Characterization and synaptic connectivity of melanopsin-containing ganglion cells in the primate retina

Melanopsin is a photopigment expressed in retinal ganglion cells, which are intrinsically photosensitive and are also involved in retinal circuits arising from rod and cone photoreceptors. This circuitry, however, is poorly understood. Here, we studied the morphology, distribution and synaptic input to melanopsin-containing ganglion cells in a New World monkey, the common marmoset (Callithrix jacchus). The dendrites of melanopsin-containing cells in marmoset stratify either close to the inner nuclear layer (outer stratifying), or close to the ganglion cell layer (inner stratifying). The dendritic fields of outer-stratifying cells tile the retina, with little overlap. However, the dendritic fields of outer-stratifying cells largely overlap with the dendritic fields of inner-stratifying cells. Thus, inner-stratifying and outer-stratifying cells may form functionally independent populations. The synaptic input to melanopsin-containing cells was determined using synaptic markers (antibodies to C-terminal binding protein 2, CtBP2, for presumed bipolar synapses, and antibodies to gephyrin for presumed amacrine synapses). Both outer-stratifying and inner-stratifying cells show colocalized immunoreactive puncta across their entire dendritic tree for both markers. The density of CtBP2 puncta on inner dendrites was about 50% higher than that on outer dendrites. The density of gephyrin puncta was comparable for outer and inner dendrites but higher than the density of CtBP2 puncta. The inner-stratifying cells may receive their input from a type of diffuse bipolar cell (DB6). Our results are consistent with the idea that both outer and inner melanopsin cells receive bipolar and amacrine input across their dendritic tree.     

Synaptic connections of the narrow-field, bistratified rod amacrine cell (AII) in the rabbit retina.

The synaptic connections of the narrow-field, bistratified rod amacrine cell (AII) in the inner plexiform layer (IPL) of the rabbit retina were reconstructed from electron micrographs of continuous series of thin sections. The AII amacrine cell receives a large synaptic input from the axonal endings of rod bipolar cells in the most vitreal region of the IPL (sublamina b, S5) and a smaller input from axonal endings of cone bipolar cells in the scleral region of the IPL (sublamina a, S1-S2). Amacrine input, localized at multiple levels in the IPL, equals the total number of synapses received from bipolar cells. The axonal endings of cone bipolar cells represent the major target for the chemical output of the AII amacrine cell: these synapses are established by the lobular appendages in sublamina a (S1-S2). Ganglion cell dendrites represent only 4% of the output of the AII amacrine and most of them are also postsynaptic to the cone bipolars which receive AII input. The AII amacrine is not presynaptic to other amacrine cells. Finally, the AII amacrine makes gap junctions with the axonal arborizations of cone bipolars that stratify in sublamina b (S3-S4) as well as with other AII amacrine cells in S5. Therefore, in the rabbit retina 1) the rod pathway consists of five neurons arranged in series: rod-->rod bipolar-->AII amacrine-->cone bipolar-->ganglion cell; 2) it seems unlikely that a class of ganglion cells exists that is exclusively devoted to scotopic functions. In ventral, midperipheral retina, about nine rod bipolar cells converge onto a single AII amacrine, but one of them establishes a much higher proportion of synaptic contacts than the rest. Conversely, each rod bipolar cell diverges onto four AII amacrine cells, but one of them receives the largest fraction of synapses. Thus, within the pattern of convergence and divergence suggested by population studies, preferential synaptic pathways are established.     

