Connections of diffuse bipolar cells in primate retina are biased against S-cones

In mammalian retina, each diffuse bipolar type stratifies in a distinct layer of the inner plexiform layer. Thus, different types of bipolar cells provide output to distinct visual pathways. Here, the question of whether diffuse bipolar cell types differ with respect to their contacts with short wavelength-sensitive (S-) cones was investigated in the retinas of a New World monkey, Callithrix jacchus, and an Old World monkey, Macaca fascicularis. Subpopulations of OFF bipolar cells were labeled with antibodies to the glutamate transporter Glt-1 and ON bipolar cells were labeled with antibodies to the alpha subunit of the Go protein (Goalpha). Two types of diffuse ON bipolar cells, DB4 and DB6, were identified with antibodies to protein kinase Calpha and CD15, respectively. Cone pedicles were labeled either with peanut agglutinin coupled to fluorescein or with antibodies to the ribbon protein, C-terminus binding protein 2. We found that immunoreactivity for Glt-1 (OFF bipolar cells) is reduced at S-cones in comparison to medium/long wavelength-sensitive (M/L-) cones. Immunoreactivity for Goalpha (ON bipolar cells) is comparable at all cone types. Nearly all M/L-cone pedicles contact the diffuse ON bipolar types DB4 and DB6, but only between 60% and 75% of the S-cone pedicles make contact. Furthermore, the number of dendritic tips of DB4 and DB6 cells at S-cone pedicles is lower than that at M/L-cone pedicles. These results suggest that there is a bias in the S-cone connectivity of diffuse bipolar cells.     

Synaptic inputs onto small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmoset retina

The inner plexiform layer of the retina contains functional subdivisions, which segregate ON and OFF type light responses. Here, we studied quantitatively the ON and OFF synaptic input to small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmosets (Callithrix jacchus). Small bistratified cells display an extensive inner dendritic tier that receives blue‐ON input from short‐wavelength‐sensitive (S) cones via blue cone bipolar cells. The outer dendritic tier is sparse and is thought to receive yellow‐OFF input from medium (M)‐ and long (L)‐wavelength‐sensitive cones via OFF diffuse bipolar cells. In total, 14 small bistratified cells from different eccentricities were analyzed. The cells were retrogradely labeled from the koniocellular layers of the lateral geniculate nucleus and subsequently photofilled. Retinal preparations were processed with antibodies against the C‐terminal binding protein 2, the AMPA receptor subunit GluR4, and/or gephyrin to identify bipolar and/or amacrine input. The results show that the synaptic input is evenly distributed across the dendritic tree, with a density similar to that reported previously for other ganglion cell types. The population of cells showed a consistent pattern, where bipolar input to the inner tier is about fourfold greater than bipolar input to the outer tier. This structural asymmetry of bipolar input may help to balance the weight of cone signals from the sparse S cone array against inputs from the much denser M/L cone array. J. Comp. Neurol. 517:655–669, 2009. © 2009 Wiley‐Liss, Inc.     

Distribution of glycine receptor subunits on primate retinal ganglion cells: a quantitative analysis

This study investigates the distribution of inhibitory neurotransmitter receptors on sensory neurons. Ganglion cells in the retina of a New World monkey, the common marmoset Callithrix jacchus, were injected with Lucifer yellow and Neurobiotin and subsequently processed with antibodies against one (alpha1), or against all subunits, of the glycine receptor, or against the anchoring protein gephyrin. Immunoreactive (IR) puncta representing glycine receptor or gephyrin clusters were found on the proximal and the distal dendrites of all ganglion cell types investigated. For both parasol and midget cells, the density of receptor clusters was greater on distal than proximal dendrites for all antibodies tested. In parasol cells the average density for the alpha1 subunit of the glycine receptor was 0.087 IR puncta/microm of dendrite, and for all subunits it was 0.119 IR puncta/microm of dendrite. Thus, the majority of glycine receptors on parasol cells contain the alpha1 subunit. For parasol cells, we estimated an average of 1.5 glycinergic synapses/100 microm2 dendritic membrane on proximal dendrites and about 9.4 glycinergic synapses/100 microm2 on distal dendrites. The segregation of receptors to the distal dendrites appears to be a common feature of inhibitory neurotransmitter input to parasol and midget cells, and might be associated with the receptive field surround mechanism.     

