Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina

Postembedding immunocytochemistry was used to determine the cellular localization of the amino acid neurotransmitters glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine in the avian retina. The through retinal pathway was glutamatergic, with all photoreceptors, bipolar cells, and ganglion cells being immunoreactive for glutamate. Bipolar cells displayed the highest level of glutamate immunoreactivity, with the cell bodies terminating just below the middle of the inner nuclear layer. All lateral elements, horizontal cells, amacrine cells, and interplexiform cells were immunoreactive for glycine or GABA. The GABAergic neurons consisted of two classes of horizontal cells and amacrine cells located in the lower part of the inner nuclear layer. GABA was also localized in displaced amacrine cells in the ganglion cell layer, and a population of ganglion cells that co-localize glutamate and GABA. Both the horizontal cells and GABAergic amacrine cells had high levels of glutamate immunoreactivity, which probably reflects a metabolic pool. At least two types of horizontal cells in the avian retina could be discriminated on the basis of the presence of aspartate immunoreactivity in the H2 horizontal cells. Glycine was contained in a subclass of amacrine cells, with their cell bodies located between the bipolar cells and GABAergic amacrine cells, two subclasses of bipolar cells, displaced amacrine cells in the ganglion cell layer, and ganglion cells that colocalize glutamate and glycine. Glycinergic amacrine cells had low levels of glutamate. We have also identified a new class of glycinergic interplexiform cell, with its stellate cell body located in the middle of the inner nuclear layer among the cell bodies of bipolar cells. Neurochemical signatures obtained by analyzing data from serial sections allowed the classification of subclasses of horizontal cells, bipolar cells, amacrine cells, and ganglion cells. © 1993 Wiley-Liss, Inc.     

Calbindin and calretinin immunoreactivity in the retina of adult and larval sea lamprey

The presence of calretinin and calbindin immunoreactivity is studied in the retina of larval and adult lamprey and their respective distributions are compared. Calretinin distribution is also studied in the retina of transforming stages. Western blot analysis in brain extracts showed a 29-kDa band with both polyclonal anti-calbindin and anti-calretinin antibodies. Calbindin and calretinin immunoreactivity has shown a partially different distribution. In the adult retina large and small bipolar cells, with respectively stratified or diffuse axons, the inner row of horizontal cells and ganglion cells and/or some amacrine cells were labeled with anti-calretinin antibody. The anti-calbindin antibody labels the same cell types except most of ganglion cells, but the label was less conspicuous. Therefore, the possible existence of these two calcium-binding proteins in the central nervous system of the sea lamprey could be discussed. In the differentiated central retina of larval lampreys, numerous calretinin immunoreactive bipolar and ganglion cells were observed, while, in the lateral retina, only ganglion cells were labeled, accordingly with the lack of differentiation of other neural cell types. CR-ir bipolar cells appeared in the retina by the stage 5 of transformation, i.e. about the time when differentiation of photoreceptors occurs. The comparison of the distribution of calretinin and calbindin between adult and larval central retina of lampreys shows striking differences that could be related to the different functionality of eyes in these two stages of the life cycle of lampreys. In addition, this is the first report on the presence of calcium-binding proteins in the larval and transforming lamprey retina, on the presence of calretinin- and calbindin-immunoreactive horizontal cells in adult lamprey retinas and on the differential stratification of bipolar cell terminals.     

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.     

Glutamate, GABA, and glycine in the human retina: an immunocytochemical investigation.