Selective synaptic connections in the retinal pathway for night vision

The mammalian retina encodes visual information in dim light using rod photoreceptors and a specialized circuit: rods→rod bipolar cells→AII amacrine cell. The AII amacrine cell uses sign-conserving electrical synapses to modulate ON cone bipolar cell terminals and sign-inverting chemical (glycinergic) synapses to modulate OFF cone cell bipolar terminals; these ON and OFF cone bipolar terminals then drive the output neurons, retinal ganglion cells (RGCs), following light increments and decrements, respectively. The AII amacrine cell also makes direct glycinergic synapses with certain RGCs, but it is not well established how many types receive this direct AII input. Here, we investigated functional AII amacrine→RGC synaptic connections in the retina of the guinea pig (Cavia porcellus) by recording inhibitory currents from RGCs in the presence of ionotropic glutamate receptor (iGluR) antagonists. This condition isolates a specific pathway through the AII amacrine cell that does not require iGluRs: cone→ON cone bipolar cell→AII amacrine cell→RGC. These recordings show that AII amacrine cells make direct synapses with OFF Alpha, OFF Delta and a smaller OFF transient RGC type that co-stratifies with OFF Alpha cells. However, AII amacrine cells avoid making synapses with numerous RGC types that co-stratify with the connected RGCs. Selective AII connections ensure that a privileged minority of RGC types receives direct input from the night-vision pathway, independent from OFF bipolar cell activity. Furthermore, these results illustrate the specificity of retinal connections, which cannot be predicted solely by co-stratification of dendrites and axons within the inner plexiform layer.     

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)     

Ultrastructural demonstration of L-glutamate decarboxylase and cysteinesulfinic acid decarboxylase in rat retina by immunocytochemistry

The gamma-aminobutyric acid (GABA) synthesizing enzyme, L-glutamate decarboxylase (GAD), and the taurine synthesizing enzyme, cysteinesulfinic acid decarboxylase (CSAD) have been localized in rat retina at the ultrastructural level by indirect immunoelectron microscopy. GAD immunoreactivity (GAD-IR) was seen only in some amacrine cells and their terminals. CSAD immunoreactivity (CSAD-IR) was found in most retinal neuronal types and their processes including photoreceptor cells (rod and cone cells), bipolar cells, amacrine cells and ganglion cells. The GAD-IR positive amacrine terminals have been found to make synaptic contact with other GAD-IR negative bipolar and amacrine terminals, and ganglion cell dendrites. Most of the GAD-IR positive terminals are presynaptic. Occasionally, GAD-IR positive amacrine terminals are postsynaptic to another amacrine terminal or ganglion cell body. In the inner plexiform layer, CSAD-IR positive amacrine terminals also make synaptic contacts with other nerve terminals, similar to that of GAD-IR positive amacrine terminals. In addition, CSAD-IR positive bipolar terminals make synaptic contact with some CSAD-IR positive as well as negative amacrine terminals. Both CSAD-IR positive amacrine and bipolar terminals are mostly presynaptic to other CSAD-IR negative terminals. In the outer plexiform layer, CSAD-IR was found to be associated with synaptic vesicles and the synaptic membrane in certain cone pedicles and rod spherules. It is concluded that only a fraction of amacrine cells in rat retina may use GABA as a neurotransmitter. The presence of CSAD-IR in some amacrine, bipolar, photoreceptor and ganglion cells in rat retina is compatible with the notion that taurine may play some important roles, such as those of neurotransmitter or neuromodulator in mammalian retina.     

Ultrastructural demonstration ofl-glutamate decarboxylase and cysteinesulfinic acid decarboxylase in rat retina by immunocytochemistry

