Structural and functional properties of homologous electrical synapses between retinal amacrine cells

Retinal amacrine cells regulate activities of retinal ganglion cells, the output neurons to higher visual centers, through cellular mechanism of lateral inhibition in the inner plexiform layer (IPL). Electrical properties of gap junction networks between amacrine cells in the IPL were investigated using combined techniques of intracellular recordings, Lucifer yellow and Neurobiotin injection, dual patch-clamp recordings and high voltage electron microscopy in isolated retinas of cyprinid fish. Six types of gap-junctionally connected amacrine cells were classified after their light-evoked responses to light flashes were recorded. Among them, gap junction networks of three types of amacrine cells were studied with structure-function correlation analysis. Cellular morphology of intercellular connections between three homologous cell classes was characterized. The interconnections between laterally extending dendrites in the IPL were localized at dendritic tip terminals. Three types of cells presented the dendrodendritic connections of tip-contact manner in the homologous cell population. High voltage as well as conventional electron microscopy revealed gap junctions between the dendritic tips of Neurobiotin-coupled cells. Receptive field properties of these amacrine cells were examined, displacing a slit of light along the distance from recording sites in the dorsal intermediate region of the retina. Receptive field size, space length constant, response latency and conduction velocity were measured. Spatial and temporal properties of receptive fields were symmetric along horizontally expanding dendrites in the dorsal retina. Simultaneous dual patch-clamp recordings revealed that the lateral gap junction connections between homologous amacrine cells expressed bidirectional electrical synapses passing Na(+) spikes. These results demonstrate that bidirectional electrical transmission in gap junction networks of these amacrine cells is symmetric along the lateral gap junction connections between horizontally extending dendrites. Lateral inhibition regulated by amacrine cells in the IPL appears to be associated with the directional extension of the dendrites and the orientation of dendrodendritic gap junctions.     

Axon-bearing amacrine cells of the macaque monkey retina

A new and remarkable type of amacrine cell has been identified in the primate retina. Application of the vital dye acridine orange to macaque retinas maintained in vitro produced a stable fluorescence in the somata of apparently all retinal neurons in both the inner nuclear and ganglion cell layers. Large somata (approximately 15-20 microns diam) were also consistently observed in the approximate center of the inner plexiform layer (IPL). Intracellular injections of horseradish peroxidase (HRP) made under direct microscopic control showed that the cells in the middle of the IPL constitute a single, morphologically distinct amacrine cell subpopulation. An unusual and characteristic feature of this cell type is the presence of multiple axons that arise from the dendritic tree and project beyond it to form a second, morphologically distinct arborization within the IPL; these cells have thus been referred to as axon-bearing amacrine cells. The dendritic tree of the axon-bearing amacrine cell is highly branched (approximately 40-50 terminal dendrites) and broadly stratified, spanning the central 50% of the IPL so that the soma is situated between the outermost and innermost branches. Dendritic field size increases from approximately 200 microns in diameter within 2 mm of the fovea to approximately 500 microns in the retinal periphery. HRP injections of groups of neighboring cells revealed a regular intercell spacing (approximately 200-300 microns in the retinal periphery), suggesting that dendritic territories uniformly cover the retina. One to four axons originate from the proximal dendrites as thin (less than 0.5 microns), smooth processes. The axons increase in diameter (approximately 1-2 microns) as they course beyond the dendritic field and bifurcate once or twice into secondary branches. These branches give rise to a number of thin, bouton-bearing collaterals that extend radially from the dendritic tree for 1-3 mm without much further branching. The result is a sparsely branched and widely spreading axonal tree that concentrically surrounds the smaller, more highly branched dendritic tree. The axonal tree is narrowly stratified over the central 10-20% of the IPL; it is approximately ten times the diameter of the dendritic tree, resulting in a 100 times greater coverage factor. The clear division of an amacrine cell's processes into distinct dendritic and axonal components has recently been observed in other, morphologically distinct amacrine cell types of the cat and monkey retina and therefore represents a property common to a number of functionally distinct cell types. It is hypothesized that the axon-bearing amacrine cells, like classical neurons,     

Presence of thyrotropin-releasing-hormone-immunoreactive (TRHir) amacrine cells in the retina of anuran and urodele amphibians

