a substantial fraction of the surface of rod bipolar terminals. The dendrites of the indoleamine-accumulating cells receive input from rod bipolars at dyad synapses, where the other postsynaptic partner is a dendrite of a narrow-field, bistratified amacrine cell; in addition, they receive amacrine cell input throughout the inner plexiform layer. The only outputs we observed are reciprocal synapses onto the rod bipolar endings. Thus, these amacrine cells appear to exert an important effect on the transmission of scotopic information through the retina.

The shapes and numbers of amacrine cells: matching of photofilled with Golgi-stained cells in the rabbit retina and comparison with other mammalian species.

Amacrine cells of the rabbit retina were studied by "photofilling" a photochemical method in which a fluorescent product is created within an individual cell by focal irradiation of the nucleus; and by Golgi impregnation. The photofilling method is quantitative, allowing an estimate of the frequency of the cells. The Golgi method shows their morphology in better detail. The photofilled sample consisted of 261 cells that were imaged digitally in through-focus series from a previous study (MacNeil and Masland [1998] Neuron 20:971-982). The Golgi material consisted of 49 retinas that were stained as wholemounts. Eleven of these subsequently were cut in vertical section. Of the many hundreds of cells stained, digital through-focus series were recorded for 208 of the Golgi-impregnated cells. The two methods were found to confirm one another: Most cells revealed by photofilling were recognized easily by Golgi staining, and vice versa. The greater resolution of the Golgi method allowed a more precise description of the cells and several types of amacrine cell were redefined. Two new types were identified. The two methods, taken together, provide an essentially complete accounting of the populations of amacrine cells present in the rabbit retina. Many of them correspond to amacrine cells that have been described in other mammalian species, and these homologies are reviewed.

Selective accumulation of diamidino yellow and chromomycin A3 by retinal glial cells.

We applied the fluorescent DNA stains diamidino yellow (DY) and chromomycin A3 to rat and rabbit retinas in vivo and in vitro. They accumulated in the nuclei of a subpopulation of cells of the inner nuclear layer. The number and distribution of the fluorochrome-accumulating cells were similar to those of the Müller glia, and double-labeling experiments showed that the cells accumulating DY or chromomycin A3 contained oriented filaments of vimentin. The fluorochromes also accumulated in the sparse astrocytes and oligodendrocytes located among the myelinated fibers of the rabbit central retina. Specific accumulation in retinal glia occurred only when the fluorochromes were applied to living retinas. If the plasma membranes were disrupted by fixation or exposure to detergent, most retinal cells were stained. This indicates that the locus of specificity is the entry of the molecules into the cells. When applied to living retinas, other DNA stains selectively accumulate in subclasses of retinal neurons. Why DNA-binding molecules should selectively cross the membranes of either retinal neurons or retinal glia remains an unsolved problem.