Scanning electron microscopy and light microscopy studies on the retina of chick embryos in cell culture

Histological and scanning electron microscopy studies (SEM) suggest a classification of neural elements into five main types: receptor cells, bipolar cells, horizontal, amacrine, and ganglion cells in dissociated cell cultures of the retina from 7 to 10 days old chick embryos. By use of SEM the development of the receptor cell inner segments was observed. At the same time many protrusions were noticed at the receptor cell surface. Specific synaptic contacts between the axons of the receptor cells and the dendrites of the bipolar neurons, as well as unspecific contacts between the pericarya of the receptor cells and other dendrites were demonstrated. The bipolar neurons showed smooth cell surfaces, however, the horizontal cells appeared rough on the cell surfaces. Müller cells were always found adjacent to the photoreceptor cells.     

Synaptic connections of starburst amacrine cells and localization of acetylcholine receptors in primate retinas

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human alpha3- and alpha5-containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210-immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways.     

Retinal structure in the smooth dogfish Mustelus canis: electron microscopy of serially sectioned bipolar cell synaptic terminals

Portions of axons of bipolar cells in the retina of the smooth dogfish Mustelus canis were sectioned serially and examined by electron microscopy. The studied axons generally could be related to a bipolar cell sub-type identified by light microscopy. Bipolar cell axons make ribbon synapses onto amacrine processes and ganglion cell dendrites, and onto ganglion cell perikarya. Bipolar cell ribbon synaptic complexes varied as to the number of post-synaptic processes (1–3) and the orientation of the ribbon with respect to the post-synaptic membrane. Amacrine processes made numerous conventional synapses onto bipolar cell axons, but reciprocal synapses between amacrine and bipolar cells constituted only 3–25% of all synapses observed. The number of ribbon synapses per unit area of bipolar cell axon membrane differed little among bipolar cell sub-classes. However, the density of amacrine cell conventional synapses was markedly lower for thin, horizontally-oriented bipolar cell axons than for axons of other bipolar cell types. Gap junctions were noted between bipolar cell axons of the same sub-type. They are structurally similar to gap junctions between horizontal cells in Mustelus retina and to those found elsewhere in the nervous system.     

Localization of GABAA receptor subunits alpha 1, alpha 3, beta 1, beta 2/3, gamma 1, and gamma 2 in the salamander retina

Electrophysiological studies have demonstrated that gamma-aminobutyric acid receptors type A (GABA(A)) mediate important information processing in the retinas of salamander and other vertebrates. The pharmacology and physiology of GABA(A) receptors depend on their subunit composition. We studied the localization of GABA(A) receptor subunit isoforms alpha(1), alpha(3), beta(1), beta(2/3) (antibody BD-17 and 62-3G1), gamma(1), and gamma(2) in salamander retina with immunocytochemical methods. All three beta-subunit antibodies labeled similarly in the outer retina, especially the inner segments and synaptic terminals of rod photoreceptors (identified with protein kinase C). Somatic labeling was observed in cell bodies of some horizontal cells, bipolar cells, amacrine cells, and cells in the ganglion cell layer (GCL). Puncta were present throughout the inner plexiform layer (IPL) for beta(1) and 62-3G1, but not for BD-17. alpha(1)-immunoreactivity (IR) stained a population of presumed OFF rod-dominated bipolar cells, including dendrites, soma, and axon terminals in the distal IPL. A subtype of GABAergic amacrine cell also expressed alpha(1)-IR, with puncta sparsely distributed at the distal and proximal margins of the IPL. Both the OPL and IPL were labeled throughout for alpha(3)-IR, as opposed to the narrow distribution of alpha(1)-IR in the IPL, suggesting that the two alpha-subunits are localized at different synaptic sites. Punctate gamma(1)-IR was observed in the OPL and IPL, whereas gamma(2) was most prominent in cone photoreceptors (identified with calbindin), including the terminal telodendria, in cell bodies of some horizontal cells, amacrine cells, cells in the GCL, and less intensely in the IPL. In addition, several subunits were present in Müller cells. The differential labeling suggests the existence of GABA(A) receptor subtypes with different subunit compositions that mediate multiple GABAergic functions in salamander retina.     