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.     

Characterization of secretagogin‐immunoreactive amacrine cells in marmoset retina

The retina contains at least 30 different types of amacrine cells but not many are well characterized. In the present study the calcium‐binding protein secretagogin was localized in a population of regular and displaced amacrine cells in the retina of the common marmoset Callithrix jacchus. Irrespective of their soma location, the dendrites of secretagogin amacrine cells occupy strata 2, 3, and 4 of the inner plexiform layer, between the two bands formed by cholinergic amacrine cells. Segretagogin amacrine cells are also immunopositive to antibodies against glutamic acid decarboxylase, suggesting that they use γ‐aminobutyric acid (GABA) as their neurotransmitter. The spatial density of secretagogin amacrine cells decreases from a peak of about 400 cells/mm² near 1 mm eccentricity to less than 100 cells/mm² in peripheral retina; these densities account for about 1% of amacrine cells in the inner nuclear layer and for up to 27% of displaced amacrine cells. The cell bodies form a regular mosaic, suggesting that they constitute a single amacrine cell population. Secretagogin cells have varicose dendrites, which are decorated with small spines. Intracellular injection of DiI into secretagogin cells revealed an average dendritic field diameter of 170 μm and an average coverage factor of 3.2. In summary, secretagogin cells in marmoset retina are medium‐field amacrine cells that share their stratification pattern with narrow‐field amacrine cells and their neurotransmitter with wide‐field amacrine cells. They may mediate spatial inhibition spanning the centralmost (on and off) bands of the inner plexiform layer. J. Comp. Neurol. 522:435–455, 2014. © 2013 Wiley Periodicals, 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.     

Depth segregation of retinal ganglion cells projecting to mouse superior colliculus

The retinotectal projections in the mouse were analyzed with injections of horseradish peroxidase into the superior colliculus and of radioactive amino acids into the eye. At least 70% of the ganglion cells, and possibly all of them, were found to project to the superior colliculus, including ganglion cells of all sizes. Small injections revealed that ganglion cells of different sizes terminate at different levels in the superior colliculus. The small ganglion cells that form the vast majority of all cells project predominantly to the upper stratum griseum superficiale. A small population of mainly medium-sized and large ganglion cells project to the deep stratum griseum superficiale and to the stratum opticum. The ipsilateral projection is restricted to the deep stratum griseum superficiale and stratum opticum and consists predominantly of medium-sized and large ganglion cells.     

Isolation of ganglion cells from the retina

A microdissection technique was developed to isolate cellular layers of the goldfish retina. Three regions of the neural retina, the photoreceptor-interneuron layer (PL), the vascular layer (VL), and the ganglion cell layer (GCL) were recovered. A cleavage plane between the PL and GCL occurred at the level of the inner plexiform region as shown by light microscopy. Scanning electron microscopy found the predominant cells of the PL to include rods and cones while the VL contained a capillary network that arose from the retinal artery. The GCL consisted of pear-shaped ganglion cells about 5 μm in size with single, unbranched axons with diameters of about 0.5 μm. This preparation was virtually free of other cell types or debris. Transmission electron microscopy demonstrated that axons of the GCL were unmyelinated in contrast to those fibers distal to the optic nerve head. On the basis of morphology, microdissection could isolate a single population of retinal neurons free of glia.Biochemical indicators were used to determine the degree of separation of the GCL from the PL and VL. Markers of the cholinergic system, acetylcholinesterase and α-bungarotoxin binding, predominate only within the PL. These findings are consistent with known histochemical features of the retina and suggest that the GCL is not contaminated by the neighboring photoreceptor-interneuron region. Ganglion cell axons distal to the optic nerve head are myelinated and contain high levels of the marker enzyme 2′,3′-cyclic nucleotide-3′-phosphodiesterase. In contrast, this enzymatic activity is not detected within the unmyelinated axons of the glia free GCL preparation.To examine retinal events during axonal regeneration, transection of ganglion cell axons was carried out at the level of the optic tract. Axotomy led to stimulated amino acid incorporation detected within the GCL and not other regions of the retina. This specific response of the GCL was observed both in vivo and in vitro indicating that isolated ganglion cells remain intact and biologically active. Microdissection provides, therefore, a population of retinal ganglion cells that may be used to analyze the biochemical events of neuronal regeneration.     

