Shifting of parvalbumin expression in the rat retina in experimentally induced diabetes

The AII amacrine cell, a unique rod signal integrator passing through the cone bipolar cell to ganglion cells, uses parvalbumin as a transducer of cytosolic calcium ion signals in the mammalian retina. For clarification of whether AII amacrine cell network contributes to the early neuropathogenesis of diabetic retinopathy, this study first analyzed alteration of parvalbumin expression in experimental diabetic retinas using immunohistochemical methods. Parvalbumin immunoreactivity was found in AII amacrine cells, some amacrine cells of a wide-field type, and displaced amacrine cells of the normal rat retina. During diabetes, cell density of each parvalbumin immunoreactive amacrine cell type showed no large changes despite decrease in immunoreactivity especially in AII amacrine cells. In addition to these parvalbumin immunoreactive amacrine cell types, a type of cone bipolar cells co-expressing glutamate transporter 1b and connecting electrically with AII amacrine cells appeared clearly by 4 weeks of diabetes, and thereafter sharply increased in number to that of AII amacrine cells. Protein levels of parvalbumin throughout the diabetic retinas also showed no large changes, except a transitional slight increase at 4 weeks of diabetes. These results suggest that the parvalbumin expression propagates from AII amacrine cells to a type of cone bipolar cell through electrical synapses due to dysfunction of biased mechanism in calcium ion buffering, caused by diabetic injury, and thus AII amacrine cells are closely involved in neuropathogenesis of ongoing diabetic retinopathy.     

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.     

Identification of a cone bipolar cell in cat retina which has input from both rod and cone photoreceptors

It has been generally accepted that rod photoreceptor cells in the mammalian retina make synaptic contact with only a single population of rod bipolar cells, whereas cone photoreceptors contact a variety of cone bipolar cells. This assumption has been challenged in rodents by reports of a type of cone bipolar cell which receives input from both rods and cones. Questions remained as to whether similar pathways are present in other mammals. We have used an antiserum against the glutamate transporter GLT1-B to visualize a population of cone bipolar cells in the cat retina which make flat contacts with axon terminals of both rod and cone photoreceptor cells. These cells are identified as OFF-cone bipolar cells and correspond morphologically to type cb1 (CBa2) cone bipolar cells which are a major source of input to OFF-beta ganglion cells in the cat retina. The GLT1-B transporter was also localized to processes making flat contacts with photoreceptor terminals in rat and rabbit retinas. Examination of tissue processed for the GluR1 glutamate receptor subunit showed that cb1 cone bipolar cells, like their rodent counterparts, express this alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-selective receptor at their contacts with rod spherules. Thus, a direct excitatory pathway from rod photoreceptors to OFF-cone bipolar cells appears to be a common feature of mammalian retinas.     

Localization of GABA, glycine, glutamate and tyrosine hydroxylase in the human retina.

A light microscope study using postembedding immunocytochemistry techniques to demonstrate the common neurotransmitter candidates gamma-aminobutyric acid (GABA), glycine, glutamate, and tyrosine hydroxylase for dopamine has been done on human retina. By using an antiserum to GABA, we found GABA-immunoreactivity (GABA-IR) to be primarily in amacrine cells lying in the inner nuclear layer (INL) or displaced to the ganglion cell layer (GCL). A few stained cells in the INL, which are probably interplexiform cells, were observed to project thin processes towards the outer plexiform layer (OPL). There were heavily stained bands of immunoreactivity in strata 1, 3 and 5 of the inner plexiform layer (IPL). An occasional ganglion cell was also GABA-IR. By using an antiserum to glycine, stained cells were observed at all levels of the INL. Most of these were amacrines, but a few bipolar cells were also glycine-IR. Displaced amacrine cells and large-bodied cells, which are probably ganglion cells, stained in the GCL. The bipolar cells that stained appeared to include both diffuse and midget varieties. The AII amacrine cell of the rod pathway was clearly stained in our material but at a lower intensity than two other amacrine cell types tentatively identified as A8 and A3 or A4. Again, there was stratified staining in the IPL, with strata 2 and 4 being most immunoreactive. An antiserum to glutamate revealed that most of the neurons of the vertical pathways in the human retina were glutamate-IR. Rod and cone photoreceptor synaptic endings labeled as did the majority of bipolar and ganglion cells. The rod photoreceptor stained more heavily than the cone photoreceptor in our material. While both midget and diffuse cone bipolar cell types were clearly glutamate-IR, rod bipolars were not noticeably stained. The most strongly staining glutamate-IR processes of the IPL lay in the outer half, in sublamina a. The antiserum to tyrosine hydroxylase (TOH) revealed two different amacrine cell types. Strongly immunoreactive cells (TOH1) had their cell bodies in the INL and their dendrites ramified in a dense plexus in stratum 1 of the IPL. Fine processes arising from their cell bodies or from the stratum 1 plexus passed through the INL to reach the OPL but did not produce long-ranging ramifications therein. The less immunoreactive amacrines (TOH2) lay in the INL, the center of the IPL or the GCL and emitted thick dendrites that were monostratified in stratum 3 of the IPL.     