The distribution of the neuroactive amino acids glutamate, GABA, and glycine in the human retina was examined in consecutive semithin sections treated with antisera specific for fixed glutamate, GABA, and glycine, respectively. Glutamate immunoreactivity was conspicuous in all photoreceptor cells (rods more strongly labelled than cones), and in a majority (85-89%) of the cells in the inner nuclear layer (INL). Rod spherules and cone pedicles showed a greater enrichment of glutamate immunoreactivity than the parent cell bodies and inner segments. Also, structures of the inner plexiform layer (IPL) were labelled. A large majority (83-91%) of cells in the ganglion cell layer (GCL) was strongly stained, as were most axons in the nerve fibre layer. Müller cell processes appeared unstained. GABA immunoreactivity was present in presumed amacrine but not in bipolar-like cells. The stained cells were restricted to the inner 1/3 of the INL and were more frequent in central than in peripheral retina (40% and 26% of all cells in the inner 1/2 of INL, respectively). GABA positive cell processes, probably originating from interplexiform cells, appeared to traverse the INL and end in the outer plexiform layer. Dense immunolabeling was found in the IPL. GABA immunoreactive cells (some also stained for glutamate) comprised 23% of all GCL cells in the peripheral retina, but only 5% in the central retina. Most of them were localized adjacent to the IPL. A few GABA positive (possibly ganglion) cells extended a single fibre toward the nerve fibre layer. Solitary GABA positive fibres were seen in this layer and in the optic nerve. Glycine immunoreactivity was observed in cells with the location typical of amacrine and bipolar (peripheral retina) cells, as well as in punctate structures of the IPL. In contrast to the GABA positive cells, the glycine positive cells were more frequent in the peripheral than in the central retina (42% and 23% of all cells in inner 1/3 of INL, respectively). A few cells in the GCL (0.5-1.5%) were glycine positive. Glutamate colocalized with GABA or glycine in a majority of the cells stained for either of these inhibitory transmitters (90-95% of the GABA positive cells, and 80-86% of the glycine positive cells, in the INL). Some bipolar cells were stained for both glutamate and glycine. Colocalization of GABA and glycine occurred in a subpopulation (3-4%) of presumed amacrine cells, about half of which was also glutamate positive.     

Amino acid signatures in the developing mouse retina

This study characterizes the developmental patterns of seven key amino acids: glutamate, γ-amino-butyric acid (GABA), glycine, glutamine, aspartate, alanine and taurine in the mouse retina. We analyze amino acids in specific bipolar, amacrine and ganglion cell sub-populations (i.e. GABAergic vs. glycinergic amacrine cells) and anatomically distinct regions of photoreceptors and Müller cells (i.e. cell bodies vs. endfeet) by extracting data from previously described pattern recognition analysis. Pattern recognition statistically classifies all cells in the retina based on their neurochemical profile and surpasses the previous limitations of anatomical and morphological identification of cells in the immature retina. We found that the GABA and glycine cellular content reached adult-like levels in most neurons before glutamate. The metabolic amino acids glutamine, aspartate and alanine also reached maturity in most retinal cells before eye opening. When the overall amino acid profiles were considered for each cell group, ganglion cells and GABAergic amacrine cells matured first, followed by glycinergic amacrine cells and finally bipolar cells. Photoreceptor cell bodies reached adult-like amino acid profiles at P7 whilst Müller cells acquired typical amino acid profiles in their cell bodies at P7 and in their endfeet by P14. We further compared the amino acid profiles of the C57Bl/6J mouse with the transgenic X-inactivation mouse carrying the lacZ gene on the X chromosome and validated this animal model for the study of normal retinal development. This study provides valuable insight into normal retinal neurochemical maturation and metabolism and benchmark amino acid values for comparison with retinal disease, particularly those which occur during development.     

Early development of the retina and pineal complex in the sea lamprey: comparative immunocytochemical study.

Lampreys have a complex life cycle, with largely differentiated larval and adult periods. Despite the considerable interest of lampreys for understanding vertebrate evolution, knowledge of the early development of their eye and pineal complex is very scarce. Here, the early immunocytochemical organization of the pineal complex and retina of the sea lamprey was studied by use of antibodies against proliferating cell nuclear antigen (PCNA), opsin, serotonin, and gamma-aminobutyric acid (GABA). Cell differentiation in the retina, pineal organ, and habenula begins in prolarvae, as shown by the appearance of PCNA-negative cells, whereas differentiation of the parapineal vesicle was delayed until the larval period. In medium-sized to large larvae, PCNA-immunoreactive (-ir) cells were numerous in regions of the lateral retina near the differentiated part of the larval retina (central retina). A late-proliferating region was observed in the right habenula. Opsin immunoreactivity appears in the pineal vesicle of early prolarvae and 3 or 4 days later in the retina. In the parapineal organ, opsin immunoreactivity was observed only in large larvae. In the pineal organ, serotonin immunoreactivity was first observed in late prolarvae in photoreceptive (photoneuroendocrine) cells, whereas only a few of these cells appeared in the parapineal organ of large larvae. No serotonin immunoreactivity was observed in the larval retina. GABA immunoreactivity appeared earlier in the retina than in the pineal complex. No GABA-ir perikaryon was observed in the retina of larval lampreys, although a few GABA-ir centrifugal fibers innervate the inner retina in late prolarvae. First GABA-ir ganglion cells occur in the pineal organ of 15-17 mm larvae, and their number increases during the larval period. The only GABA-ir structures observed in the parapineal ganglion of larvae were afferent fibers, which appeared rather late in development. The time sequence of development in these photoreceptive structures is rather different from that observed in teleosts and other vertebrates. This suggests that the unusual development of the three photoreceptive organs in lampreys reflects specialization for their different functions during the larval and adult periods.     