The γ-aminobutyric acid (GABA) synthesizing enzyme,l-glutamate decarboxylase (GAD), and the taurine synthesizing enzyme, cysteinesulfinic acid decarboxylase (CSAD) have been localized in rat retina at the ultrastructural level by indirect immunoelectron microscopy. GAD immunoreactivity (GAD-IR) was seen only in some amacrine cells and their terminals. CSAD immunoreactivity (CSAD-IR) was found in most retinal neuronal types and their processes including photoreceptor cells (rod and cone cells), bipolar cells, amacrine cells and ganglion cells. The GAD-IR positive amacrine terminals have been found to make synaptic contact with other GAD-IR negative bipolar and amacrine terminals, and ganglion cell dendrites. Most of the GAD-IR positive terminals are presynaptic. Occasionally, GAD-IR positive amacrine terminals are postsynaptic to another amacrine terminal or ganglion cell body. In the inner plexiform layer, CSAD-IR positive amacrine terminals also make synaptic contacts with other nerve terminals, similar to that of GAD-IR positive amacrine terminals. In addition, CSAD-IR positive bipolar terminals make synaptic contact with some CSAD-IR positive as well as negative amacrine terminals. Both CSAD-IR positive amacrine and bipolar terminals are mostly presynaptic to other CSAD-IR negative terminals. In the outer plexiform layer, CSAD-IR was found to be associated with synaptic vesicles and the synaptic membrane in certain cone pedicles and rod spherules. It is concluded that only a fraction of amacrine cells in rat retina may use GABA as a neurotransmitter. The presence of CSAD-IR in some amacrine, bipolar, photoreceptor and ganglion cells in rat retina is compatible with the notion that taurine may play some important roles, such as those of neurotransmitter or neuromodulator in mammalian retina.     

The rod pathway in the rabbit retina: a depolarizing bipolar and amacrine cell

Anatomical and electrophysiological techniques were combined to study the morphology, synaptic connections, and response properties of two neurons in the rod pathway of the rabbit retina: the rod bipolar cell and the narrow-field, bistratified (NFB) amacrine cell. Rod bipolars receive synaptic input from rod cells in the outer plexiform layer (OPL), where their dendrites end as central elements in the invaginating synapse of rod spherules. Their main synaptic output in the inner plexiform layer (IPL) is onto NFB amacrine cells and at least one other type of amacrine, which in turn feeds a reciprocal synapse back onto the bipolar endings. Rod bipolars, or a variety of them, respond to diffuse, white light stimulation with a transient-sustained depolarization dominated by rods; with high-intensity flashes, they generate a secondary depolarization at off, which is homologous to the rod aftereffect of horizontal cells, although opposite in polarity. NFB amacrine cells receive synaptic input from rod bipolars, cone bipolars, and other types of amacrine cells; they are presynaptic to ganglion cell dendrites and communicate via gap junctions with other processes, whose parent neuron has not yet been identified. They respond to light with a triphasic potential, characterized by a depolarizing transient at on, followed by a sustained plateau phase, and finally by a hyperpolarizing transient at off. Threshold of their responses is the same as in the depolarizing rod bipolars and saturation is reached with nearly the same stimulus intensity in both neurons. Furthermore, NFB amacrine cells exhibit a depolarizing rod aftereffect at the termination of high-intensity flashes. Thus, this amacrine cell type is inserted in series along the rod pathway in the rabbit retina and modulates the transfer of scotopic signals from rod bipolars to ganglion cells.     

Ectopic retinal ON bipolar cell synapses in the OFF inner plexiform layer: Contacts with dopaminergic amacrine cells and melanopsin ganglion cells

A key principle of retinal organization is that distinct ON and OFF channels are relayed by separate populations of bipolar cells to different sublaminae of the inner plexiform layer (IPL). ON bipolar cell axons have been thought to synapse exclusively in the inner IPL (the ON sublamina) onto dendrites of ON‐type amacrine and ganglion cells. However, M1 melanopsin‐expressing ganglion cells and dopaminergic amacrine (DA) cells apparently violate this dogma. Both are driven by ON bipolar cells, but their dendrites stratify in the outermost IPL, within the OFF sublamina. Here, in the mouse retina, we show that some ON cone bipolar cells make ribbon synapses in the outermost OFF sublayer, where they costratify with and contact the dendrites of M1 and DA cells. Whole‐cell recording and dye filling in retinal slices indicate that type 6 ON cone bipolars provide some of this ectopic ON channel input. Imaging studies in dissociated bipolar cells show that these ectopic ribbon synapses are capable of vesicular release. There is thus an accessory ON sublayer in the outer IPL. J. Comp. Neurol. 517:226‐244, 2009. © 2009 Wiley‐Liss, Inc.     

