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Two bilaterally symmetric eyes arise from the anterior neural plate in vertebrate embryos. An interesting question is whether both eyes share a common developmental origin or they originate separately. We report here that the expression pattern of a new gene ET reveals that there is a single retina field which resolves into two separate primordia, a suggestion supported by the expression pattern of the Xenopus Pax-6 gene. Lineage tracing experiments demonstrate that retina field resolution is not due to migration of cells in the median region to the lateral parts of the field. Removal of the prechordal mesoderm led to formation of a single retina both in chick embryos and in Xenopus explants. Transplantation experiments in chick embryos indicate that the prechordal plate is able to suppress Pax-6 expression. Our results provide direct evidence for the existence of a single retina field, indicate that the retina field is resolved by suppression of retina formation in the median region of the field, and demonstrate that the prechordal plate plays a primary signaling role in retina field resolution.

The localization of gamma-aminobutyric acid (GABA)- and L-glutamate 1 carboxy-lyase (GAD)-immunoreactive neurons was compared in the skate, frog, pigeon, chicken, rabbit, and man. Horizontal cells show both GABA and GAD immunoreactivity in the skate, frog, and bird. Certain amacrine cells show GABA and GAD immunoreactivity in all species. The distribution of GABA- and GAD-immunoreactive cell bodies and cell processes was very similar, if not identical, in the skate and man. In the other species, cell populations with GAD immunoreactivity also showed GABA immunoreactivity. However, in the bird, frog, and rabbit, the GABA-immunoreactive amacrine cells were at least twice as numerous as the GAD-immunoreactive cells. In birds, the distributions of the GAD and GABA immunoreactivities were different in the sublayers of the inner plexiform layer. The reason for the difference is currently unknown. GABA-immunoreactive bipolar-like cells were seen in the frog.

The regional distribution and cellular location of GABA-synthesizing enzyme, L-glutamate decarboxylase (GAD), GABA degrading enzyme, GABA-transaminase (GABA-T), taurine synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSAD), aspartate and glutamate converting enzyme, aspartate aminotransferase (AAT), and somatostatin have been visualized in the rat retina by immunocytochemical methods. GAD immunoreactivity was found to be concentrated in the inner plexiform layer. A moderate to weak staining of GAD was found in the inner nuclear layer. The distribution of GABA-T immunoreactivity was similar to that of GAD with the exception that a weak to moderate staining of GABA-T was also observed in the outer plexiform layer. CSAD immunoreactivity was seen in every layer with the heaviest staining in the inner plexiform layer, and moderate staining in the inner and outer nuclear layers and ganglion cell layer. AAT immunoreactivity was mostly concentrated in the outer nuclear layer; there was weak staining in the inner nuclear layer and inner and outer plexiform layer. Dense somatostatin staining was seen in the inner plexiform layer and moderate staining was present in the inner nuclear layer, outer plexiform layer and ganglion cell layer. These findings suggest that in rat retina, GABA-containing cells occur in some types of amacrine cells only, while taurine and somatostatin appear in both amacrine and horizontal cells. AAT immunoreactivity was primarily associated with the photoreceptor cells suggesting that AAT may be used as a marker for aspartergic/glutamergic cells and their endings in the central nervous system.

The comparative distribution of 3H-GABA uptake and glutamic acid decarboxylase immunoreactivity (GAD-IR) in amacrine cells of goldfish retina was studied simultaneously by using a combined autoradiographic/immunocytochemical technique in order to determine the degree of colocalization of these two markers of GABAergic neurons; 3H-GABA was taken up most intensely by large pyriform Ab amacrine cells (3% of inner nuclear layer (INL) somata), and less intensely by smaller, polyform amacrine cells (12% of INL somata), cell bodies in the ganglion cell layer (one-half as common as Ab cells), and cell bodies in the inner plexiform layer (very rare). GAD-IR was observed in 25% of amacrine cells in the INL, the vast majority of which were polyform in shape, and cell bodies in the ganglion cell layer. Twice as many cells were labeled for GAD-IR as for 3H-GABA uptake. Of the cells that took up 3H-GABA, colocalization of 3H-GABA uptake and GAD-IR was observed in 90% of the polyform amacrine cells, 80% of the cells in the ganglion cell layer, and none of the pyriform Ab amacrine cells or cells in the inner plexiform layer. We suggest that the polyform cells compose the major population of GABAergic amacrine cells in the goldfish retina, rather than the pyriform Ab cells. GABAergic displaced amacrine cells or ganglion cells are also indicated by our data. The implications of these data with regard to the physiology of the goldfish retina are discussed as well.