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.     

Spatial density and immunoreactivity of bipolar cells in the macaque monkey retina.

The anatomical substrates of spatial and color vision in the primate retina are investigated by measuring the immunoreactivity and spatial density of bipolar, amacrine and horizontal cells in the inner nuclear layer of the macaque monkey retina. Bipolar cells can be distinguished from amacrine and horizontal cells by their differential immunoreactivity to antisera against glutamate, glycine, GABA, parvalbumin, calbindin (CaBP D-28K), and the L7 protein from mouse cerebellum. The spatial density of bipolar cells is compared to the densities of photoreceptors and ganglion cells at different retinal eccentricities. In the centralmost 2 mm, cone bipolar cells outnumber ganglion cells by about 1.4:1. The density of cone bipolar cells is thus high enough to allow for input to different (parasol and midget) ganglion cell classes by different (diffuse and midget) bipolar cell classes. The density gradient of cone bipolar cells follows closely that of ganglion cells in central retina but falls less steeply in peripheral retina. This suggests that the convergence of cone signals to the receptive fields of ganglion cells in the peripheral retina occurs in the inner plexiform layer. The density of cone bipolar cells is 2.5-4 times that of cones at all eccentricities studied, implying that cone connectivity to bipolar cells remains constant throughout the retina. Different subgroups of bipolar cells are distinguished by their relative immunoreactivity to the different antisera. All rod and cone bipolar cells show moderate to strong glutamate-like immunoreactivity. The bipolar cells that show weak to moderate GABA-like immunoreactivity are also labeled with the antiserum to the L7 protein and are thus identified as rod bipolar cells. Nearly half of all cone bipolar cells showed glycine-like immunoreactivity. The results suggest that the inhibitory neurotransmitter candidates GABA and glycine are segregated respectively in rod and cone bipolar cell pathways. A diffuse, cone bipolar cell type can be identified by the anti-parvalbumin and the anti-calbindin antisera. All horizontal cells show parvalbumin-like immunoreactivity. Nearly all amacrine cells show GABA-like or glycine-like immunoreactivity; a variety of subpopulations also show immunoreactivity to one or more of the other markers used.     

Agonist-stimulated cobalt uptake provides selective visualization of neurons expressing AMPA- or kainate-type glutamate receptors in the retina

Fast-acting excitatory neurotransmission in the retina is mediated primarily by glutamate, acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) -selective and kainate-selective receptors. To localize these sites of action, cat retinas were stimulated with either AMPA or kainate and processed for histochemical visualization of cobalt uptake through calcium-permeable channels. Treatment with both agonists resulted in staining of A- and B-type horizontal cells and several types of OFF cone bipolar cells; there was no evidence for staining of ON cone bipolar cells or rod bipolar cells. The subpopulations of OFF cone bipolar cells differed in their responses with two distinct types that stained heavily with cobalt after exposure to AMPA and three different types that were preferentially labeled after exposure to kainate. Although many amacrine and ganglion cells appeared to respond to both agonists, AII amacrine cells were stained after stimulation by AMPA but not by kainate. The OFF cone bipolar cells that exhibit AMPA-stimulated cobalt uptake were found to have a high level of correspondence with cells that show immunocytochemical staining for the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3. Similarly, the cone bipolar cells exhibiting kainate-stimulated cobalt uptake resemble those that are immunoreactive for the kainate subunit GluR5. The results indicate that, whereas many retinal neurons express both AMPA and kainate receptors, AII amacrine cells and subpopulations of OFF cone bipolar cells are limited to the expression of either AMPA or kainate receptors. This differential expression may contribute to the unique character of transmission by these cell types.     