The presence of thyrotropin-releasing-hormone-immunoreactive (TRH-ir) amacrine cells in the retina of amphibians is reported for the first time. The anuran and urodele retinas studied exhibit major differences in the distribution of TRH-ir cells. In the two urodele species investigated, most TRH-ir amacrine cells were located in the ganglion cell layer (GCL). These pear-shaped cells originate a dense TRH-ir dendritic plexus in strata 4-5 of the inner plexiform layer (IPL). A small number of TRH-ir amacrine cells were observed in the inner nuclear layer (INL). Most of these INL TRH-ir cells were multipolar neurons with radiating dendrites that originate a loose plexus in the IPL stratum 1. In the three anuran species investigated, most TRH-ir amacrine cells were located in the INL. Distribution of TRH-ir processes in the IPL of anurans was not so clearly layered as in urodeles, dendrites being observed throughout strata 1-5. In the toad retina THR-ir material was also observed in the outer plexiform layer, which suggests that toads may have some TRH-ir interplexiform neurons. In the frog and toad, TRH-ir fibers were also observed in the optic nerve, although their origin could not be ascertained. The number of TRH-ir amacrine cells per whole retina was higher in anurans than in urodeles, though urodeles have higher cell densities. The marked differences in distribution of TRH-ir amacrine cells observed between anurans and urodeles, and among the three anuran species, suggest different functions of TRH in retinal processing, perhaps related to the different specializations of the visual systems of these species.     

Light- and electron-microscopic analysis of vasoactive intestinal polypeptide-immunoreactive amacrine cells in the guinea pig retina

Vasoactive intestinal polypeptide (VIP) is a neuroactive substance that is expressed in both nonmammalian and mammalian retinas. This study investigated the morphology and synaptic connections of VIP-containing neurons in the guinea pig retina by immunocytochemistry, by using antisera against VIP. Specific VIP immunoreactivity was localized to a population of wide-field and regularly spaced amacrine cells with processes ramifying mainly in strata 1 and 2 of the inner plexiform layer (IPL). Double-label immunohistochemistry demonstrated that all VIP-immunoreactive cells possessed gamma-aminobutyric acid immunoreactivity. The synaptic connectivity of VIP-immunoreactive amacrine cells was identified in the IPL by electron microscopy. The VIP-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in strata 1 to 3 of the IPL. The most frequent postsynaptic targets of VIP-immunoreactive amacrine cells were other amacrine cell processes in strata 1 to 3 of the IPL. Synaptic outputs to bipolar cells were also observed in strata 1 to 3 of the IPL. In addition, ganglion cell dendrites were also postsynaptic to VIP-immunoreactive neurons in the sublamina a of the IPL. These studies show that one type of VIP-immunoreactive amacrine cells make contact predominantly with other amacrine cell processes. This finding suggests that VIP-containing amacrine cells may influence inner retinal circuitry, thus mediating visual processing.     

Amacrine cells in the tiger salamander retina: morphology, physiology, and neurotransmitter identification.

Amacrine cells of the vertebrate retina comprise multiple neurochemical types. Yet details of their electrophysiological and morphology properties as they relate to neurotransmitter content are limited. This issue of relating light responsiveness, dendritic projection, and neurotransmitter content has been addressed in the retinal slice preparation of the tiger salamander. Amacrine cells were whole-cell clamped and stained with Lucifer yellow (LY), then processed to determine their immunoreactivity (IR) to GABA, glycine, dopamine or tyrosine hydroxylase (TOH), and glucagon antisera. Widefield, ON-OFF amacrine cells were glycine-IR. The processes of these cells extended laterally in the inner plexiform layer (IPL) from 250-600 microns. They were either multistratified in the IPL or monostratified near the IPL midline. Three multistratified ON-OFF narrowfield glycine-IR cells also were found. Four types of ON amacrine cells were found to be GABA-IR; all types had their processes concentrated in the proximal IPL (sublamina b). Type I cells were narrowfield (approximately 100 microns) with a compact projection. Type II cells were widefield (220-300 microns) with a sparse projection. Type III cells had an asymmetrical projection and varicose processes. Type IV cells were pyriform and monostratified in sublamina b. One narrowfield ON-OFF amacrine cell, with processes broadly distributed in the middle of the IPL, was GABA-IR. This cell appeared similar to an ON-OFF cell that was glycine-IR and may comprise a type in which GABA and glycine colocalize. Another class of amacrine cell, with processes forming a major plexus along the distal border of the IPL and a lesser plexus in the proximal IPL, produced slow responses at light ON and OFF; these cells were dopamine/TOH-IR. A narrowfield class of transient ON-OFF amacrine cell, with processes ramifying throughout both sublaminae a and b of the IPL, were glucagon-IR; these cells appeared to be dye-coupled at the soma. We have shown that, with respect to GABA, glycine, dopamine, and glucagon, salamander amacrine cells fall into rather discrete groups on the basis of ramification patterns in the IPL and responses to photic stimulation. The physiological, structural, and neurochemical diversity of amacrine cells is indicative of multiple and complex roles in retinal processing.     