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

The localization of gamma-aminobutyric acid (GABA) neurons in the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (L-glutamate I-carboxy-lyase, EC 4.1.1.15) and by [3H]GABA uptake autoradiography. When Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Intravitreally administered [3H]GABA produced a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.

Two procedures have been used to test for the colocalization of different neuroanatomical gamma-aminobutyric acid (GABA)ergic markers within the same neurons in primate retina. First, sequential immunocytochemical processing of sections was done using antisera to glutamic acid decarboxylase and to GABA, and these antisera were visualized by peroxidase-antiperoxidase and fluorescein isothiocyanate techniques respectively. Colocalization of both antisera was found within the same neuron cell bodies. In the second experiment, immunocytochemical staining using GABA antiserum was performed on retinal tissue that had been previously incubated in vitro for neuronal uptake of [3H]muscimol. Both markers colocalized in 70% of the labeled cell body population.

An EM-autoradiographical/immunocytochemical technique was used to study amacrine cell synapses onto mb1 bipolar cell terminals in goldfish retina. Tissue was double labeled for [3H]GABA uptake and glutamate decarboxylase (GAD) immunolocalization. Nearly 90% of the amacrine cell synaptic processes onto both proximal and distal halves of mb1 terminals were labeled with either [3H]GABA or GAD-immunoreactivity (IR). Proximal half: 73% of the amacrine synapses were labeled with [3H]GABA uptake and 82% with GAD-IR; 88% of [3H]GABA labeled contacts were double labeled. Distal half: 17% of the amacrine synapses were labeled with [3H]GABA uptake and 67% with GAD-IR; 63% of [3H]GABA labeled contacts were double labeled. After consideration of the possible sources of [3H]GABA labeled synapses onto mb1 terminals, we concluded that the synaptic terminals of pyriform Ab amacrine cells double label for [3H]GABA and GAD-IR despite our previous report that Ab cell bodies do not stain for anti-catfish brain GAD antiserum. We suggest that Ab cells contain isoenzymes of GAD which differ in subcellular distribution, thereby accounting for the differential staining of the cell bodies and dendrites obtained with the GAD antiserum we used.

Ultrastructural analysis of the inner synaptic layer of goldfish retina, using a double-label technique, showed that [3H]GABA uptake and glutamic acid decarboxylase (GAD)-immunoreactivity (IR) occurred in different neuronal processes in most cases. Many [3H]GABA-accumulating processes were found surrounding GAD-IR processes, though not necessarily in a postsynaptic relationship. Co-localization of [3H]GABA uptake and GAD-IR occurred only when one GAD-IR process was juxtaposed to another GAD-IR process. This study suggests that [3H]GABA uptake may be a poor marker for GABA releasing neurons.

The presence of glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was investigated in neuroretina sections from hatching quail embryos by immunocytochemistry. The photoreceptors were found to be intensely immunoreactive to anti-GAD antiserum and to two distinct anti-GABA antisera.

GABA immunoreactivity was studied and compared with GAD immunoreactivity in the retinae of baboon, cynomolgus monkey and man. The central and peripheral parts of the retinae were investigated separately in cynomolgus monkey and in man. The same kinds of structures were stained with both antisera. Cells with a position corresponding to amacrine cells were stained, as well as processes in the inner plexiform layer and some cells in the ganglion cell layer. The outer plexiform layer and some cells with the position and configuration of horizontal cells also appeared immunoreactive. Staining was also observed in bipolar-like cells, in man most clearly when using the GABA antiserum in sections from the central parts of the retina. It is possible that horizontal cells, as well as bipolar-like cells, may play a previously unsuspected role in GABAergic transmission in the primate retina.

Rabbit retina was used as a model to study the possible role of taurine in the retina. The taurine-synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSAD), is localized immunohistochemically using specific antibodies against CSAD. The CSAD-immunoreactivity appears to be most prominent in the inner nuclear layer (INL) and ganglion cell layer (GCL). The inner plexiform layer (IPL), the outer nuclear layer and outer plexiform layer are sporadically stained. The CSAD-positive neurons include some amacrine cells and probably the bipolar cells in the INL and some large and small ganglion cells in the GCL. Autoradiographic studies reveal that the uptake of [3H]taurine is most prominent in the INL. The IPL and GCL, as well as the Müller cells, also show a moderate degree of [3H]taurine accumulation. In conclusion, we have demonstrated the presence of the taurine-synthesizing enzyme and uptake systems in rabbit retina. Based on the above evidence, we propose that taurine may be used by some neurons, presumably amacrine cells, as a transmitter in the rabbit retina.