Amacrine Cells In Rabbit Retina Research Articles

Development of excitatory and inhibitory neurotransmitters in transitory cholinergic neurons, starburst amacrine cells, and GABAergic amacrine cells of rabbit retina, with implications for previsual and visual development of retinal ganglion cells

Starburst amacrine cells (SACs), the only acetylcholine (ACh)-releasing amacrine cells (ACs) in adult rabbit retina, contain GABA and are key elements in the retina's directionally selective (DS) mechanism. Unlike many other GABAergic ACs, they use glutamic acid decarboxlyase (GAD)(67), not GAD(65), to synthesize GABA. Using immunocytochemistry, we demonstrate the apoptosis at birth (P0) of transitory putative ACs that exhibit immunoreactivity (IR) for the ACh-synthetic enzyme choline acetyltransferase (ChAT), GAD(67), and the GABA transporter, GAT1. Only a few intact, displaced ChAT-immunoreactive SAC bodies are detected at P0. At P2, ChAT-IR is detected in the two narrowly stratified substrata of starburst dendrites in the inner plexiform layer (IPL). Quantitative analysis reveals that in the first postnatal week, only a small fraction of SACs cells express ChAT- and GABA-IR. Not until the end of the second week are they expressed in all SACs. At P0, a three-tiered stratification of GABA-IR is present in the IPL, entirely different from the adult pattern of seven substrata, emerging at P3-P4, and optimally visualized at P13. At P0, GAD(65) is detectable in normally placed AC bodies. At P1, GAD(65)-IR appears in dendrites of nonstarburst GABAergic ACs, and by P5 is robust in the adult pattern of four substrata in the IPL. GAD(65)-IR never co-localizes with ChAT-IR. In a temporal comparison of our data with physiological, pharmacological, and ultrastructural studies, we suggest that transitory ChAT-immunoreactive cells share with SACs production of stage II (nicotinic) waves of previsual synchronous activity in ganglion cells (GCs). Further, we conclude that (1) GAD(65)-immunoreactive, non-SAC GABAergic ACs are the most likely candidates responsible for the suppression of stage III (muscarinic/AMPA-kainate) waves and (2) DS responses first appear in DS GCs, when about 50% of SACs express ChAT- and GABA-IR, and in 100% of DS GCs, when expression occurs in all SACs.

The Synaptic Mechanism of Direction Selectivity in Distal Processes of Starburst Amacrine Cells

Patch-clamp recordings revealed that distal processes of starburst amacrine cells (SACs) received largely excitatory synaptic input from the receptive field center and nearly purely inhibitory inputs from the surround during both stationary and moving light stimulations. The direct surround inhibition was mediated mainly by reciprocal GABA(A) synapses between opposing SACs, which provided leading and prolonged inhibition during centripetal stimulus motion. Simultaneous Ca(2+) imaging and current-clamp recording during apparent-motion stimulation further demonstrated the contributions of both centrifugal excitation and GABA(A/C)-receptor-mediated centripetal inhibition to the direction-selective Ca(2+) responses in SAC distal processes. Thus, by placing GABA release sites in electrotonically semi-isolated distal processes and endowing these sites with reciprocal GABA(A) synapses, SACs use a radial-symmetric center-surround receptive field structure to build a polar-asymmetric circuitry. This circuitry may integrate at least three levels of interactions--center excitation, surround inhibition, and reciprocal inhibitions that amplify the center--surround antagonism-to generate robust direction selectivity in the distal processes.

Directional Selectivity Is Formed at Multiple Levels by Laterally Offset Inhibition in the Rabbit Retina

The excitatory and inhibitory inputs to directionally selective (DS) ganglion cells are themselves directionally selective. Directionality is achieved because excitation is reduced during null-direction movement along a GABAergic pathway. Inhibition is reduced during preferred-direction movement along a pathway that includes cholinergic synapses. Both excitation and inhibition are made directional by laterally offset inhibitory signals similar to the spatial offset of the direct inhibitory input to the DS cell dendrites. Thus, spatially offset lateral inhibition generates directionality at three different levels in the DS circuitry. We also found that for stimuli falling within the dendritic field, cholinergic input is delivered to the OFF but not the ON dendrites. Cholinergic pathways from outside the dendritic field reach both ON and OFF dendrites, but both of these pathways are normally inactivated by GABAergic synapses.