Immunocytochemical localization of the glutamate transporter GLT-1 in goldfish (Carassius auratus) retina

Glutamate is the major excitatory neurotransmitter in the retina of vertebrates. Electrophysiological experiments in goldfish and salamander have shown that neuronal glutamate transporters play an important role in the clearance of glutamate from cone synaptic clefts. In this study, the localization of the glutamate transporter GLT-1 has been investigated immunocytochemically at the light and electron microscopical levels in the goldfish retina using a GLT-1-specific antibody. GLT immunoreactivity (IR) was observed at the light microscopical level in Müller cells, bipolar cells, the outer plexiform layer (OPL), and the inner plexiform layer (IPL). At the electron microscopical level, membrane-bound and cytoplasmic GLT-IR in the OPL was located in finger-like protrusions of the cone terminal located near the invaginating postsynaptic processes of bipolar and horizontal cells. GLT-IR was not observed in the vicinity of synaptic ribbons. This location of GLT-1 allows modulation of the glutamate concentration in the synaptic cleft, thereby shaping the dynamics of synaptic transmission between cones and second-order neurons. In the inner IPL, GLT-IR was observed in the cytoplasm and was membrane bound in mixed rod/cone bipolar cell terminals and cone bipolar cell terminals. The membrane-bound GLT-1 was generally observed at some distance from the synaptic ribbon. The morphology of the bipolar cell terminal together with the localization of GLT-1 suggests that at least these glutamate transporters are not primarily involved in rapid uptake of glutamate release by the bipolar cells. The GLT-IR in the cytoplasm of Müller cells was located throughout the entire goldfish retina from the outer limiting membrane to the inner limiting membrane. The location of GLT-1 in Müller cells is consistent with the role of Müller cells in converting glutamate to glutamine.     

Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses

Melanopsin is a novel opsin synthesized in a small subset of retinal ganglion cells. Ganglion cells expressing melanopsin are capable of depolarizing in response to light in the absence of rod or cone input and are thus intrinsically light sensitive. Melanopsin ganglion cells convey information regarding general levels of environmental illumination to the suprachiasmatic nucleus, the intergeniculate leaflet, and the pretectum. Typically, retinal ganglion cells communicate information to central visual structures by receiving input from retinal photoreceptors via bipolar and amacrine cells. Because melanopsin ganglion cells do not require synaptic input to generate light-induced signals, these cells need not receive synapses from other neurons in the retina. In this study, we examined the ultrastructure of melanopsin ganglion cells in the mouse retina to determine the type (if any) of synaptic input these cells receive. Melanopsin immunoreaction product was associated primarily with the plasma membrane of (1) perikarya in the ganglion cell layer, (2) dendritic processes in the inner plexiform layer (IPL), and (3) axons in the optic fiber layer. Melanopsin-immunoreactive dendrites in the inner (ON) region of the IPL were postsynaptic to bipolar and amacrine terminals, whereas melanopsin dendrites stratifying in the outer (OFF) region of the IPL received only amacrine terminals. These observations suggested that rod and/or cone signals may be capable of modifying the intrinsic light response in melanopsin-expressing retinal ganglion cells.     

Localization of neuropeptide Y1 receptor immunoreactivity in the rat retina and the synaptic connectivity of Y1 immunoreactive cells

Neuropeptide Y (NPY), an inhibitory neuropeptide expressed by a moderately dense population of wide-field amacrine cells in the rat retina, acts through multiple (Y1-y6) G-protein-coupled receptors. This study determined the cellular localization of Y1 receptors and the synaptic connectivity of Y1 processes in the inner plexiform layer (IPL) of the rat retina. Specific Y1 immunoreactivity was localized to horizontal cell bodies in the distal inner nuclear layer and their processes in the outer plexiform layer. Immunoreactivity was also prominent in cell processes located in strata 2 and 4, and puncta in strata 4 and 5 of the IPL. Double-label immunohistochemical experiments with calbindin, a horizontal cell marker, confirmed Y1 immunostaining in all horizontal cells. Double-label immunohistochemical experiments, using antibodies to choline acetyltransferase and vesicular acetylcholine transporter to label cholinergic amacrine cell processes, demonstrated that Y1 immunoreactivity in strata 2 and 4 of the IPL was localized to cholinergic amacrine cell processes. Electron microscopic studies of the inner retina showed that Y1-immunostained amacrine cell processes and puncta received synaptic inputs from unlabeled amacrine cell processes (65.2%) and bipolar cell axon terminals (34.8%). Y1-immunoreactive amacrine cell processes most frequently formed synaptic outputs onto unlabeled amacrine cell processes (34.0%) and ganglion cell dendrites (54.1%). NPY immunoreactivity in the rat retina is distributed primarily to strata 1 and 5 of the IPL, and the present findings, thus, suggest that NPY acts in a paracrine manner on Y1 receptors to influence both horizontal and amacrine cells.     