A double-label study demonstrating that all serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina express GABA-like immunoreactivity.

A previous study localized serotonin-like immunoreactivity to amacrine cell populations in the larval tiger salamander retina. The present double-label immunocytochemical analysis of the tiger salamander retina was performed to determine if gamma-aminobutyric acid (GABA)-like immunoreactivity is expressed by serotonin-immunoreactive amacrine cells. More than 3,000 serotonin-amacrine cells were observed in double-label preparations, and all were found to express GABA-like immunoreactivity. This finding extends previous studies of serotonin-GABA coexistence in the retina by providing the first report of the co-localization of endogenous serotonin and GABA-like compounds in a retinal neuron.     

Immunochemical localization of calretinin in the retina of the turbot (Psetta maxima) during development

The expression of the calcium-binding protein calretinin was analysed by immunohistochemistry techniques in the retina of turbot (Psetta maxima) from embryonic to juvenile stages. Calretinin immunoreactivity was first detected in retinae from newly hatched larvae, in which the anlage of the inner plexiform layer and a subset of amacrine and ganglion cells displayed a faint immunolabelling. First appearance of photoreceptors during larval life coincided with an increase in the intensity of the labelling. During subsequent larval development, the expression of calretinin affected distinctive retinal components. The inner plexiform layer, optic fiber layer, and a population of amacrine and ganglion cells were invariably labelled. Occasional bipolar cells were labelled at the end of the larval period. By metamorphosis, calretinin is sequentially expressed in horizontal cells, and bipolar immunoreactive cells become numerous. The pattern of calretinin immunoreactivity of the inner plexiform layer changes from the larval to juvenile period. In all cases, calretinin immunoreactivity exhibited variations between the peripheral retina, which contains the most recently differentiated retinal components, and the remainder of the differentiated retina. Our results suggest that the progressive expression of calretinin in the turbot retina appears associated with some degree of neuronal differentiation. Once the definitive pattern of calretinin immunoreactivity is established in the turbot retina, both similarities and differences with the calretinin location in the retina of other vertebrates can be demonstrated.     

Immunocytochemical localization of kainate-selective glutamate receptor subunits GluR5, GluR6, and GluR7 in the cat retina

Localizations of the kainate-selective glutamate receptor subunits GluR5, 6, and 7 were studied in the cat retina by light and electron microscopic immunocytochemistry. GluR5 immunoreactivity was observed in the cell bodies and dendrites of numerous cone bipolar cells and ganglion cells. The labeled cone bipolar cells make basal or flat contacts with cone pedicles in the outer plexiform layer, leading to their identification as OFF-center bipolar cells. Reaction product within the inner plexiform layer was observed in processes of ganglion cells at their sites of input from cone bipolar cells. Staining for GluR6 was localized to A- and B-type horizontal cells, numerous amacrine cells, and an occasional cone bipolar cell. The larger ganglion cells were also immunoreactive. As with other GluR molecules, labeling was usually confined to one of the two postsynaptic elements at a cone bipolar dyad contact. Immunoreactivity for GluR7 was very limited and was seen only in a few amacrine and displaced amacrine cells. Findings of this study are consistent with a major role for kainate receptors in mediating OFF pathways in the outer retina with participation in both OFF and ON pathways in the inner retina.     

Co-localization of carnosine and glutamate in photoreceptors and bipolar cells of the frog retina

Immunocytochemical methods were used to visualize carnosine (β-alanyl- -histidine)-like immunoreactivity (-LI) in the frog retina and to compare its localization with that of glutamate. Carnosine-LI was conspicuous in photoreceptors and bipolar cells. The axon terminals of labelled bipolar cells formed five bands in the inner plexiform layer. A few presumed amacrine and ganglion cells, as well as Müller cell endfeet, were also labelled. Post-embedding immunocytochemistry revealed particularly high levels of glutamate-LI in the synaptic axon terminals of bipolar cells, with a mean gold particle density 5 × higher than that of amacrine cells. Photoreceptor terminals were also labelled, but with a labelling intensity about half that of bipolar cells. Labelling of serial semithin sections showed co-localization of carnosine and glutamate in photoreceptors and bipolar cells. These findings are consistent with the notion that glutamate is the neurotransmitter of neuronal elements that transfer information vertically through the retina. We propose that carnosine may modulate GABA and/or glutamate receptors by virtue of its ability to chelate Zn²⁺ and other ions.     