Identification of cholinoceptive glycinergic neurons in the mammalian retina.

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs.

NADPH-diaphorase (nitric oxide synthase)-reactive amacrine cells of rabbit retina: Putative target cells and stimulation by light

In the mammalian retina there are two populations of nitric oxide synthase-containing amacrine cells that stain with the nicotinamide adenine dinucleotide phosphate-diaphorase reaction. To determine the response of these neurons to light, immunoreactivity to Fos proteins was used as a marker of synaptic activation. Fos immunoreactivity is absent in dark-adapted retinas, but 70% of large, Type I nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells and 5–10% of the smaller but more numerous Type II nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells contain Fos proteins after light stimulation. To localize putative cellular targets of nitric oxide in the retina, retinas were stained immunocytochemically for cyclic GMP after the local administration of the nitric oxide donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine. Both compounds induce strong cyclic GMP immunoreactivity in ON cone bipolar cells. The data suggest that the light-induced inward current in ON cone bipolar cells is enhanced by a nitric oxide-cyclic GMP pathway and that the major source of nitric oxide is the nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells in the rabbit retina.

Dendritic co-stratification of ON and ON-OFF directionally selective ganglion cells with starburst amacrine cells in rabbit retina.

The morphology, dendritic branching patterns, and dendritic stratification of retinal ganglion cells have been studied in Golgi-impregnated, whole-mount preparations of rabbit retina. Among a large number of morphological types identified, two have been found that correspond to the morphology of ON and ON-OFF directionally selective (DS) ganglion cells identified in other studies. These cells have been characterized in the preceding paper in terms of their cell body size, dendritic field size, and branching pattern. In this paper, the two kinds of DS ganglion cell are compared in terms of their levels of dendritic stratification. They are compared with each other and also with examples of class III.1 cells, defined in the preceding paper with reference to our previous studies. Studies employing computer-aided, 3D reconstruction of dendritic trees, as well as analysis of a pair of ON DS and ON-OFF DS ganglion cells with overlapping dendritic trees show that the two types of DS ganglion cell partly co-stratify in the middle of sublamina b (stratum 4). The report that some ON DS ganglion cells extend a few dendrites into sublamina a is confirmed. The study of pairs of ON-OFF DS ganglion cells and starburst amacrine cells with overlapping dendritic trees reveals a precise co-stratification of these two cell types, and many points of close apposition of starburst boutons with ON-OFF DS ganglion cell dendrites in both sublaminae of the inner plexiform layer (IPL). This is confirmed by high-resolution light microscopy and by electron microscopy. It is possible to conclude, therefore, that ON DS are also partly co-stratified with type b starburst (cholinergic) amacrine cells, and are apparently also partly co-stratified with type a starburst amacrine cells, when occasional dendrites rise to that level. The co-stratification of the two kinds of DS ganglion cell is consistent with the sharing of some inputs in common, including some cone bipolar cell inputs. The co-stratification of both with starburst amacrine cells agrees with the physiological demonstration of the powerful pharmacological effects upon ON and ON-OFF DS ganglion cells reported for cholinergic agonists. The major difference in the dendritic stratification of bistratified ON-OFF DS ganglion cells and generally unistratified ON DS ganglion cells is consistent with the bisublaminar organization of ON and OFF pathways in the IPL. The problem of occasional branches of ON DS cells in sublamina a is discussed in terms of a threshold for OFF responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyaxonal amacrine cells of rabbit retina: size and distribution of PA1 cells.

