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.

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.

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.

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.

Confronting complexity: strategies for understanding the microcircuitry of the retina.

The mammalian retina contains upward of 50 distinct functional elements, each carrying out a specific task. Such diversity is not rare in the central nervous system, but the retina is privileged because its physical location, the distinctive morphology of its neurons, the regularity of its architecture, and the accessibility of its inputs and outputs permit a unique variety of experiments. Recent strategies for confronting the retina's complexity attempt to marry genetic approaches to new kinds of anatomical and electrophysiological techniques.

Light-evoked responses of bipolar cells in a mammalian retina.

We recorded light-evoked responses from rod and cone bipolar cells using patch-clamp techniques in a slice preparation of the rat retina. Rod bipolar cells responded to light with a sustained depolarization (ON response) followed at light offset by a slight hyperpolarization. ON and OFF cone bipolar cells were encountered, both with diverse temporal properties. The responses of rod bipolar cells were composed primarily of two components, a nonspecific cation current and a chloride current. The chloride current was reduced greatly in axotomized cells and could be suppressed by coapplication of the GABA(A) antagonist bicuculline and the GABA(C) antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid. This suggests that it largely reflects feedback from GABAergic amacrine cells. The response latency of intact rod bipolar cells was shorter than that of the axotomized cells, and the sensitivity curve covered more than twice the dynamic range. Application of the GABA receptor antagonists partially mimicked the effects of axotomy. These findings suggest that functional properties of the axon terminal system-notably synaptic feedback from amacrine cells-play an important role in defining the response properties of mammalian bipolar cells.

Spatial order within but not between types of retinal neurons.

We studied the mosaics of six types of retinal neurons, asking how the position of a cell relates to the positions of other cells of that same type and also to cells of different types. Every neuron studied was found to be nonrandomly positioned: Cells of a particular type were evenly spaced. However, all cells were positioned randomly with respect to members of the other cell classes. This was true even when the cells were known to be synaptically connected. It is consistent with a concept of developmental pattern formation in which (i) the number of cells of a particular type and their laminar distribution are specified, and (ii) the final spatial position of each cell is controlled exclusively by a rule that prevents cells of the same type from being positioned close to each other. This sequence would imply that a cell's final position is independent of the cell's position at the time of its specification, and we suggest a reason why, in laminar structures containing many cell types, it might be desirable for this to be so.

Amacrine, ganglion, and displaced amacrine cells in the rabbit retina express nicotinic acetylcholine receptors.

Acetylcholine (ACh) in the vertebrate retina affects the response properties of many ganglion cells, including those that display directional selectivity. Three beta and eight alpha subunits of neuronal nicotinic acetylcholine receptors (nAChRs) have been purified and antibodies have been raised against many of them. Here we describe biochemical and immunocytochemical studies of nAChRs in the rabbit retina. Radioimmunoassay and Western blot analysis demonstrated that many of the nAChRs recognized by a monoclonal antibody (mAb210) contain beta2 subunits, some of which are in combination with alpha3 and possibly other subunits. MAb210-immunoreactive cells in the inner nuclear layer (INL) were 7-14 microm in diameter and were restricted to the innermost one or two tiers of cells, although occasional cells were found in the middle of the INL. At least 60% of the cells in the ganglion cell layer (GCL) in the visual streak displayed mAb210 immunoreactivity; these neurons ranged from 7-18 microm in diameter. The dendrites of cells in both the INL and GCL could sometimes be followed until they entered one of two dense, poorly defined, bands of processes in the inner plexiform layer (IPL) that overlap the arbors of the cholinergic starburst cells. Parvalbumin and serotonin-positive neurons did not exhibit nAChR immunoreactivity. Although the level of receptor expression appeared to be low, mAb210 immunoreactivity was observed in some of the ChAT-positive (starburst) amacrine cells.

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.

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.

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.

Action potentials in the dendrites of retinal ganglion cells.

The somas and dendrites of intact retinal ganglion cells were exposed by enzymatic removal of the overlying endfeet of the Müller glia. Simultaneous whole cell patch recordings were made from a ganglion cell's dendrite and the cell's soma. When a dendrite was stimulated with depolarizing current, impulses often propagated to the soma, where they appeared as a mixture of small depolarizations and action potentials. When the soma was stimulated, action potentials always propagated back through the dendrite. The site of initiation of action potentials, as judged by their timing, could be shifted between soma and dendrite by changing the site of stimulation. Applying QX-314 to the soma could eliminate somatic action potentials while leaving dendritic impulses intact. The absolute amplitudes of the dendritic action potentials varied somewhat at different distances from the soma, and it is not clear whether these variations are real or technical. Nonetheless, the qualitative experiments clearly suggest that the dendrites of retinal ganglion cells generate regenerative Na+ action potentials, at least in response to large direct depolarizations.

The major cell populations of the mouse retina.