Melanopsin-expressing ganglion cells in human retina: Morphology,distribution, and synaptic connections

Melanopsin-expressing retinal ganglion cells are intrinsically photosensitive cells that are involved in non-image forming visual processes such as the pupillary light reflex and circadian entrainment but also contribute to visual perception. Here we used immunohistochemistry to study the morphology, density, distribution, and synaptic connectivity of melanopsin-expressing ganglion cells in four post mortem human donor retinas. Two types of melanopsin-expressing ganglion cells were distinguished based on their dendritic stratification near either the outer or the inner border of the inner plexiform layer. Outer stratifying cells make up on average 60% of the melanopsin-expressing cells. About half of the melanopsin-expressing cells (or 80% of the outer stratifying cells) have their soma displaced to the inner nuclear layer. Inner stratifying cells have their soma exclusively in the ganglion cell layer and include a small proportion of bistratified cells. The dendritic field diameter of melanopsin-expressing cells ranges from 250 (near the fovea) to 1,000 µm in peripheral retina. The dendritic trees of outer stratifying cells cover the retina independent of soma location. The dendritic fields of both outer and inner stratifying cells show a high degree of overlap with a coverage factor of approximately two. Melanopsin-expressing cells occur at an average peak density of between ∼20 and ∼40 cells/mm² at about 2 mm eccentricity, the density drops to below ∼10 cells/mm² at about 8 mm eccentricity. Both the outer and inner stratifying dendrites express postsynaptic density (PSD95) immunoreactive puncta suggesting that they receive synaptic input from bipolar cells.     

Immunocytochemical identification and analysis of the diffuse bipolar cell type DB6 in macaque monkey retina

The distribution and morphology of CD15‐immunoreactive bipolar cells were studied in the retina of macaque monkey. Labelled cells have a large dendritic tree contacting several cones and a narrowly stratified axon terminal that ends deep in the inner plexiform layer, close to the ganglion cell layer. The morphology of the labelled cells corresponds to that of the diffuse bipolar cell type named DB6 by Boycott & Wässle (1991; Eur. J. Neurosci., 3 ,1069). We conclude that CD15 is a marker for DB6 bipolar cells, enabling the quantitative analysis of the distribution and connectivity of this diffuse bipolar cell type.     

Retinal ganglion cells projecting to the accessory optic system in the rat

The present data identify the distribution and morphological features of a homogeneous group of rat retinal ganglion cells. These cells were labelled after injection of either horseradish peroxidase or a fluorescent tracer, Fast Blue, into the medial terminal nucleus (MTN) of the accessory optic system. After retrograde fluorescent labelling, MTN-projecting retinal ganglion cells were intracellularly injected with Lucifer Yellow to reveal their complete dendritic morphology. There were on average 1,750 MTN-projecting cells fairly evenly distributed over the entire retinal ganglion cell layer. Their density ranged from 40-49 cells/mm2 in superior retina to 10-19 cells/mm2 towards the peripheral regions of both inferior and superior retina. The area of highest density formed a nasal-temporal band suggestive of a visual streak. Soma diameters ranged from 8.7 to 14.5 micron centrally and from 9.9 to 17.1 microns peripherally. Maximal dendritic field diameter ranged from 431 to 644 micron and averaged 516 micron with no obvious eccentricity dependence. The majority of MTN-projecting cells were bistratified. Dendrites stratified predominantly in the inner sublamina of the inner plexiform layer (IPL) with a varying number of branches from the remaining dendrites contained within the outer IPL, both strata presumably corresponding to the electrophysiologically determined on-off dichotomy. Cells projecting to the MTN were characterised by higher-order dendritic branching patterns that resulted in a dense dendritic tree with minor dendritic overlap. The slender dendrites had a beaded appearance and displayed spiny protrusions. The dendritic coverage of 5-6, stratification pattern, and overall morphological appearance of rat MTN-projecting cells renders them suitable candidates for on-direction--selective cells shown electrophysiologically to be linked with the MTN of the accessory optic system.     