Type 1 polyaxonal (PA1) amacrine cells have been identified previously in rabbit retina, and their morphological characteristics have been described in detail in the preceding paper. Like other polyaxonal amacrine cells they bear distinct dendritic and axonal branching systems, the latter of which originates in two to six thin, branching axons which emerge from or near to the cell body. Unlike other types of polyaxonal amacrine cells, however, their branching is stratified at the a/b sublaminar border and their cell bodies are most often displaced interstitially in the inner plexiform layer (IPL). This report emphasizes quantitative features of the population of PA1 cells, documented in Golgi-impregnated and Nissl-stained retinas, and provides further evidence in Nissl preparations for the amacrine-cell nature of polyaxonal amacrine cells. The cell bodies of Golgi-impregnated PA1 amacrine cells are relatively large: 12-15 microns in equivalent diameter over the range extending from the visual streak 6 mm into ventral retina. Over the same range, dendritic trees are 400-800 microns in equivalent diameter, but they are much smaller than the axonal arborizations, which extend up to and perhaps beyond 2 mm from the cell body. Interstitial cell bodies appropriate to PA1 cells have been identified in Nissl-stained, whole-mounted rabbit retinas. In the plane of the retina, these are comparable in area to smaller medium-size ganglion cells, but their very pale Nissl staining, high nuclear/cytoplasmic ratio, and absence of nucleolar staining are all characteristics of amacrine cells. Interstitial displacement of presumed PA1 cells is rare in the visual streak, and the frequency of interstitial cells reaches a peak between 1 and 2 mm ventral to the streak. Counts in Nissl-stained retinas and estimates from nearest neighbor analyses in these and in Golgi-impregnated retinas indicate a density of PA1 cells in the range of 15-16 cells/mm2 at about 2 mm ventral to the streak, when an estimated 25% shrinkage of the material is taken into account. Dendritic field overlap, based upon this estimate, is calculated to be about fourfold, while a lower bound to estimates of the overlap of axonal arborizations is nearly an order of magnitude higher. Many similarities are noted in a qualitative and quantitative comparison of PA1 amacrine cells in rabbit and monkey retinas. In assessing the contribution of the structural organization of PA1 amacrine cells to their possible functional role(s), it is notable that their appearance conforms not to amacrine cells as commonly viewed, but to a more conventional model of neuronal dynamic polarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyaxonal amacrine cells of rabbit retina: PA2, PA3, and PA4 cells. Light and electron microscopic studies with a functional interpretation.

Polyaxonal (PA) amacrine cells are a new class of amacrine cell bearing one to six branching, axon-like processes that emerge from the cell body or dendritic trees within 50 microns of the cell body. These slender processes of uniform caliber branch at right angles and in many respects closely resemble the axons of Golgi type II cells found elsewhere in the brain. Of the four types of polyaxonal amacrine cell that we have recognized in rabbit retina, two have been described previously in brief communications. One of these, the PA1 amacrine cell with its interstitially displaced cell body, located in the inner plexiform layer (IPL), has been analyzed extensively in two preceding reports. This paper concerns PA2, PA3, and PA4 amacrine cells. Type 2 polyaxonal (PA2) amacrine cells, identified in Golgi preparations of whole-mounted rabbit retinas, have smaller cell bodies (9-14 microns) than the other three types and these are always displaced to the ganglion cell layer (GCL) or the inner border of the inner plexiform layer (IPL). The dendritic fields of PA2 cells are also smaller than those of other PA amacrine cells, and most of their sparse dendritic branching is narrowly stratified at the border of strata (S) 4 and 5. Some members of this more heterogeneous amacrine cell "type" are bistratified, however, and more highly branched with terminal branches rising to end in S1. PA2 amacrine cells bear a scattering of small dendritic spines and may also exhibit complex dendritic appendages arising at the ends of terminal branches in proximal regions of the dendritic tree. PA2 cells emit one to three axons from the proximal dendritic tree, and about half of the cells bear a single axon. Type 3 polyaxonal (PA3) amacrine cells resemble PA1 cells in the large size of their cells bodies (11-16 microns) and dendritic fields, but differ from the latter in placement of cell bodies, which is in the GCL, and dendritic and axonal stratification, which is multistratified, ranging from S4 to S1, with a concentration in S3 or S4 and a variable contribution to S1. PA3 cells differ from PA1 cells in several other respects, including dendritic branching which occurs at higher frequency and is biased toward temporal retina, and in characteristic bristling dendritic spines, clustered in the intermediate regions of the dendritic tree, that are longer, more variable in appearance and more tightly clustered than the small, uniform spines of PA1 cells that are clustered on proximal dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyaxonal amacrine cells of rabbit retina: morphology and stratification of PA1 cells.

