Robert Gábriel

Serotonin synthesis and accumulation by neurons of the anuran retina.

Serotonin-synthesizing and serotonin-accumulating neurons were studied in the retinas of Xenopus laevis and Bufo marinus. All previously identified cell types exhibiting serotonin-like immunoreactivity (SLI) were labeled by intravitreal injection of 5,7-dihydroxytryptamine (5,7-DHT). They included two amacrine cell types (large and small) in both species, and one bipolar cell type in Xenopus. Incubation of retinas in culture medium in the ambient light reduced SLI in amacrine cells and enhanced the labeling in bipolar cells. After incubation, some photoreceptor cell bodies and large numbers of outer segments also displayed SLI in both species. Incubation with the serotonin-uptake inhibitor, fluoxetine, reduced immunolabeling in bipolar cells and outer segments to the level in the untreated retinas. Both large SLI and 5,7-DHT-accumulating amacrine cells in Xenopus and Bufo were labeled with an antibody raised against phenylalanine hydroxylase (PH), which binds to tryptophan 5-hydroxylase, one of the synthesizing enzymes for serotonin. Small SLI and 5,7-DHT-accumulating amacrine cells in both species represented two populations, one with and the other without PH-like immunoreactivity (PH-LI). The anti-PH antibody failed to label any SLI or 5,7-DHT-accumulating bipolar cells in Xenopus. These observations indicate that all large and some small SLI amacrine cells in the retinas of Xenopus and Bufo synthesize serotonin, while other small SLI amacrine, bipolar and photoreceptor cell bodies, and outer segments only accumulate serotonin.

GABA-like immunoreactive neurons in the retina of Bufo marinus: evidence for the presence of GABA-containing ganglion cells

gamma-Aminobutyric acid (GABA)-like immunoreactive (IR) neurons in the retina of the cane toad Bufo marinus were revealed using immunohistochemistry on retinal wholemount preparation and sectioned material. GABA-IR neurons included horizontal, bipolar and amacrine cells in the inner nuclear layer and small to medium sized cells in the ganglion cell layer. A few IR axons were seen in the optic fiber layer of the retina. Following the injection of the carbocyanine dye, DiI into the optic tectum ganglion cells were retrogradely filled. A small population of DiI-filled ganglion cells (2.8%) was found to be GABA-IR. GABA-IR neurons in the ganglion cell layer without DiI label were considered to be displaced amacrine cells of which 45.3% were GABA positive. It is proposed that GABA-containing ganglion cells may form an inhibitory projection to visual centers of the anuran brain.

Immunocytochemical localization of parvalbumin- and neurofilament triplet protein immunoreactivity in the cat retina : colocalization in a subpopulation of AII amacrine cells

Using antibodies against parvalbumin and neurofilament triplet protein, colocalization of these two neuronal markers was revealed in all of type A horizontal cells and alpha ganglion cells and in a small number of AII amacrine cells of the cat retina. Besides the double-labeled neurons, parvalbumin alone was present in type B horizontal cells, in small numbers of starburst- and A13-like amacrine cells and in the somata of unidentified ganglion cells. The processes of the double- or single-labeled amacrine cells did not have a continuous retinal cover. Although the parvalbumin- and neurofilament-immunolabeled amacrine cells belonged to groups of neurons with well-defined cell morphologies, their neurochemical features differed from other AII, starburst and A13 amacrine cells. The presence of these cells may be due to an accidental expression of an unusual combination of neurochemical features during retinal development. It is also possible that these cells support the functioning of ganglion cells with rarely occurring complex receptive fields.

Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells

SummaryNeuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.

Synaptic contacts of serotonin-like immunoreactive and 5,7-dihydroxytryptamine-accumulating neurons in the anuran retina

The synapses of serotonin-like immunoreactive retinal neurons were studied in Bufo marinus and Xenopus laevis and those of 5,7-dihydroxytryptamine-labelled cells in Xenopus. Immunoreactivity to serotonin was mostly confined to amacrine cells. Synapses formed by profiles of labelled cells were almost uniformly distributed in the inner plexiform layer in both species. Interamacrine synapses were the most frequent, and in some cases two labelled amacrine cell profiles made a gap junction. Some of the labelled amacrine cells synapsed on to presumed ganglion cell dendrites and onto bipolar cell terminals. Labelled bipolar cell terminals synapsed on to non-labelled amacrine cell dendrites and received inputs both from labelled and non-labelled amacrine cells. Labelled bipolar cell profiles were not observed in the outer plexiform layer. After preloading and photoconversion of 5,7-dihydroxytryptamine in the Xenopus retina, labelled bipolar cell dendrites in the outer plexiform layer were observed to be postsynaptic to cone pedicles and less frequently to rods and horizontal cells. In the inner plexiform layer, synapse types formed by labelled bipolar cells were similar to those with serotonin immunoreactivity. The frequency of synapses formed by 5,7-dihydroxytryptamine-labelled amacrine cells increased, compared with serotonin immunocytochemistry. Labelled amacrine cells synapsed mostly with non-labelled amacrine cells, although the ratio of contacts formed by two labelled profiles increased. Synapses from labelled amacrine cell dendrites to non-labelled bipolar cell terminals and from non-labelled bipolar cell terminals to labelled amacrine cell profiles increased in number, while those from labelled amacrine cells to presumed ganglion cell dendrites decreased. The quantitative data obtained by the two approaches enabled us to propose different neuronal circuits for serotonin-synthesizing and -accumulating neurons of the Xenopus retina.