Distribution of muscarinic acetylcholine receptors on processes of isolated retinal cells

Binding of propylbenzilylcholine mustard, a muscarinic acetylcholine receptor antagonist, to isolated retinal cells was examined with light microscopic autoradiography. Dissociation of the adult tiger salamander retina yielded identifiable rod, cone, horizontal, bipolar, amacrine/ganglion, and Müller cells. Preservation of fine structure was assessed with conventional electron microscopy. For all cell types, the plasmalemma was intact and free of adhering debris; in addition, presynaptic ribbon complexes were present in photoreceptor and bipolar axon terminals indicating that synaptic structures were retained. Specific binding to cell bodies and processes was analyzed separately by using morphometric and statistical techniques. The highest grain densities occurred on processes of amacrine/ganglion cells and axons and 2 degrees and 3 degrees dendrites of bipolar neurons. Bipolar cells, however, seemed to be a heterogeneous population because there was great variation in the density of binding sites on both their axons and distal dendrites. Intermediate levels of binding were found on bipolar 1 degree dendrites and horizontal cells. No specific binding was detected on Müller cells and most parts of photoreceptors. Comparisons between cells showed that grain densities were similar for bipolar axons and amacrine/ganglion cell processes but bipolar dendrites were richer in binding sites than horizontal cell dendrites. Thus, muscarinic receptors in the salamander retina are located on amacrine/ganglion, bipolar, and horizontal cells and primarily confined to the processes which compose the two synaptic layers. In the inner plexiform layer, muscarinic receptors reside on processes from all three inner retinal neurons: in the outer synaptic layer, receptors are only on second-order cells and are more numerous along bipolar than horizontal cell dendrites.     

Connections of two types of flat cone bipolars in the rabbit retina

The synaptic connections of two types of cone bipolar cells in the rabbit retina were studied with the electron microscope after labeling in vitro with 4′,6-diamidino-2-phenylindole (DAPI), intracellular injection with Lucifer Yellow, and photooxidation (Mills and Massey [1992] J. Comp. Neurol. 321:133). Both types of bipolars belong to the flat variety, because they make basal junctions with a group of four to ten neighboring cone pedicles. One cell type has an axonal arborization that occupies strata 1 through 3 of the inner plexiform layer (IPL). At ribbon synaptic junctions, it is presynaptic to ganglion cell dendrites and to reciprocal dendrites belonging to narrow-field bistratified (AII) amacrine cells. In addition, it contacts and is contacted by other amacrine cell processes of unknown origin. The other cell type has an axonal arborization entirely confined to stratum 2 of the IPL; it is pre- or postsynaptic to a pleomorphic population of amacrine cell processes, and, in particular, it receives input from the lobular appendages of AII. Thus, these two bipolar types probably belong to the off-variety because they make basal junctions with cone photoreceptors and send their axon to sublamina α of the IPL, which is occupied by the dendrites of off-ganglion cells. They are also part of the rod pathway because they receive input from AII amacrine cells. © 1996 Wiley-Liss, Inc.     

Electron microscopic analysis of the rod pathway of the rat retina

Two immunocytochemical markers were used to label the rod pathway of the rat retina. Rod bipolar cells were stained with antibodies against protein kinase C and AII-amacrine cells with antibodies against parvalbumin. The synaptic circuitry of rod bipolars in the inner plexiform layer (IPL) was studied. Rod bipolar cells make approximately 15 ribbon synapses (dyads) in the IPL. Both postsynaptic members of the dyads are amacrine cells; one is usually the process of an AII-amacrine cell and the other one frequently provides a reciprocal synapse. No direct output from rod bipolar cells into ganglion cells was found. AII-amacrine cells make chemical output synapses with cone bipolar cells and ganglion cells in sublamina a of the IPL. They make gap junctions with cone bipolar cells and other AII-amacrine cells in sublamina b of the IPL. The rod pathway of the rat retina is practically identical to that of the cat and of the rabbit retina. It is very likely that this circuitry is a general feature of mammalian retinal organization. © Wiley-Liss, Inc.     