We report a quantitative analysis of the major populations of cells present in the retina of the C57 mouse. Rod and cone photoreceptors were counted using differential interference contrast microscopy in retinal whole mounts. Horizontal, bipolar, amacrine, and Müller cells were identified in serial section electron micrographs assembled into serial montages. Ganglion cells and displaced amacrine cells were counted by subtracting the number of axons in the optic nerve, learned from electron microscopy, from the total neurons of the ganglion cell layer. The results provide a base of reference for future work on genetically altered animals and put into perspective certain recent studies. Comparable data are now available for the retinas of the rabbit and the monkey. With the exception of the monkey fovea, the inner nuclear layers of the three species contain populations of cells that are, overall, quite similar. This contradicts the previous belief that the retinas of lower mammals are "amacrine-dominated", and therefore more complex, than those of higher mammals.

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.

ON direction-selective ganglion cells in the rabbit retina: dendritic morphology and pattern of fasciculation.

ON direction-selective (DS) ganglion cells were identified by electrophysiological recordings in DAPI labeled, isolated rabbit retinas. Their responses to a flashing spot were sustained. Their responses to moving stimuli were strong in the preferred direction and weak in the null direction. Injection of the recorded cells with Lucifer yellow revealed that the cells had a distinct dendritic morphology, consistent with that described previously (Buhl & Peichl, 1986; Amthor et al., 1989; Famiglietti, 1992a). When neighboring cells were injected, an extensive dendritic co-fasciculation was observed. The pattern of fasciculation restricts the possible synaptic connections of the ON DS cell.

Retinal direction selectivity after targeted laser ablation of starburst amacrine cells.

Directionally selective retinal ganglion cells respond strongly when a stimulus moves in their preferred direction, but respond little or not at all when it moves in the opposite direction. This selectivity represents a classic paradigm of computation by neural microcircuits, but its cellular mechanism remains obscure. The directionally selective ganglion cells receive many synapses from a type of amacrine cell termed 'starburst' because of its regularly spaced, evenly radiating dendrites. Starburst amacrine cells have a synaptic asymmetry that has been proposed as the source of the directional response in the ganglion cells. Here we report experiments that make this unlikely, and offer an alternative concept of the function of starburst cells. We labelled starburst cells in living retinas, then killed them by targeted laser ablation while recording from individual directionally selective ganglion cells. Ablating starburst cells revealed no asymmetric contribution to the ganglion cell response. Instead of being direction discriminators, the starburst cells appear to potentiate generically the responses of ganglion cells to moving stimuli. The origin of direction selectivity probably lies with another type of amacrine cell.

The number of unidentified amacrine cells in the mammalian retina.

The three largest known populations of amacrine cells in the rabbit retina were stained with fluorescent probes in whole mounts and counted at a series of retinal eccentricities. The retinas were counterstained using a fluorescent DNA-binding molecule and the total number of nuclei in the inner nuclear layer were counted in confocal sections. From the total number of inner nuclear layer cells and the known fraction of them occupied by amacrine cells, the fraction of amacrine cells made up by the stained populations could be calculated. Starburst cells made up 3%, indoleamine-accumulating cells made up 4%, and AII cells made up 11% of all amacrine cells. By referring four smaller populations of amacrine cells to the number of indoleamine-accumulating cells, they were estimated to make up 4% of all amacrine cells. Thus, 78% of all amacrine cells in the rabbit's retina are known only from isolated examples, if at all. This proportion is similar in the retinas of the mouse, cat, and monkey. It is likely that a substantial fraction of the local circuit neurons present in other regions of the central nervous system are also invisible as populations to current techniques.

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.

Responses to light of starburst amacrine cells.

1. Starburst amacrine cells were studied using whole cell patch recording. Displaced starburst cells were labeled in rabbit retinas by intraocular injection of 4,6-diamidino-2-phenylindole. The retinas were isolated and maintained in vitro. The inner limiting membrane and Müller cell endfeet were removed mechanically from small areas above the starburst cell bodies, allowing an unimpeded approach under visual control to the cells. A total of 104 cells was studied. 2. In voltage-clamp recordings, the cells responded to light with slow, graded inward and outward currents on which were superimposed smaller, rapid inward currents. The rapid inward currents appeared to be postsynaptic currents. 3. The receptive fields of the cells were mapped using small spots. They had an on-center, off-surround organization. Visualizing the dendrites by including Lucifer yellow in the patch pipette showed that the receptive fields' centers closely approximated the dendritic spread of the neurons. 4. The cells' responses to movement were tested with smooth movements or with two-spot apparent motion. No directional preference was seen for spots swept across the whole receptive field, for centrifugal movements, or for centripetal movements. 5. Bath-applied tetrodotoxin (TTX) or intracellularly applied lidocaine N-ethyl bromide (QX-314) had no effect on any component of the spontaneous or light-evoked activity. Depolarization of the cell bodies by injected current showed evidence of active conductances, but they were unaffected by TTX or QX-314. 6. 6-Cyano-7-nitroquionxyline-2,3-dione eliminated the small rapid currents, indicating that they depend on alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid/kainate glutamate receptors. 7. Because it is unlikely that we voltage clamped the distalmost dendrites of these wide-field cells, uncertainties remain about rapid electrical events occurring in the dendrites. From a functional point of view, though, the fact that slow responses to distal photic stimulation were recorded at the soma suggests that the starburst cells could in principle integrate inputs across fairly substantial fractions of their total dendritic arbors. The extent to which this actually occurs remains to be learned.