‘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.     

Dendritic tree structure and dendritic hypertrophy during growth of the crucian carp eye

The areas of the ganglion cell dendritic trees were determined in Golgistained, flatmounted retinas of crucian carp ranging in age from one summer to 7 years. The dendritic trees of small ganglion cells (S-GC), forming the majority of retinal ganglion cells, add new branches as the retina grows. The increase in dendritic tree area exactly compensates for the decrease in ganglion cell density during growth of the eye so that the number of dendritic trees covering a particular point remains constant. While the retinal diameter increases by a factor of 2.5, the mean diameter of the S-GC dendritic fields increases by a factor of 1.9 and the visual angle covered by one S-GC dendritic tree decreases from 1.6° to 1.2°. The number of branching points of the S-GC dendrites is significantly higher in the ventral retina than in the dorsal. In general the dendrites of the S-GCs tend to grow towards the retinal margin. Dendritic orientation patterns of large (LGC) and large displaced (LDGC) ganglion cells closely resemble those of the amacrines, being oriented parallel to the retinal margin over a wide peripheral region, while the SGCs rapidly lose their tangential orientation. The dendrites of the SGCs are restricted mainly to the proximal sublayer of the inner plexiform layer, suggesting they are ON-cells, while LGC, LDGC, and amacrine cell dendrites are distributed in depth bimodally. As determined from Golgi-stained sections the crucian carp has the same basic IPL organization as the carp and cat.     

Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina

Horizontal, bipolar, and amacrine cells in the zebrafish retina were morphologically characterized using DiOlistic techniques. In this method, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-coated microcarriers are shot at high speed onto the surfaces of living retinal slices where the DiI then delineates axons, somata, and dendrites of isolated neurons. Zebrafish retinal somata were 5-10 microm in diameter. Three horizontal cell types (HA-1, HA-2, and HB) were identified; dendritic tree diameters averaged 25-40 microm. HA somata were round. Cells classified as HA-2 were larger than HA-1 cells and possessed an axon. HB somata were flattened, without an axon, although short fusiform structure(s) projected from the soma. Bipolar cells were separated into 17 morphological types. Dendritic trees ranged from 10 to 70 microM. There were six B(on) types with axon boutons only in the ON sublamina of the inner plexiform layer (IPL), and seven B(off) types with axon boutons or branches only in the OFF sublamina. Four types of bistratified bipolar cells displayed boutons in both ON and OFF layers. Amacrine cells occurred in seven types. A(off) cells (three types) were monostratified and ramified in the IPL OFF sublamina. Dendritic fields were 60-150 microM. A(on) pyriform cells (three types) branched in the ON sublamina. Dendritic fields were 50-170 microM. A(diffuse) cells articulated processes in all IPL strata. Dendritic fields were 15-90 microM. These findings are important for studies examining signal processing in zebrafish retina and for understanding changes in function resulting from mutations and perturbations of retinal organization.     

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: 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)     

A distinct type of displaced ganglion cell in a mammalian retina.