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.     

The spatial relationship between Müller cell processes and the photoreceptor output synapse.

Glutamate is the neurotransmitter released by photoreceptors in the retina. The postsynaptic action of glutamate is terminated partly by uptake into glial (Müller) cells. The anatomical distribution of Müller cell processes around the synaptic terminals of photoreceptors was investigated electron microscopically in the tiger salamander retina. Müller cells wrap around the synaptic terminals of both rods and cones and come within 1-3 microns of the sites of glutamate release, close enough to contribute to terminating the synaptic action of glutamate.     

Development of the rhesus monkey retina: II. A three-dimensional analysis of the sequences of synaptic combinations in the inner plexiform layer

The inner plexiform layer (IPL) of the retina provides a useful model for ultrastructural analysis of synaptic development. In primates, the IPL consists of numerous combinations of neuronal contacts that assume the morphological configuration of either conventional or ribbon synapses. We have determined the sequential development of these combinations by analyzing serial electron microscopic sections from fetal rhesus monkeys. Our analysis reveals an orderly emergence of various pre- and postsynaptic elements: (1) patches of dense filamentous membrane first appear on the dendrites of ganglion (G) cells; (2) membrane densities on ganglion cell dendrites then become apposed to amacrine (A) cell processes still lacking their own membrane densities and synaptic vesicles; (3) amacrine cell processes acquire membrane specializations associated with vesicles at the sites apposing ganglion cell dendrites, thereby establishing the first morphologically complete, A----G subtype of conventional synapse; (4) pairs of amacrine cell processes form A----A subtypes of conventional synapses; (5) next, monad ribbon synapses are established between bipolar (B) and ganglion or amacrine cell processes (B----G; B----A); (6) the three subclasses of dyad ribbon synapses (B----GG; B----GA; B----AA) are subsequently formed by the introduction of additional amacrine or ganglion cell processes in the dyad synapse; (7) concurrently, processes of some amacrine and interplexiform (I) cells form a feedback circuit with bipolar cell axons (A----B; I----B), thereby completing the synaptic microcircuitry of the IPL. Present findings provide evidence that the sequence of synaptic differentiation in the IPL proceeds from the postsynaptic to the presynaptic site. Furthermore, lateral interactions (A----G and A----A) are established prior to the formation of the "straight signal" pathway from photoreceptors (P) via bipolar cells to ganglion cells (P----B----G). Observed developmental events provide new insight into the order of establishment of local neuronal circuits in the primate retina.     

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.     

Dopamine D1-receptor immunolocalization in goldfish retina.

Dopamine, a neuromodulator in the vertebrate retina, is involved in numerous functions related to light adaptation. However, unlike in mammals, localization of retinal D1-dopamine receptors in nonmammalian vertebrates has been hampered due to a lack of antisera. To address this problem, an antiserum against the 18 C-terminal amino acids of the goldfish D1 receptor (gfD1r) was generated in chicken eggs and tested in retinae of goldfish and rat, and rat caudate putamen, by using immunoblots and light microscopic immunocytochemistry. No labeling was observed in any tissue or immunoblots with preabsorbed gfD1r antiserum. Immunoblot analysis of goldfish retina revealed a single band at about 101 kDa. The patterns of gfD1r immunoreactivity (gfD1r-IR), found in rat caudate putamen and rat retina were virtually identical to that previously reported with other D1-receptor ligands and antisera. In goldfish retina, gfD1r-IR was most intense over cell bodies in the ganglion cell layer, amacrine cells in the proximal inner nuclear layer (INL), and bipolar cells in the distal INL. Weaker gfD1r-IR was observed over horizontal cell bodies and both plexiform layers. Müller cells and axons of cone photoreceptors were labeled as well. Double labeling showed that all protein kinase C-immunoreactive bipolar cells (ON type) were gfD1r-IR on the soma, axon terminal, and dendrites. All glutamate decarboxylase-immunoreactive (i.e., gamma-aminobutyric acid utilizing) amacrine cells and horizontal cells were gfD1r-IR. Retinal D1r distribution is more extensive than dopamine neuron innervation, but is consistent with physiologic estimates of dopamine function, suggestive of both wiring and volume transmission of dopamine in the retina. The gfD1r antiserum displays cross-reactivity to dopamine receptors in a mammal and a nonmammal and should prove useful in future studies of dopaminergic systems.     