Connectivity of glycine immunoreactive amacrine cells in the cat retina

The synaptic relationships of glycine immunoreactive amacrine cells in the cat retina were studied through the use of postembedding immunogold techniques. Glycine immunoreactive amacrine cells were found to synapse extensively with other amacrines and ganglion cells, particularly in strata 1-3 of the inner plexiform layer. This contrasts with GABA immunoreactive amacrine cells which provide major input to bipolar cells in strata 3-5. Glycine containing amacrine terminals exhibited diversity with respect to the morphology of their synaptic vesicles. The three types of terminals which could be distinguished were characterized by small pleomorphic (32-35 nm), medium-sized flattened (38-45 nm), or larger rounded (48-55 nm) vesicles. Comparison of retinal sections processed for glycine immunoreactivity with adjacent sections stained for GABA reactivity revealed a colocalization of glycine and GABA in 3% of the cells in the amacrine layer and approximately 40% of the cells in the ganglion cell layer. The amacrine terminals in which glycine and GABA were colocalized typically contained the small pleomorphic type of vesicles.     

Identification and characterization of an aquaporin 1 immunoreactive amacrine-type cell of the mouse retina

Using immunocytochemistry, a type of amacrine cell that is immunoreactive for aquaporin 1 was identified in the mouse retina. AQP1 immunoreactivity was found in photoreceptor cells of the outer nuclear layer (ONL) and in a distinct type of amacrine cells of the inner nuclear layer (INL). AQP1-immunoreactive (IR) amacrine cell somata were located in the INL and their processes extended through strata 3 and 4 of the inner plexiform layer (IPL) with thin varicosities. The density of the AQP1-IR amacrine cells increased from 100/mm(2) in the peripheral retina to 350/mm(2) in the central retina. The AQP1-IR amacrine cells comprise 0.5% of the total amacrine cells. The AQP1-IR amacrine cell bodies formed a regular mosaic, which suggested that they represent a single type of amacrine cell. Double labeling with AQP1 and glycine, gamma-aminobutyric acid (GABA) or GAD(65) antiserum demonstrated that the AQP1-IR amacrine cells expressed GABA or GAD(65) but not glycine. Their synaptic input was primarily from other amacrine cell processes. They also received synaptic inputs from a few cone bipolar cells. The primary synaptic targets were ganglion cells, followed by other amacrine cells and cone bipolar cells. In addition, gap junctions between an AQP1-IR amacrine process and another amacrine process were rarely observed. In summary, a GABAergic amacrine cell type labeled by an antibody against AQP1 was identified in the mouse retina and was found to play a possible role in transferring a certain type of visual information from other amacrine or a few cone bipolar cells primarily to ganglion cells.     

Diversity of glycine receptors in the mouse retina: localization of the alpha2 subunit

Gamma-aminobutyric acid (GABA) and glycine are the major inhibitory neurotransmitters in the retina, glycine being produced in approximately half of all amacrine cells. Whereas retinal cell types expressing the glycine receptor (GlyR) alpha1 and alpha3 subunits have been mapped, the role of the alpha2 subunit in retinal circuitry remains unclear. By using immunocytochemistry, we localized the alpha2 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by doubly labeling sections for GlyR alpha2 and bassoon (a presynaptic marker) or gephyrin (a postsynaptic marker). Synapses containing GlyR alpha2 were rarely found on ganglion cell dendrites but were observed on bipolar cell axon terminals and on amacrine cell processes. Recently, an amacrine cell type has been described that is immunopositive for glycine and for the vesicular glutamate transporter vGluT3. The processes of this cell type were presynaptic to GlyR alpha2 puncta, suggesting that vGluT3 amacrine cells release glycine. Double labeling of sections for GlyR alpha1 and GlyR alpha2 subunits showed that they are clustered at different synapses. In sections doubly labeled for GlyR alpha2 and GlyR alpha3, approximately one-third of the puncta were colocalized. The most abundant GlyR subtype in retina contains alpha3 subunits, followed by those containing GlyR alpha2 and GlyR alpha1 subunits.     