The morphology, dendritic stratification and laminal position of the soma of retinal ganglion cells were analyzed in Golgi preparations and in other rabbit retinas containing cells backfilled from the superior colliculus. Only one type, among 40 Golgi-impregnated types identified, always had its cell body displaced to the amacrine cell sublayer of the inner nuclear layer. The displaced ganglion cell of rabbit retina has a small cell body, very wide dendritic field with sometimes unbranched dendrites extending up to a millimeter from the cell body. The dendritic tree is narrowly stratified just under the amacrine cell bodies in stratum 1, and therefore does not co-stratify with starburst (cholinergic) amacrine cells, but rather with dopaminergic amacrine cells. Its correlate among ganglion cells backfilled from tectum is apparently a very sparse population of small-bodied cells mixed with a variable population of misplaced ganglion cells of varying size and type. The authentic displaced ganglion cell of rabbit retina, unlike the large displaced ganglion cell of birds, is apparently not a directionally selective ganglion cell, and its functional role in vision is presently unknown.     

Neuropeptide Y- and substance P-like immunoreactive amacrine cells in the retina of the developing Xenopus laevis.

The development of neuropeptide Y-like (NPY-LI) and substance P-like (SP-LI) immunoreactive neurons was studied in retinas of Xenopus laevis from young tadpole through to adult animals. In adult retina these neuropeptides are present in wide-field amacrine cells located in the inner nuclear layer and the ganglion cell layer of the retina. Retinal wholemount preparations and sectioned material showed that immunoreactive cells appeared during early larval life and NPY-LI occurred earlier than SP-LI cells. The primary dendritic branching of NPY-LI neurons appeared from early larval life whilst SP-LI was evident in dendrites from mid-larval stages. In postmetamorphic animals the numbers of immunoreactive cells increased in proportion to retinal area growth with a relatively constant cell density of about 35 cells/mm2 for SP-LI and 45 cells/mm2 for NPY-LI. The maturation of dendritic morphology of both NPY- and SP-LI amacrine cells appeared later in larval development than the appearance of immunoreactivity in cell somas. However, the sequence of expression of NPY- or SP-LI and their dendritic maturation was different for the two classes of amacrine cells. It is suggested that the maturation of dendritic fields of amacrine cells is complete just prior to metamorphosis, consistent with the postmetamorphic onset of electrophysiological features of ganglion cells attributed to amacrine cells.     

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.     

Characterization of genetically labeled catecholamine neurons in the mouse retina

Mouse neurons were labeled transgenically with red fluorescent protein (RFP) driven by the tyrosine hydroxylase (TH) promoter and observed in living retinas and brain slices. Two types of retinal amacrine cells expressed TH::RFP. One type had large cell bodies, processes that ramified in S1 of the inner plaxiform layer (IPL) and were TH immunoreactive, identifying them as dopaminergic neurons. A second type had smaller somas, ramified in S3 and lacked TH. Dopaminergic cells had large dendritic fields and exceptionally long axon-like processes, whereas type 2 cells were more compact. Neither cell type exhibited tracer coupling. Thus, murine retinal dopaminergic neurons exhibit functional anatomy similar to their primate counterparts and TH::RFP mice are useful for in situ characterization of catecholaminergic neurons.     

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 microns. 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.     

Morphology and physiology of the polyaxonal amacrine cells in the rabbit retina.

We examined the morphology and physiological response properties of the axon-bearing, long-range amacrine cells in the rabbit retina. These so-called polyaxonal amacrine cells all displayed two distinct systems of processes: (1) a dendritic field composed of highly branched and relatively thick processes and (2) a more extended, often sparsely branched axonal arbor derived from multiple thin axons emitted from the soma or dendritic branches. However, we distinguished six morphological types of polyaxonal cells based on differences in the fine details of their soma/dendritic/axonal architecture, level of stratification within the inner plexiform layer (IPL), and tracer coupling patterns. These morphological types also showed clear differences in their light-evoked response activity. Three of the polyaxonal amacrine cell types showed on-off responses, whereas the remaining cells showed on-center responses; we did not encounter polyaxonal cells with off-center physiology. Polyaxonal cells respected the on/off sublamination scheme in that on-off cells maintained dendritic/axonal processes in both sublamina a and b of the IPL, whereas processes of on-center cells were restricted to sublamina b. All polyaxonal amacrine cell types displayed large somatic action potentials, but we found no evidence for low-amplitude dendritic spikes that have been reported for other classes of amacrine cell. The center-receptive fields of the polyaxonal cells were comparable to the diameter of their respective dendritic arbors and, thus, were significantly smaller than their extensive axonal fields. This correspondence between receptive and dendritic field size was seen even for cells showing extensive homotypic and/or heterotypic tracer coupling to neighboring neurons. These data suggest that all polyaxonal amacrine cells are polarized functionally into receptive dendritic and transmitting axonal zones.     