Structure of the macroglia of the retina: sharing and division of labour between astrocytes and Müller cells.

A detailed comparison is made between astrocytes and Müller cells of the cat's retina, with emphasis on their structural specialisations. Evidence is presented that astrocytes and Müller cells both contribute to the formation of the inner glia limitans of the retina, the glia limitans of vessels, and the glial sheaths of neurones. In particular, it was noted that both astrocytes and Müller cells wrap bundles of ganglion cells axons, that both contribute processes to the glial convergence on the initial segments and node-like structures of axons, and that both wrap the somas of neurones in the ganglion cell layer. Further, it was noted that adherent junctions form between astrocytes, between Müller cells, and between astrocytes and Müller cells, but not between these cells and neurones, or among neurones. These similarities suggest that astrocytes and Müller cells function interchangeably in many respects, and we suggest that they be regarded as variants of macroglia. Quantitative differences between astrocytes and Müller cells were noted in their ensheathment of neurones. In particular, the glial sheaths around the somas of ganglion cells are formed predominantly by Müller cells, and the glial processes attached to node-like specialisations of their axons are formed mainly by astrocytes. One qualitative difference was noted between the two cell classes. The gap junctions which form between astrocytes do not form between Müller cells or between cells of the two classes. From these differences, and previously established features of their shape, orientation, distribution and origin, a hypothesis is developed of the specialisation of macroglia represented by Müller cells.     

Spatial and temporal patterns of proliferation and differentiation in the developing turtle eye

Here we show for the first time different aspects of the pattern of neurogenesis in the developing turtle retina by using different morphological and molecular clues. We show the chronotopographical fashion of occurrence of three major aspects of retinal development: (1) morphogenesis of the optic primordia and emergence of the different retinal layers, (2) the temporal progression of neurogenesis by the cessation of proliferative activity, and (3) the apparition and cellular localization of different antigens and neuroactive substances. Retinal cells were generated in a conserved temporal order with ganglion cells born first, followed by amacrine, photoreceptor, horizontal and bipolar/Müller cells. While eventually expressed in many types of retinal neurons, Islet1 was permanently expressed in differentiating and mature ganglion cells. Calbindin-immunoreactive elements were found in the ganglion cell layer and the inner nuclear layer. Interestingly, at later stages the amount of expressing cells in these layers was reduced dramatically. On the contrary, the number of calbindin-immunoreactive photoreceptors increased as development proceeded. In addition, calretinin expressing cells were prominent in the horizontal cell bodies, and their processes extending into the outer plexiform layer were also strongly labeled. Finally, the synthesis of gamma-aminobutyric acid (GABA) was detected in developing and matured horizontal and amacrine cells. All these maturational features began in the dorso-central area, in a region slightly displaced towards the temporal retina.     

Patterns of glutamate immunoreactivity in the goldfish retina

Postembedding silver-intensified immunogold procedures reveal high levels of glutamate immunoreactivity in "vertical" elements of the goldfish retina: (1) Red-sensitive and green-sensitive cones display strong glutamate immunoreactivity, especially in their synaptic terminals, but blue-sensitive cones are poorly immunoreactive. (2) All type Mb (on-center) and Ma (off-center) mixed rod-cone bipolar cells and all identifiable cone bipolar cells are highly glutamate immunoreactive. We find no evidence for bipolar cells that lack glutamate immunoreactivity. (3) The majority of the somas in the ganglion cell layer and certain large cells of the amacrine cell layer resembling displaced ganglion cells are strongly glutamate immunoreactive. (4) Despite their high affinity symport of acidic amino acids, the endogenous levels of glutamate in Müller's cells are among the lowest in the retina. (5) GABAergic neurons possess intermediate levels of glutamate immunoreactivity. Quantitative immunocytochemistry coupled with digital image analysis allows estimates of intracellular glutamate levels. Photoreceptors and bipolar and ganglion cells contain from 1 to 10 mM glutamate. The bipolar and ganglion cell populations maintain high intracellular glutamate concentrations, averaging about 5 mM, whereas red-sensitive and green-sensitive cones apparently maintain lower levels. Importantly, photoreceptor glutamate levels are extremely volatile, and in vitro maintenance is required to preserve cone glutamate immunoreactivity in the goldfish. GABAergic horizontal and amacrine cells contain about 0.3-0.7 mM glutamate, which matches the values predicted from the Km of glutamic acid decarboxylase. Müller's cells and non-GABAergic amacrine cells contain less than 0.1 mM glutamate. Though Müller's cells are known to possess potent glutamate symport, they clearly possess equally potent mechanisms for maintaining low intracellular glutamate concentrations.     