Synaptic connectivity of the diffuse bipolar cell type DB6 in the inner plexiform layer of primate retina

Diffuse bipolar cells in primate retina receive synaptic input from multiple cones and provide output to ganglion cells. Diffuse bipolar cells can be subdivided into six types (DB1-DB6) according to the stratification of their axon terminals in the inner plexiform layer, but their synaptic connectivity in the inner plexiform layer is not well understood. Here the stratification and synaptic connectivity of DB6 axon terminals were studied in the retinae of New World (marmoset) and Old World (macaque) monkeys. Immunohistochemical markers were applied to retinal sections. The sections were analyzed by confocal and deconvolution light microscopy as well as electron microscopy. The DB6 cells were identified with antibodies against CD15; rod bipolar cells were identified with antibodies against protein kinase Calpha (PKCalpha); and AII amacrine cells were identified with antibodies against calretinin. The axons of DB6 and rod bipolar cells occupy distinct regions in stratum 5 of the inner plexiform layer. The distal processes of calretinin-labeled AII cells are usually closely associated with rod bipolar axons but sometimes also with DB6 axons. Pre-embedding immunoelectron microscopy showed that the vast majority (over 86%) of the synaptic output of DB6 cells is onto amacrine cell processes, whereas less than 14% goes to ganglion cell processes. In double-labeled preparations DB6 axons occasionally made output onto calretinin-labeled amacrine processes. Thus it is possible that AII cells receive some input from DB6 cells.     

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.     

Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina

Amacrine cells comprise ～30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ‐aminobutyric acid (GABA)ergic wide‐field amacrine cells have recently been studied; however, with the exception of the rod pathway‐specific AII amacrine cell, the connectivity of glycinergic small‐field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small‐field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A‐type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway‐specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A‐type ganglion cells. J. Comp. Neurol. 523:1529–1547, 2015. © 2015 Wiley Periodicals, Inc.     

Microcircuitry of bipolar cells in cat retina

We have studied 15 bipolar neurons from a small patch (14 X 120 micron) of adult cat retina located within the area centralis. From electron micrographs of 189 serial ultrathin sections, the axon of each bipolar cell was substantially reconstructed with its synaptic inputs and outputs by means of a computer-controlled reconstruction system. Based on differences in stratification, cytology, and synaptic connections, we identified eight different cell types among the group of 15 neurons: one type of rod bipolar and seven types of cone bipolar neurons. These types correspond to those identified by the Golgi method and by intracellular recording. Those bipolar cell types for which we reconstructed three or four examples were extremely regular in form, size, and cytology, and also in the quantitative details of their synaptic connections. They appeared quite as specific in these respects as invertebrate "identified" neurons. The synaptic patterns observed for each type of bipolar neuron were complex but may be summarized as follows: the rod bipolar axon ended in sublamina b of the inner plexiform layer and provided major input to the AII amacrine cell. The axons of three types of cone bipolar cells also terminated in sublamina b and provided contacts to dendrites of on-beta and other ganglion cells. All three types, but especially the Cb1, received gap junction contacts from the AII amacrine cell. Axons of four types of cone bipolar cells terminated in sublamina a of the inner plexiform layer and contacted dendrites of off-beta and other ganglion cells. One of these cone bipolar cell types, CBa1, made reciprocal chemical contacts with the lobular appendage of the AII amacrine cell. These results show that the pattern of cone bipolar cell input to beta (X) and probably alpha (Y) ganglion cells is substantially more complex than had been suspected. At least two types of cone bipolar contribute to each type of ganglion cell where only a single type had been anticipated. In addition, many of the cone bipolar cell pathways in the inner plexiform layer are available to the rod system, since at least four types of cone bipolar receive electrical or chemical inputs from the AII amacrine cell. This may help to explain why, in a retina where rods far outnumber the cones, there should be so many types of cone bipolar cells.     