Synaptic contacts between an identified type of ON cone bipolar cell and ganglion cells in the mouse retina

We surveyed the potential contacts between an identified type of bipolar cell and retinal ganglion cells in the mouse. By crossing two existing mouse strains (line 357 and line GFP-M), we created a double transgenic strain in which GFP is expressed by all members of a single type of ON cone bipolar cell and a sparse, mixed population of retinal ganglion cells. The GFP-expressing bipolar cells appear to be those termed CB4a of Pignatelli & Strettoi [(2004) J. Comp. Neurol., 476, 254-266] and type 7 of Ghosh et al. [(2004) J. Comp. Neurol., 469, 70-82 and J. Comp. Neurol., 476, 202-203]. The labelled ganglion cells include examples of most or all types of ganglion cells present in the mouse. By studying the juxtaposition of their processes in three dimensions, we could learn which ganglion cell types are potential synaptic targets of the line 357 bipolar cell. Of 12 ganglion cell types observed, 10 types could be definitively ruled out as major synaptic targets of the line 357 bipolar cells. One type of monostratified ganglion cell and one bistratified cell tightly cofasciculate with axon terminals of the line 357 bipolar cells. Double labelling for kinesin II demonstrates colocalization of bipolar cell ribbons at the sites of contact between these two types of ganglion cell and the line 357 bipolar cells.     

Synaptic inputs to two types of koniocellular pathway ganglion cells in marmoset retina

The retinal connectivity of the diverse group of cells contributing to koniocellular visual pathways (widefield ganglion cells) is largely unexplored. Here we examined the synaptic inputs onto two koniocellular-projecting ganglion cell types named large sparse and broad thorny cells. Ganglion cells were labeled by retrograde tracer injections targeted to koniocellular layer K3 in the lateral geniculate nucleus in marmosets (Callithrix jacchus) and subsequently photofilled. Retinal preparations were processed with antibodies against the C-terminal binding protein 2, the AMPA receptor subunit GluR4, and against CD15 to identify bipolar (excitatory) and/or antibodies against gephyrin to identify amacrine (inhibitory) input. Large sparse cells are narrowly stratified close to the ganglion cell layer. Broad thorny ganglion cells are broadly stratified in the center of the inner plexiform layer. Bipolar input to large sparse cells derives from DB6 and maybe other ON bipolar types, whereas that to broad thorny cells derives from ON and OFF bipolar cell types. The total number of putative synapses on broad thorny cells is higher than the number on large sparse cells but the density of inputs (between 2 and 5 synapses per 100 μm(2) dendritic area) is similar for the two cell types, indicating that the larger number of synapses on broad thorny cells is attributable to the larger membrane surface area of this cell type. Synaptic input density is comparable to previous values for midget-parvocellular and parasol-magnocellular pathway cells. This suggests functional differences between koniocellular, parvocellular, and magnocellular pathways do not arise from variation in synaptic input densities.     

A monoclonal antibody specific for retinal ganglion cells of mammals

The development of specific markers for retinal ganglion cells is an area of great interest in retinal research. In this study we report on a monoclonal antibody (AB5) which specifically labels ganglion cells in rabbit, cat and monkey, as well as a variety of other mammalian species. Labelling of ganglion cells was also observed in isolated cell preparations of rabbit retina.