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.     

Kainate activation of horizontal, bipolar, amacrine, and ganglion cells in the rabbit retina

Patterns of excitation in populations of retinal bipolar, amacrine, and ganglion cells were mapped by activating alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA) and kainate (KA) receptors with KA in the presence of the channel-permeant guanidinium analogue 1-amino-4-guanidobutane (AGB). Registered serial thin sections were probed with immunoglobulins targeting AGB, glutamate, glycine, and gamma-aminobutyric acid (GABA) to visualize KA-evoked responses and the neurochemical signatures of distinct cell types. OFF-center cone bipolar cells and both type A and type B horizontal cells were strongly activated by KA. ON-center cone bipolar cells displayed weak AGB signals that arose at least partially, if not entirely, from coupling with KA-responsive glycinergic amacrine cells, whereas rod bipolar cells exhibited no detectable AGB permeation after KA activation. GABA-positive amacrine cells displayed a range of KA responses, some possessing little AGB signal even after strong KA activation, whereas all identifiable glycine-positive amacrine cells were driven by KA. Quantitative agonist responsivities of cells in the ganglion cell layer revealed that starburst amacrine cells are the most KA-responsive cell type in that layer. Ganglion cells varied in KA responsivity across morphologic subtypes, with a large alpha-like ganglion cell group the being the most KA responsive. Some ganglion cells displayed weak KA responses, even with saturating doses, that may have been be due to an absence of AMPA/KA receptors or to the existence of AGB-impermeant AMPA/KA receptor complexes.     

Light and electron microscopy of the ground squirrel retina: Functional considerations

Light and electron microscopy of Golgi-impregnated ground squirrel retinas have revealed a range of morphological subtypes of bipolar, amacrine, and ganglion cells. There are at least seven subtypes of bipolar cells. Those subtypes in which the somata were high (sclerad) in the inner nuclear layer (3 subtypes) had axon terminals low (vitread) in the inner plexiform layer, and those with somata low in the inner nuclear layer (4 subtypes) had axon terminals high in the inner plexiform layer. The bipolar subtypes with high axon terminals made flat contacts with receptor cells, whereas all but one of the bipolar subtypes with low axon terminals made ribbon-related contacts with receptor cells. There are at least five subtypes of amacrine cells. The two subtypes which the Golgi method revealed most frequently were a broad-field, unistratified neuron with a dendritic spread in excess of 1,000 m?m and a narrow-field, diffuse neuron with a dendritic spread of about 30 m?m. The broad-field, unistratified cell had the lowest proportion of amacrine vs. bipolar cell synaptic input of the amacrine subtypes (43%), whereas the narrow-field, diffuse cell had one of the greatest proportions of amacrine cell input (96%). There are at least 15 subtypes of ganglion cells. The proportion of synaptic inputs to these cells ranged from 21% to 100% amacrine cell synapses. An attempt has been made to relate this new knowledge of retinal circuitry to the physiological output of the ganglion cells.     

Analysis of bipolar and amacrine populations in marmoset retina

About 15 parallel ganglion cell pathways transmit visual signals to the brain, but the interneuron (bipolar and amacrine) populations providing input to ganglion cells remain poorly understood in primate retina. We carried out a quantitative analysis of the inner nuclear layer in the retina of the marmoset (Callithrix jacchus). Vertical Vibratome sections along the horizontal meridian were processed with immunohistochemical markers. Image stacks were taken with a confocal microscope, and densities of cell populations were determined. The density of flat midget bipolar cells fell from 15,746 cells/mm² at 1 mm (8 deg) to 7,827 cells/mm² at 3 mm (25 deg). The rod bipolar cell density fell from 8,640 cells/mm² at 1 mm to 4,278 cells/mm² at 3 mm, but the ratio of the two bipolar cell types did not change with eccentricity. The amacrine cell density ranged from 30,000 cells/mm² at 8 deg to less than 15,000 cells/mm² at 25 deg, but throughout the retina, the ratio of glycinergic to γ‐aminobutyric acid (GABA)ergic to amacrine cells remained relatively constant. The fractions of rod bipolar, cone bipolar, amacrine, Müller, and horizontal cells of all cells in the inner nuclear layer were comparable in central and peripheral retina. Marmosets had lower proportions of midget bipolar and rod bipolar in comparison with macaque. These differences were correlated with differences in rod and cone densities between the two species and did not reflect fundamental differences in the wiring between the two species. J. Comp. Neurol. 523:313–334, 2015. © 2014 Wiley Periodicals, 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.     