Structure and functional connections of presynaptic terminals in the vertebrate retina revealed by activity-dependent dyes and confocal microscopy

The fluorescent dyes sulforhodamine 101 (SR 101) and FM1-43 were used as activity-dependent dyes (ADDs) to label presynaptic terminals in the retinas of a broad range of animals, including amphibians, mammals, fish, and turtles. The pattern of dye uptake was studied in live retinal preparations by using brightfield, fluorescence, and confocal microscopy. When bath-applied to the retina-eyecup, these dyes were avidly sequestered by the presynaptic terminals of virtually all rods, cones, and bipolar and amacrine cells; ganglion cell dendrites and horizontal cells lacked significant dye accumulation. Other structures stained with these dyes included pigment epithelial cells, cone outer segments, and Müller cell end-feet. Studies of dye uptake in dark- and light-adapted preparations showed significant differences in the dye accumulation pattern in the inner plexiform layer (IPL), suggesting a dynamic, light-modulated control of endocytotic activity. Presynaptic terminals in the IPL could be segregated on the basis of volume: bipolar varicosities in the IPL were typically larger than those of amacrine cells. The combination of retrograde labeling of ganglion cells and presynaptic terminal labeling with ADDs served as the experimental preparation for three-dimensional reconstruction of both structures, based on dual detector, confocal microscopy. Our results demonstrate a new approach for studying synaptic interactions in retinal function. These findings provide new insights into the likely number and position of functional connections from amacrine and bipolar cell terminals onto ganglion cell dendrites.     

Development of serotoninergic chick retinal neurons

Numerous neurotransmitters have been studied in detail in the developing retina. Almost all known neurotransmitters and neuromodulators were demonstrated in vertebrate retinas using formaldehyde-induced fluorescence, uptake autoradiography or immunohistochemistry procedures. Serotoninergic (5HT) amacrine neurons were described in the inner nuclear layer (INL) of the retina with their dendrites spreading within the inner plexiform layer (IPL). The present work describes the morphological pattern of development of serotoninergic amacrine neurons with a stratified dendritic branching pattern in the chick retina from embryonic day 12 to postnatal day 7. Serotoninergic-bipolar neurons are also described. 5HT-amacrine neurons have round or pear-shaped somata and primary dendritic trees oriented toward the IPL that runs through the INL, showing several varicosities. Secondary dendrites then go through the INL, without any collateral branch. At the outer and inner margin of the IPL the primary and secondary dendrites originate an outer and an inner serotoninergic network, respectively. When the primary dendritic tree reaches the IPL it deflects laterally in sublayer 1—the outer serotoninergic network. Tertiary branches then arise from the secondary dendrite and deflect in the innermost sublayer of the IPL— the inner serotoninergic network. The final pattern of branching of 5HT amacrine cells was present at embryonic day 14 and was completely developed at hatching. Serotoninergic (5HT) bipolar neurons were also present in the INL at hatching. They are weakly immunoreactive and are probably a subset of bipolar cells that accumulate serotonin from the intersynaptic cleft and are not ‘‘true’’ 5HT neurons.     

Dendritic morphology of dopaminergic cells revealed by intracellular injection of Lucifer yellow in fixed carp retina

The dendritic morphology of dopaminergic cells in carp retinas was investigated by identifying their fluorescent cell bodies in isolated, aldehyde-fixed preparations and injecting them iontophoretically with Lucifer yellow CH (LY) under microscopic control. The LY-injected cells were examined in flatmount and in radial cryosections. The cell bodies were located at the inner margin of the inner nuclear layer (the amacrine cell sublayer), and gave rise to 3-5 primary dendrites that branched repeatedly within the inner plexiform layer to form a narrow-field, diffusely branched dendritic tree. In some cases, a distal process was found to extent to the outer plexiform layer, representing the interplexiform type of cells. The 59 filled cells had roughly round or oval dendritic fields covering an area of 0.102 +/- 0.003 mm2 (361 +/- 57 micron in diameter) in the intermediate retinal region. The density of dopaminergic cells in this region was 31 cells/mm2 and, therefore, the dendritic field coverage of these cells approximately 3.0.