Polyaxonal amacrine cells are a new class of amacrine cell bearing one to six branching, axon-like processes, closely resembling the axons of Golgi type II cells found elsewhere in the central nervous system. Of the four types of polyaxonal amacrine cell that we have recognized in rabbit retina, three have been described previously in brief communications, and one is the subject of this paper. Type 1 polyaxonal (PA1) amacrine cells have larger cell bodies than most amacrine cells in Golgi preparations, averaging about 13 microns in diameter. These are typically positioned interstitially in the middle of the inner plexiform layer (IPL), although some are also found in the amacrine and ganglion cell layers. Axons and dendrites are broadly stratified in the middle of the IPL, in the vicinity of the a/b sublaminar border. Sparsely branching dendrites have a conventional appearance, branching at a narrow angle, and giving rise to smaller daughter branches, which taper gradually toward their termination. An unusual feature of the dendrites is the zig-zag course of some terminal branches. Clusters of small, pedunculated spines are common on proximal dendrites, and spines are virtually absent on axons. Axons emerge from proximal dendrites within 50 microns of the soma, and more rarely from the soma, in a tapering initial segment, commonly interrupted by one or two large swellings. Subsequent branching is at a wide angle, and the fine caliber is maintained in the transition from parent to daughter branches. The uniform thickness of the axonal branches is interrupted at intervals by boutons en passant. Although the extent of the dendritic tree is large, exceeding 500 microns in radial extent from the cell body, for cells a few millimeters distant from the visual streak, the axonal tree is much larger, and its radial extent is measured in millimeters. PA1 amacrine cells are believed to be polarized in their functional organization, with a primarily recipient dendritic tree and a primarily transmissive axonal tree. PA1 amacrine cells co-stratify with nab cone bipolar cells and with certain small tufted amacrine and ganglion cells at the a/b sublaminar border. The co-stratification of both axons and dendrites at the a/b sublaminar border of the IPL suggests that PA1 amacrine cells are important modulators of neural activity in the middle of the IPL, affecting both ON and OFF responses, and perhaps ON-OFF cells selectively.

Synaptic organization of starburst amacrine cells in rabbit retina: Analysis of serial thin sections by electron microscopy and graphic reconstruction

The synaptic organization of starburst amacrine cells was studied by electron microscopy of individual or overlapping pairs of Golgi-impregnated cells. Both type a and type b cells were analyzed, the former with normally placed somata and dendritic branching in sublamina a, and the latter with somata displaced to the ganglion cell layer and branching in sublamina b. Starburst amacrine cells were thin-sectioned horizontally, tangential to the retinal surface, and electron micrographs of each section in a series were taken en montage. Cell bodies and dendritic trees were reconstructed graphically from sets of photographic montages representing the serial sections. Synaptic inputs from cone bipolar cells and amacrine cells are distributed sparsely and irregularly all along the dendritic tree. Sites of termination include the synaptic boutons of starburst amacrine cells, which lie at the perimeter of the dendritic tree in the "distal dendritic zone." In central retina, bipolar cell input is associated with very small dendritic spines near the cell body in the "proximal dendritic zone." The proximal dendrites of type a and type b cells generally lie in planes or "strata" of the inner plexiform layer (IPL), near the margins of the IPL. The boutons and varicosities of starburst amacrine cells, distributed int he distal dendritic zone, lie in the "starburst substrata," which occupy a narrow middle region in each of the two sublaminae, a and b, in rabbit retina. As a consequence of differences in stratification, proximal and distal dendritic zones are potentially subject to different types of input. Type b starburst amacrines do not receive inputs from rod bipolar terminals, which lie mainly in the inner marginal zone of the IPL (stratum 5), but type a cells receive some input from the lobular presynaptic appendages of rod amacrine cells in sublamina a, at the border of strata 1 and 2. There is good correspondence between boutons or varicosities and synaptic outputs of starburst amacrine cells, but not all boutons gave ultrastructural evidence of presynaptic junctions. The boutons and varicosities may be both pre- and postsynaptic. They are postsynaptic to cone bipolar cell and amacrine cell terminals, and presynaptic primarily to ganglion cell dendrites. In two pairs of type b starburst amacrine cells with overlapping dendritic fields, close apposition of synaptic boutons was observed, raising the possibility of synaptic contact between them. The density of the Golgi-impregnation and other technical factors prevented definite resolution of this question. No unimpregnated profiles, obviously amacrine in origin, were found postsynaptic to the impregnated starburst boutons.(ABSTRACT TRUNCATED AT 400 WORDS)