Spatial and Temporal Patterns of Neurogenesis in the Chick Retina

Chick embryo retinas were labelled in ovo by single injections of [3H]thymidine at selected times between days 2 and 12 of incubation. Embryos were later removed, at different stages of development, and the retinas processed for autoradiography of either serial sections or dissociated cell preparations. Analysis of unlabelled cells shows that neurogenesis starts, on day 2 of incubation, in a dorsotemporal area of the central retina, close to the posterior pole and to the optic nerve head. A gradient of neurogenesis spreads from this central area to the periphery, where neurogenesis ends, shortly after day 12, when the last few bipolar cells withdraw from the cell cycle. Additional dorsal-to-ventral and temporal-to-nasal gradients can be discerned in our autoradiographs. In all retinal sectors, ganglion cells start first to withdraw from the cell cycle, followed, with substantial overlapping, by amacrine, horizontal, photoreceptor plus Müller, and bipolar neuroblasts. Ganglion cells are also the first to reach the 50% level of unlabelled cells, followed this time by horizontal, photoreceptor, amacrine, Müller and bipolar cells. Finally, 100% levels of unlabelled cell populations are attained simultaneously by ganglion, horizontal and photoreceptor cells, followed by amacrine, then by Müller, and last by bipolar cells. Although all classes of neurons, in varying proportions, are being produced most of the time, our results also demonstrate that, in any given retinal area, the first cells leaving the cycle are determined to become ganglion cells, and the last ones bipolar cells, and not other types.     

Rod bipolar cells in the macaque monkey retina: immunoreactivity and connectivity.

Rod bipolar cells in the macaque monkey retina were labeled by three antibodies: an antibody against the alpha- and beta-subspecies of protein kinase C (PKC), a polyclonal antiserum against the L7 protein from mouse cerebellum, and a monoclonal antibody against rabbit olfactory bulb (MAb 115A 10). The MAb 115A10 antibody also labeled some cone bipolar and some amacrine cells. The antibody against PKC was used to study the synaptic connectivity of rod bipolar cells. Reconstructions of 28 rod spherules showed that usually two and up to four rod bipolar processes invaginate each rod spherule. Six rod bipolar axons in the inner plexiform layer were reconstructed; they all showed the same pattern of connectivity. Synaptic output at rod bipolar dyads usually was onto two amacrine cell profiles: one that resembled the All amacrine cell and another that frequently made a reciprocal synapse. Rod bipolar cells did not contact ganglion cells. Synaptic input to rod bipolar cells came from reciprocal amacrine cells at dyads and other amacrine cells. In these respects, the rod pathway in the monkey is very similar to that described in cat and rabbit. The density of rod bipolar cells was determined and compared with the density of rods. There is a maximum of 15,000-20,000 rod bipolar cells/mm2 at 1-3 mm eccentricity, close to where rod density is maximum. Rod density is 10 times higher than rod bipolar cell density within 2 mm of the fovea, and 30 times higher at 15 mm eccentricity. This change in relative density is compensated by an increase in the number of rods contacted by individual rod bipolar cells (seen in Golgi-stained whole-mount retina) so that the number of rod bipolar terminal boutons in each rod photoreceptor remains relatively constant with changing eccentricity. We estimate that each rod bipolar cell is contacted by about 20 rods at 2-4 mm eccentricity and about 60 rods at 6-7 mm eccentricity.     