Localization of immunoreactive cholecystokinin precursor to amacrine cells and bipolar cells of the macaque monkey retina

We used antisera that recognized precursors of the neuropeptide cholecystokinin extended at the carboxyl terminus in an immunocytochemical study of the macaque retina. A subpopulation of bipolar cells with long, obliquely oriented dendrites was labeled. Their axons terminated exclusively in the fifth stratum of the inner plexiform layer, where they contacted processes of amacrine and ganglion cells. Based on their morphology, these cells appeared to be the type that contacts short-wavelength cones selectively. Two types of amacrine cells were also labeled, and processes from both types formed dense plexuses in the second and fourth strata of the inner plexiform layer. The majority of their synaptic connections were with other amacrine cells, but they had more contacts with bipolar cell axons and retinal ganglion cell dendrites than any other peptidergic cells in the macaque retina. We studied extracts of macaque retina with gel-filtration chromatography and radioimmunoassays to confirm our immunohistochemical results. We found cholecystokinin octapeptide and other immunoreactive forms that were amidated at their carboxyl termini and were therefore likely to be biologically active. Unlike most other regions of the CNS, however, the retina had relatively low concentrations of amidated forms, and forms with extended carboxyl termini that are presumably their precursors were far more abundant. These findings suggest that the rate of cholecystokinin synthesis in the retina is quite high, as we would expect if the peptide were found in tonically active neurons.     

Synaptic connections of DB3 diffuse bipolar cell axons in macaque retina

In primate retinas, the dendrites of DB3 diffuse bipolar cells are known to receive inputs from cones. The goal of this study was to describe the synaptic connections of DB3 bipolar cell axons in the inner plexiform layer. DB3 bipolar cells in midperipheral retina were labeled with antibodies to calbindin, and their axons were analyzed in serial, ultrathin sections by electron microscopy. Synapses were found almost exclusively at the axonal varicosities of DB3 axon terminals. There were 2.14 synaptic ribbons per varicosity. There were 33 varicosities per DB3 cell, giving an average of 71 ribbons per axon terminal. Because there were 1.5 postsynaptic ganglion cell dendrites per DB3 axonal varicosity, we estimate that there is at least 1 synapse per varicosity onto a parasol ganglion cell dendrite. There were 3.4 input synapses from amacrine cells per axonal varicosity. Among these were feedback synapses to the DB3 bipolar cell axon varicosities, which were made by 47% of the postsynaptic amacrine cell processes. Some of the feedback synapses could be from amacrine cells immunoreactive for cholecystokinin precursor or choline acetyltransferase, because both types of amacrine cells costratify with parasol cells and are known to be presynaptic to bipolar cells. AII amacrine cells were both presynaptic and postsynaptic to DB3 axons, a finding consistent with the large rod input to parasol ganglion cells reported in physiological experiments. DB3 bipolar cell axons also made frequent contacts with neighboring DB3 axons, and gap junctions were always found at these sites.     

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.     

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.     

Electron microscopic analysis of amacrine and bipolar cell inputs on Y-, X- and W-cells in the cat retina

In the cat retina, bipolar and amacrine cell inputs were analyzed electron microscopically in 5 ganglion cells (two Y-cells, two X-cells and one W-cell) that were well-isolated and had clear morphological features. For Y- and X-cells, subtypes of a and b were further identified according to the sublamina of the inner plexiform layer in which their dendrites extended. Y-a and Y-b ganglion cells had large somas, thick axons, and several thick dendrites that branched extensively with a large dendritic field. X-a and X-b cells had medium-sized somas, medium-sized axons and extremely narrow dendritic fields. The W-cell studied had a medium-sized soma, a medium-sized axon, and extremely thin dendrites that extended widely. For each of the 5 ganglion cells, ultrathin serial sections were made to study relative occurrence of amacrine and bipolar synapses in whole length of dendrites. About 50% of the terminals were bipolar in the Y-a and Y-b cell dendrites, 36-38% in the X-a and X-b cell dendrites, whereas only 19.7% were bipolar in the W cell dendrites. Bipolar terminals tended to make synaptic contacts with the distal dendrites of Y- and W-cells.