Amacrine cells of the rhesus monkey retina

Amacrine cells of the rhesus monkey, Macaca mulatta, were studied in 38 retinas Golgi-impregnated as whole, flat preparations. By using criteria of dendritic morphology, span of arborization, and level of arborization in the inner plexiform layer, 26 types of amacrine cell ranging in size of dendritic span from 30 microns to 2 mm were identified and listed in increasing size of dendritic span. In some instances, different cell types could be grouped together due to similar morphological features. For example, 1 group, "knotty amacrine cells," has small cell bodies and a profusion of small, varicose, intertwined processes that span up to 30 microns and are essentially monostratified, but each of the 3 types ends in different strata. Another group is 2 types with about 20 fine radiating processes spanning 1 mm that possess some prominent varicosities. One of these has all of its processes terminating in the innermost stratum of the inner plexiform layer ("spidery"-type 2 amacrine cells). The other with predominantly similarly ending processes has some that also terminate in the outermost stratum ("spidery"-type 1 amacrines). These 2 cell types likely correspond to the type 1 and type 2 indolamine-accumulating amacrine cells in rabbit retina. Other types are individuals which cannot be grouped together but resemble familiar types in cat retina (AII and A13). Other types can be correlated with their putative neurotransmitter (type 1 CA-dopamine) or transmitter/drug receptor ("spiny"-benzodiazepine receptor) phenotype. Many types as yet have no known correlate from other Golgi studies or clues as to transmitter or receptor phenotype. This study provides evidence for an unprecedented number of amacrine cell types in the primate retina. The similar morphologies of different types of amacrine cell types within a group suggest other common features within these groups such as neurotransmitter phenotype.

Immunocytochemical staining of cholinergic amacrine cells in rabbit retina

Cholinergic neurons of rabbit retina were labelled with an antibody against choline acetyltransferase, the synthesizing enzyme for acetylcholine. Two populations of cells are immunoreactive. Type a cell bodies lie in the nuclear layer (INL), their dendrites branching narrowly in sublamina a of the inner plexiform layer (IPL), while type b cell bodies lie in the ganglion cell layer (GCL) with dendrites branching in sublamina b of the IPL. The irregular networks of clustered immunoreactive dendrites are similar, but not identical, in the two sublaminae. Type b cells are more numerous than type a cells in central retina. No axons were stained. It appears that the immunoreactive neurons are normally placed and displaced starburst/cholinergic amacrine cells.

Starburst amacrine cells in cat retina are associated with bistratified, presumed directionally selective, ganglion cells

Starburst amacrine cells of cat retina are similar in form, though more delicate and less profusely branched, when compared to the starburst/cholinergic amacrine cells of rabbit retina, as identified in Golgi preparations. In both species, type a cells branch in the middle of sublamina a of the inner plexiform layer (IPL), but type b (displaced) starburst amacrine cells of cat branch near the a/b sublaminar border (stratum 3) of the IPL, not in the middle of sublamina b (stratum 4), as do those of rabbit. Nevertheless, in each species, this starburst substratum in sublamina b coincides with the sublamina b-level branching of a bistratified ganglion cell, which in rabbit retina shows directionally selective responses. It is proposed that starburst amacrine cells of cat retina are cholinergic and, as in rabbit retina, make selective connections with on-off directionally selective ganglion cells.

NADPH diaphorase histochemistry in the rabbit retina

NADPH diaphorase activity has been shown by histochemical staining to co-localize with markers for selective neurotransmitter candidates in various regions of the rat brain. The rabbit retina was therefore examined to determine if the technique stains a selective population of retinal neurons as well. Whole retinas of adult, male, pigmented rabbits are incubated with a specific reaction mixture containing nitro blue tetrazolium as the electron acceptor. Dark blue reaction product is deposited in two populations of cell bodies near the inner border of the inner nuclear layer (INL). One cell type is larger and more darkly stained than the second. The larger cells have 2–4 tapering primary dendrites which branch sparsely in the inner plexiform layer (IPL) and which can be traced for up to 500 μm. The second cell type has smaller and more lightly stained somata. In retinal cross sections, a dense layer of varicose fibers is seen in the middle (sublamina 3) of the IPL; these fibers arise at least in part from the larger, darkly stained cell bodies. A less dense plexus of fibers is stained at the outer margin (sublamina 1) of the IPL, and occasional varicosities are seen in the inner sublaminas (4 and 5) of the IPL. NADPH diaphorase histochemistry, therefore, selectively stains at least two subtypes of amacrine cells in rabbit retina. Although a definite identification of the transmitter content of these cells cannot be made, diaphorase histochemistry provides, in the retina, a remarkably convenient method for achieving Golgi-like images of morphologically distinct neuronal populations.