Localization of glycine receptor alpha subunits on bipolar and amacrine cells in primate retina

The major inhibitory neurotransmitter glycine is used by about half of the amacrine cells in the retina. Amacrine cells provide synaptic output to bipolar, ganglion, and other amacrine cells. The present study investigated whether different bipolar and amacrine cell types in the primate retina differ with respect to the expression of glycine receptor (GlyR) subtypes. Antibodies specific for the alpha1, alpha2, and alpha3 subunits of the GlyR were combined with immunohistochemical markers for bipolar and amacrine cells and applied to vertical sections of macaque (Macaca fascicularis) and marmoset (Callithrix jacchus) retinae. For all subunits, punctate immunoreactivity was expressed in the inner plexiform layer. The GlyRalpha2 immunoreactive (IR) puncta occur at the highest density, followed by GlyR(alpha)3 and GlyR(alpha)1 IR puncta. Postembedding electron microscopy showed the postsynaptic location of all subunits. Double immunofluorescence demonstrated that the three alpha subunits are clustered at different postsynaptic sites. Two OFF cone bipolar cell types (flat midget and diffuse bipolar DB3), are predominantly associated with the alpha1 subunit. Two ON bipolar cell types, the DB6 and the rod bipolar cell, are predominantly associated with the alpha2 subunit. The glycinergic AII amacrine cell is presynaptic to the alpha1 subunit in the OFF-sublamina, and postsynaptic to the alpha2 subunit in the ON-sublamina. Another putative glycinergic cell, the vesicular glutamate transporter 3 cell, is predominantly presynaptic to the alpha2 subunit. The dopaminergic amacrine cell expresses the alpha3 subunit at a low density.     

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.     

AII amacrine cells in the mammalian retina show disabled-1 immunoreactivity

Disabled 1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by Reelin, which controls cell positioning in the developing brain. It has been localized to AII amacrine cells in the mouse and guinea pig retinas. This study was conducted to identify whether Dab1 is commonly localized to AII amacrine cells in the retinas of other mammals. We investigated Dab1-labeled cells in human, rat, rabbit, and cat retinas in detail by immunocytochemistry with antisera against Dab1. Dab1 immunoreactivity was found in certain populations of amacrine cells, with lobular appendages in the outer half of the inner plexiform layer (IPL) and a bushy, smooth dendritic tree in the inner half of the IPL. Double-labeling experiments demonstrated that all Dab1-immunoreactive amacrine cells were immunoreactive to antisera against calretinin or parvalbumin (i.e., other markers for AII amacrine cells in the mammalian retina) and that they made contacts with the axon terminals of the rod bipolar cells in the IPL close to the ganglion cell layer. Furthermore, all Dab1-labeled amacrine cells showed glycine transporter-1 immunoreactivity, indicating that they are glycinergic. The peak density was relatively high in the human and rat retinas, moderate in the cat retina, and low in the rabbit retina. Together, these morphological and histochemical observations clearly indicate that Dab1 is commonly localized to AII amacrine cells and that antiserum against Dab1 is a reliable and specific marker for AII amacrine cells of diverse mammals.     

Distribution of two splice variants of the glutamate transporter GLT1 in the retinas of humans, monkeys, rabbits, rats, cats, and chickens

Antibodies have been generated against two carboxyl-terminal splice variants of the glutamate transporter GLT1, namely, the originally described version of GLT1 and GLT1-B, and labelling has been examined in multiple species, including chickens and humans. Although strong specific labelling was observed in each species, divergent patterns of expression were noted. Moreover, each antibody was sensitive to the phosphorylation state of the appropriate protein, because chemical removal of phosphates using alkaline phosphatase revealed a broader range of labelled elements in most cases. In general, GLT1-B was present in cone photoreceptors and in rod and cone bipolar cells in the retinas of rabbits, rats, and cats. In the cone-dominated retinas of chickens and in marmosets, GLT1-B was associated only with cone photoreceptors, whereas, in macaque and human retinas, GLT1-B was associated with bipolar cells and terminals of photoreceptors. In some species, such as cats, GLT-B was also present in horizontal cells. By contrast, GLT1 distribution varied. GLT1 was associated with amacrine cells in chickens, rats, cats, and rabbits and with bipolar cells in marmosets and macaques. In the rat retina, rod photoreceptor terminals also contained GLT1, but this was evident only in enzymatically dephosphorylated tissues. We conclude that the two variants of GLT1 are present in all species examined but are differentially distributed in a species-specific manner. Moreover, each cell type generally expresses only one splice variant of GLT1.