Synaptic inputs to physiologically identified retinal X-cells in the cat.

The cat retina contains a number of different classes of ganglion cells, each of which has a unique set of receptive field properties. The mechanisms that underlie the functional differences among classes, however, are not well understood. All of the afferent input to retinal ganglion cells are from bipolar and amacrine cell terminals in the inner plexiform layer, suggesting that the physiological differences among cat retinal ganglion cells might be due to differences in the proportion of input that they receive from these cell types. In this study, we have combined in vivo intracellular recording and labeling with subsequent ultrastructural analysis to determine directly the patterns of synaptic input to physiologically identified X-cells in the cat retina. Our primary aim in these analyses was to determine whether retinal X-cells receive a characteristic pattern of bipolar and amacrine cell input, and further, whether the functional properties of this cell type can be related to identifiable patterns of synaptic input in the inner plexiform layer. We reconstructed the entire dendritic arbor of an OFF-center X-cell and greater than 75% of the dendritic tree of an ON-center X-cell and found that 1) both ON- and OFF-center X-cells are contacted with approximately the same frequency by bipolar and amacrine cell terminals, 2) each of these input types is distributed widely over their dendritic fields, and 3) there is no significant difference in the pattern of distribution of bipolar and amacrine cell synapses onto the dendrites of either cell type. Comparisons of the inputs to the ON- and the OFF-center cell, however, did reveal differences in the complexity of the synaptic arrangements found in association with the two neurons; a number of complex synaptic arrangements, including serial amacrine cell synapses, were found exclusively in association with the dendrites of the OFF-center X-cell. Most models of retinal X-cell receptive fields, because their visual responses share a number of features with those of bipolar cells, have attributed X-cell receptive field properties to their bipolar cell inputs. The data presented here, the first obtained from analyzing the inputs to the entire dendritic arbors of retinal X-cells, demonstrate that these retinal ganglion cells receive nearly one-half of their input from amacrine cells. These results clearly indicate that further data concerning the functional consequences of amacrine cell input are needed to understand more fully visual processing in the X-cell pathway.     

Aspartate aminotransferase-like immunoreactivity in the guinea pig and monkey retinas

The excitatory amino acids, aspartate and glutamate, have been proposed as retinal neurotransmitters. Aspartate aminotransferase (AAT) is an enzyme which is involved in the routine metabolism of these amino acids and may be involved in the specific synthesis of glutamate and/or aspartate for use as a neurotransmitter. On the basis of the hypothesis that increased levels of aspartate aminotransferase may reflect a transmitter role for aspartate and/or glutamate, we have localized aspartate aminotransferase in the guinea pig and cynamolgus monkey retinas with light and electron microscopic immunohistochemical techniques. AAT-like immunoreactivity is localized to the cones of guinea pig retina and to monkey rods. Both species contain a subpopulation of immunoreactive amacrine cells as well as a subpopulation of immunoreactive cells in the ganglion cell layer. Immunostaining is seen in bipolar cells and terminals in the monkey but not in the guinea pig retina. We have performed quantitative analysis of the immunoreactive staining in the outer plexiform layer and described the synaptic organization of immunoreactive processes in the inner plexiform layer (IPL). Labeled amacrine processes in both species form synaptic contacts predominantly to and from bipolar terminals in the inner third of the IPL and to and from other amacrine and small unidentified processes in the outer portion of the IPL. The majority of labeled bipolar terminals in the monkey retina are seen in the inner third of the IPL where they synapse exclusively onto amacrine processes. Labeled bipolar terminals in the outer third of the IPL occasionally synapse onto ganglion processes.     