Colocalization of GAD-like immunoreactivity and 3H-GABA uptake in amacrine cells of rabbit retina.

Rabbit retinas were double labeled to determine the degree of colocalization of glutamic-acid-decarboxylase-like immunoreactivity (GAD-like IR) and 3H-GABA uptake using light (LM) and electron microscopic (EM) autoradiography. Both GAD-like IR and 3H-GABA uptake were found in amacrine cell bodies in the inner nuclear layer (INL) as well as in cell bodies in the ganglion cell layer (GCL), and throughout the inner plexiform layer. GAD-like IR was found in 32% of the amacrine cells in the INL, 86% of which also showed 3H-GABA uptake; 3H-GABA uptake was observed in 38% of the amacrine cells. However, only 72% of these cells showed GAD-like IR. Labeled cells in the GCL were only 10-15% as common as similarly labeled cells in the INL. As in the INL, all GAD-positive cells in the GCL were double labeled, but only 53% of the cells taking up 3H-GABA were double labeled. We suggest that labeled cells in the GCL were ganglion cells rather than displaced amacrine cells. Cells, in both the INL and GCL, that showed 3H-GABA uptake but no GAD-like IR had a higher average grain density than double-labeled cells, indicating that uptake by these cells was specific. The relevance to GABAergic function of 3H-GABA uptake without an indication of GAD-like IR is yet to be determined. Statistical analysis at the EM level showed that one-third of the GAD-positive synaptic terminals of amacrine cells were double labeled after a 4-month exposure. Longer exposures at the EM level should reveal a higher percentage of GAD-positive terminals because at the LM level, one-half of the double-labeled cell bodies were "lightly" labeled with grains. The high degree of colocalization of GAD-like IR and 3H-GABA uptake suggests that both markers may be useful for labeling GABAergic neurons in the rabbit retina.

Starburst amacrine cells: morphological constancy and systematic variation in the anisotropic field of rabbit retinal neurons

Starburst amacrine cells of rabbit retina have been characterized previously in terms of their highly distinctive and regular dendritic geometry. They have been identified as probable cholinergic neurons of the retina and have been shown to direct output solely to ganglion cells. The objectives of this paper are to chart the variation of starburst amacrine cells across the retina, to register the morphological features which are held constant for individual cells, and to examine factors which may remain invariant for the population with change in retinal position. Starburst amacrine cells occur as two completely segregated mirror-symmetrical populations, type a and type b cells, separately serving OFF and ON pathways, respectively. They are treated here as two distinct subpopulations with very similar features. A characteristic morphological feature of both types, related to branching pattern and best seen in flat view, is the location of boutons in the distal annular zone. This is the effective zone of synaptic output, which is constant at 50 to 60% of dendritic field area, regardless of the cell's retinal location. Both type a and type b cells exhibit systematic increase in cell body size and dendritic field diameter, and systematic decrease in frequency of branching and of synaptic boutons with perpendicular distance from the visual streak. These rates of increase or decrease fall off considerably at distances greater than about 1.5 mm dorsal and ventral to the visual streak, but at this distance, the dendritic field diameters of cells in dorsal retina are about 65% larger than the diameters of cells in ventral retina. When type a and type b cells are closely compared, they are seen to differ in several respects. Branching patterns of type a and type b cells differ slightly, the latter being more highly branched, and the normalized branching frequency histograms, characteristic for each type, remain constant with changing retinal position. At the same retinal location type a cells always have larger dendritic field diameters than type b cells. This difference is significant in ventral retina, out to a distance of at least 4.5 mm from the streak. The maximum percentage difference in size occurs not at mid-visual streak, but about 1.5 mm ventral to the streak. The population statistics of dendritic field overlap and areal dendritic coverage have been calculated using published data on cell densities. It is concluded that overlap is extraordinarily high (k greater than 25), more than 10 times that calculated for retinal ganglion cells.(ABSTRACT TRUNCATED AT 400 WORDS)