Morphologic and neurochemical target selectivity of regenerating adult photoreceptors in vitro

Regenerating adult central nervous system (CNS) neurons must re-establish synaptic circuits in an environment very different from that present during development. However, the complexity of CNS circuitry has made it extremely difficult to assess the selectivity and mechanisms of synaptic regeneration at the cellular level in vivo. The synaptic preferences of adult photoreceptors were examined by using a defined cell culture system known to support regenerative process growth, presynaptic varicosity formation, and establishment of functional synapses. Immunolabeling for synaptic vesicle protein 2 and ultrastructural analysis demonstrated that cell-cell contacts made by photoreceptors were synaptic in nature. Target selectivity was determined by quantitative analysis of contacts onto normal and novel target cell types in cultures in which opportunities to contact all retinal cell types were present. Target cells were identified by morphology and immunolabeling for the amino acid neurotransmitters glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine. Regenerating photoreceptors showed a strong preference for novel multipolar cell targets (amacrine and ganglion cells) over normal photoreceptor, horizontal, and bipolar cell targets. Additionally, photoreceptors were selective for targets containing the transmitter GABA. These results indicate first, that the normal synaptic partners for photoreceptors are not intrinsically the optimal targets for regenerative synapse formation, and second, that GABA may modulate synaptic targeting by adult photoreceptors. © 1996 Wiley-Liss, Inc.     

Immunohistochemical localization of calbindin-D28K and calretinin in the lamprey retina

Calbindin-D28K and calretinin are homologous cytosolic calcium binding proteins localized in many retinal neurons from different species. In this report, location of cells immunoreactive to both proteins was investigated in the retina of the lamprey, Lampetra fluviatilis. This organism constitutes one of the older representative vertebrates and possesses a peculiar organization, probably unique: two-thirds of the ganglion cells are in the classical amacrine cell layer and the nerve fiber layer is located in the scleral part of the inner plexiform layer. Calbindin-like immunoreactivity was demonstrated in large bipolar cells and in cell bodies located in the inner retina. Although the distinction between labelled ganglion cells and labelled amacrine cells was rendered difficult, we hypothetized that the majority of calbindin-immunoreactive cells observed in the inner retina are ganglion cells, because of the high number of labelled fibers in the nerve fiber layer. Calretinin-like immunoreactivity was detected in both large and small bipolar cells, and also in cells located in the inner retina. Since few calretinin-immunoreactive fibers were observed in the nerve fiber layer, we assume that the latter category of cells are amacrine cells. Horizontal cells were both negative for calbindin and calretinin-like immunoreactivities. Calbindin and calretinin, which are present in cones from many species, could not be detected in the photoreceptor layer favouring the rod-dominated lamprey retina. Although their distribution differs from those observed in most vertebrates, the present results indicate the good conservation of both calcium binding proteins in the retina during the vertebrate evolution. © Wiley-Liss, Inc.     

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.     

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.     

Localization of aspartate-like immunoreactivity in the retina of the turtle (Pseudemys scripta).

Aspartate has been reported to be a putative excitatory neurotransmitter in the retina, but little detailed information is available concerning its anatomical distribution. We used an antiserum directed against an aspartate-albumin conjugate to analyze the anatomy, dendritic stratification, and regional distribution of cell types with aspartate-like immunoreactivity in the turtle retina. The results showed dramatic differences in immunoreactivity in the peripheral versus the central retina. Strong aspartate-like immunoreactivity was shown in the peripheral retina, with many well-labeled processes in the inner plexiform layer. Many bipolar, horizontal, amacrine, and ganglion cells, some photoreceptors, and some unidentified cells were strongly immunoreactive in the peripheral retina. In contrast, although the central retina showed well-labeled horizontal cells, there was only light labeling in the inner plexiform layer with weakly immunoreactive amacrine and ganglion cells and no labeled bipolar cells. There were several strongly immunoreactive efferent nerve fibers which left the optic nerve head and arborized extensively in the retina. At the electron microscopic level, electron-dense reaction product was associated with synaptic vesicles at bipolar and amacrine cell synapses in the inner plexiform layer. These results suggest that aspartate may be involved in many diverse synaptic interactions in both the outer plexiform layer and the inner plexiform layer of the turtle 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.