Localization of hydroxyindole-O-methyltransferase-like immunoreactivity in photoreceptors and cone bipolar cells in the human retina: a light and electron microscope study

The localization of the melatonin-synthesizing enzyme hydroxyindole-O-methyltransferase (HIOMT) was examined by light and electron microscopic immunocytochemistry in the human retina. HIOMT-like immunoreactivity was observed in the photoreceptor layers and the inner nuclear layer (INL). The immunoreactive cells in the INL were more numerous in the central retina than in the peripheral retina and sent processes to both the outer plexiform and inner plexiform layers. The HIOMT immunoreactivity in the inner plexiform layer (IPL) appeared as punctate terminals in the proximal and distal one-thirds of that layer. At the ultrastructural level, HIOMT-like immunoreactivity was localized to the cytoplasm of rod and cone photoreceptors and to a population of cone bipolar cells. HIOMT-immunoreactive bipolar cell dendrites were observed to make both invaginating and flat synaptic contacts with cone pedicles. No immunoreactive invaginating contacts in rod spherules were observed. HIOMT immunoreactivity was observed in the bipolar cell cytoplasm in the INL, and in the bipolar synaptic terminals in the IPL. These terminals contained synaptic ribbons, which formed synaptic contacts with unlabeled cells in the IPL. HIOMT radioenzymatic assays confirmed the presence of HIOMT in the human retina. Average HIOMT activity of eight donors was determined to be 15.0 pmol/mg protein/hour +/- 7.2 S.D. The ultrastructural localization of HIOMT observed in this study, combined with reports from other laboratories, suggests that the cytoplasm of the photoreceptors and a population of cone bipolar cells may be the sites of melatonin synthesis in the human retina.     

Off-alpha and OFF-beta ganglion cells in cat retina: II. Neural circuitry as revealed by electron microscopy of HRP stains

An OFF-center alpha and an OFF-center beta ganglion cell in cat retina, which had been recorded from and intracellularly stained with horseradish peroxidase (HRP) were examined by serial section electron microscopy. We counted synapses and identified presynaptic neurons to the HRP-stained cells in 20 μm radial slices through the centers of their dendritic trees. Presynaptic amacrine and bipolar cells were identified on cytological criteria known from previous studies. The OFF-beta cell with a 62 μm dendritic arbor, restricted to S1 and S2 (sublamina a) of the inner plexiform layer (IPL), received 38% bipolar and 62% amacrine cell synapses. The bipolar input was from both cb1 and cb2 cone bipolar types. Input from three distinct amacrine cell types occurred upon the dendrites, namely from: (1) AII amacrine lobular appendages, (2) large pale amacrine profiles (possibly A2 or A3 cells), and (3) small, dark amacrine types (possibly A8 cells). Large pale amacrine profiles (possibly A13) were found on the cell body and apical dendrite in sublamina b of the IPL. In addition, several amacrine profiles synapsed directly on the sides and base of the cell body in the ganglion cell layer. We estimate that the complete dendritic tree of this beta cell received about 1,000 synapses contributed by 12–14 bipolar cells, 7–10 AII amacrines and 28–41 other amacrine cells. The OFF-alpha cell had a dendritic tree size of 680 × 920 μm. A 250 μm length of two major dendrites stratifying narrowly in S2 of the IPL was reconstructed. Amacrine cells provided most of the synaptic input (80%). This input came from: (1) AII amacrine lobular appendages, (2) amacrines exhibiting large, pale synaptic profiles (possibly A2 or A3 cells), (3) pale amacrines with large mitochondria and a few neurotubules (unknown type), and (4) densely neurotubule-filled amacrine profiles (possibly A19 cells). A large pale amacrine cell type (possibly A13) provided synaptic input to the cell body as a serial synaptic intermediary with rod bipolar cells. Cone bipolar synapses were from only one type of cone bipolar, the cb2 type and formed 20% of the total synaptic input. We estimate that a minimum of 142 bipolar cells, 256 AII amacrine cells and 1,011 other amacrine cells, altogether providing 6,000–10,000 synapses, converged on the dendritic tree of this OFF-alpha cell. © 1993 Wiley-Liss, Inc.     

Connections of indoleamine-accumulating cells in the rabbit retina

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