Neuron-specific enolase-like immunoreactivity in the vertebrate retina : selective labelling of Müller cells in Anura

SummaryNeuron-specific enolase (NSE) immunocytochemistry was carried out in retinae of goldfish, axolotl, clawed frog, cane toad, lizard, chick, guinea-pig, rabbit, rat, cat and human. With the exception of Anura, strong immunoreactivity was seen in the large ganglion, amacrine cells and horizontal cells of the retina in all of the other species. Photoreceptors were found to be labelled in the rat and human retina and only one cone type in rabbit. Photoreceptor pedicles and ellipsoids were stained in the goldfish and the somata and inner segments of some photoreceptors in axolotl. In the axolotl retina, besides neurons, Müller cells (MCs) were also immunolabelled. In the retina of the cane toad and the clawed frog MCs were the only stained elements. Similarly in other parts of the central nervous system of the cane toad, glial elements of the optic tectum and spinal cord were immunoreactive. In contrast, in the peripheral nervous system, neurons of the 1st sympathetic ganglion and the 2nd dorsal root ganglion were labelled. In double-labelling experiments, glial fibrillary acidic protein and NSE showed colocalisation both in the glial elements of the optic tectum and spinal cord and in MCs of the retina of the cane toad.

Morphology and distribution of Müller cells in the retina of the toad Bufo marinus

We have previously shown that an antibody against neuron-specific enolase (NSE) selectively labels Müller cells (MCs) in the anuran retina (Wilhelm et al. 1992). In the present study the light- and electron-microscopic morphology of MCs and their distribution were described in the retina of the toad, Bufo marinus, using the above antibody. The somata of MCs were located in the proximal part of the inner nuclear layer and were interconnected with each other by their processes. The MCs were uniformly distributed across the retina with an average density of 1500 cells/mm2. Processes of MCs encircled the somata of photoreceptor cells isolating them from each other by glial sheath, except for those of the double cones. Some of the photoreceptor pedicles remained free of glial sheath. Electron-microscopic observations confirmed that MC processes provide an extensive scaffolding across the neural retina. At the outer border of the ganglion cell layer these processes formed a non-continuous sheath. The MC processes traversed through the ganglion cell layer and spread beneath it between the neuronal somata and the underlying optic axons. These processes formed a continuous inner limiting membrane separating the optic fibre layer from the vitreous tissue. Neither astrocytic nor oligodendrocytic elements were found in the optic fibre layer. The significance of the uniform MC distribution and the functional implications of the observed pattern of MC scaffolding are discussed.

Synaptic contacts of tyrosine hydroxylase-immunoreactive elements in the inner plexiform layer of the retina of Bufo marinus.

SummaryTyrosine hydroxylase (TH) immunocytochemistry was utilized to quantify dopaminergic synapses in the inner plexiform layer of the retina of Bufo marinus. Since dopaminergic cells have bistratified dendritic arborisation in the inner plexiform layer, attention was given to the segregation of synapses between the scleral and the vitreal sublaminae. Light-microscopically, a more elaborate dendritic branching was observed in the scleral than in the vitreal sublamina. In contrast, about 55% of synapses occurred in the vitreal one fifth of the inner plexiform layer, 30% in the scleral fifth, and 15% in the intermediate laminae. Input sources and output targets showed only minor quantitative differences between sublaminae 1 and 5. TH-immunoreactive processes were found in presynaptic (62.8%) and postsynaptic (37.2%) positions. Synapses to the stained dendrites derived from bipolar (40.4%) and amacrine (59.6%) cells, whereas outputs from the TH-positive processes were directed to amacrine cells (56.8%) and to small and medium-sized dendrites (35.4%); at least some of these can be considered as ganglion cell dendrites. TH-positive profiles neither formed synapses with each other nor were presynaptic to bipolar cell terminals. Junctional appositions of the immunoreactive profiles were occasionally seen on non-stained amacrine and ganglion cell dendrites in the scleral sublamina of the inner plexiform layer and on optic axons in the optic fibre layer. Although dopaminergic cells are mainly involved in amacrine-amacrine interactions, inputs from bipolar terminals and outputs to ganglion cell dendrites were also substantial, suggestive of a role also in vertical information processing.

The number and distribution of bipolar to ganglion cell synapses in the inner plexiform layer of the anuran retina

The main route of information flow through the vertebrate retina is from the photoreceptors towards the ganglion cells whose axons form the optic nerve. Bipolar cells of the frog have been so far reported to contact mostly amacrine cells and the majority of input to ganglion cells comes from the amacrines. In this study, ganglion cells of frogs from two species (Bufo marinus, Xenopus laevis) were filled retrogradely with horseradish peroxidase. After visualization of the tracer, light-microscopic cross sections showed massive labeling of the somata in the ganglion cell layer as well as their dendrites in the inner plexiform layer. In cross sections, bipolar output and ganglion cell input synapses were counted in the electron microscope. Each synapse was assigned to one of the five equal sublayers (SLs) of the inner plexiform layer. In both species, bipolar cells were most often seen to form their characteristic synaptic dyads with two amacrine cells. In some cases, however, the dyads were directed to one amacrine and one ganglion cell dendrite. This type of synapse was unevenly distributed within the inner plexiform layer with the highest occurrence in SL2 both in Bufo and Xenopus. In addition, SL4 contained also a high number of this type of synapse in Xenopus. In both species, we found no or few bipolar to ganglion cell synapses in the marginal sublayers (SLs 1 and 5). In Xenopus, 22% of the bipolar cell output synapses went onto ganglion cells, whereas in Bufo this was only 10%. We conclude that direct bipolar to ganglion cell information transfer exists also in frogs although its occurrence is not as obvious and regular as in mammals. The characteristic distribution of these synapses, however, suggests that specific type of the bipolar and ganglion cells participate in this process. These contacts may play a role in the formation of simple ganglion cell receptive fields.