‘Starburst’ amacrine cells and cholinergic neurons: mirror-symmetric ON and OFF amacrine cells of rabbit retina

Golgi-impregnated ‘starburst’ amacrine cells share significant morphological features with cholinergic neurons in rabbit retina. They are mirror-symmetrical about the a/b (OFF/ON) sublaminar border of the inner plexiform layer. Type a starburst amacrines have cell bodies in the amacrine cell layer and dendrites in sublamina a, while type b cells have their cell bodies in the ganglion cell layer and dendrites in sublamina b of the inner plexiform layer (IPL). The two levels of narrow dendritic stratification are precisely those demonstrated by Masland and Mills for cholinergic amacrine cells. The morphological evidence indicates that the duality of ON and OFF pathways is served separately by type b (displaced) and type a starburst amacrine cells, respectively.

The cholinergic amacrine cells of rabbit retina receive on and off input: An analysis of [ 3H]-ACh release using 2-amino-4-phosphonobutyric acid (APB) and chloride free medium

Using the in vitro rabbit eye-cup we have examined the light-evoked release of ACh from cholinergic amacrine cells under conditions known to eliminate On responses in the retina. APB (100 μM), which blocks the photoreceptor/depolarizing bipolar cell synapse, reduced the light-evoked release of ACh by 80% but a small light-evoked response remained, which was potentiated by bicuculline. Depolarizing bipolar cells are also Cl − dependent. Cl − free medium caused a tenfold Ca 2+-dependent increase in the release of ACh but some small light evoked release remained. These results indicate that the cholinergic amacrine cells receive On and Off input. Our findings are consistent with anatomical and electrophysiological evidence which suggests that the displaced cholinergic amacrine cells are On cells and the conventionally placed cholinergic amacrine cells are Off cells.

A tonic gamma-aminobutyric acid-mediated inhibition of cholinergic amacrine cells in rabbit retina

Using the in vivo rabbit eyecup, we have studied the light-evoked release of acetylcholine (ACh) which is presumed to indicate the activity of cholinergic amacrine cells. Gamma-Aminobutyric acid (GABA) inhibited the light-evoked release of ACh (IC50 congruent to 1 mM), but the GABA antagonists bicuculline (5 micro M) and picrotoxin (20 micro M) potentiated the light-evoked release and markedly increased the resting release of ACh. This bicuculline/picrotoxin-evoked release was calcium dependent and the effects of bicuculline, but not picrotoxin, were blocked by muscimol, a potent GABA agonist. Muscimol also inhibited the light-evoked release of ACh (IC50 less than 1 micro M) and was at least 1000 times more potent than GABA. Nipecotic acid (1 mM), a GABA transport blocker, also inhibited the light-evoked release of ACh, but the effect was slow in onset and recovery was prompt. We conclude that the cholinergic amacrine cells of rabbit retina are inhibited by GABA. The relatively weak action of GABA, compared to muscimol, may be due to the presence of avid GABA transport systems. We ascribe the excitatory effects of bicuculline and picrotoxin to the antagonism of endogenous GABA, suggesting that the cholinergic cells are influenced by a tonic release of GABA. This is consistent with the effects of nipecotic acid. Although we are unable to specify the synaptic arrangements involved, we suggest that the most likely interaction is directly between GABA amacrine cells and the cholinergic amacrine cells and/or their presumed bipolar cell inputs.

Acetylcholinesterase: method for demonstration in amacrine cells of rabbit retina.

The activity of acetylcholinesterase in the inner plexiform layer of the rabbit retina was not affected detectably by prior section of the optic nerve. After the animals were treated with diisopropyl phosphorofluoridate, acetylcholinesterase reappeared in the somata of the amacrine cells and in certain cells of the ganglion cell layer before it reappeared in the inner plexiform fibers. This confirms the normal presence of acetylcholinesterase at the former site. The possible role of acetylcholine in intraretinal transmission is considered.