All indoleamine-accumulating cells in the rabbit retina contain GABA.

The indoleamine-accumulating amacrine cells of the rabbit retina are wide-field and numerous. They form a dense plexus in sublamina 5 of the inner plexiform layer where they make reciprocal synapses with rod bipolar cells. To provide a quantitative test for the colocalization of serotonin (5-HT) and gamma-aminobutyric acid (GABA) in the rabbit retina, we designed two parallel double-label experiments. In the first series, the indoleamine-accumulating cells were labeled with 5,7-dihydroxytryptamine (5,7-DHT), which was subsequently visualized by photooxidation in the presence of diaminobenzidine. This was combined with autoradiography for 3H-muscimol. In the second and complementary series, 3H-5-HT uptake was combined with postembedding GABA immunocytochemistry. These two experiments provided essentially identical results: over 98% of the indoleamine-accumulating amacrine cells were double-labeled. This means that, within the limit of experimental error, all the indoleamine-accumulating amacrine cells are GABAergic. The indoleamine-accumulating amacrine cells account for 15-20% of a large diverse group of GABA amacrine cells. In addition, the rare type 3 indoleamine-accumulating cells and fine processes running in the optic fiber layer were double-labeled. If there is insufficient 5-HT to support a transmitter role in the rabbit retina, our results suggest that the indoleamine-accumulating cells may use GABA as a neurotransmitter. Thus, rod bipolar cells, in common with other bipolar cell types, receive extensive negative feedback at GABA-mediated reciprocal synapses.     

Electron microscopical observations on the indoleamine-accumulating neurons and their synaptic connections in the retina of the cat

The distribution of indoleamine-accumulating amacrine cells and their synaptic connections in the retina of the cat were analyzed in the fluorescence, phase-contrast, and electron microscopes. The findings were compared to recently characterized morphological subclasses of amacrine cells. The indoleamine-accumulating neurons were visualized after labeling with an exogenous indoleamine, 5, 6-dihydroxytryptamine. The intravitreal injection of the labeling drug was preceded by treatment with the neurotoxic dopamine-analogue, 6-hydroxydopamine, in order to destroy the otherwise interfering dopaminergic processes. The analysis in the fluorescence and phase-contrast microscopes confirmed earlier reports that the indoleamine-accumulating cell bodies and processes have a distribution consistent with that of amacrine cells. A stratified branching pattern of the indoleamine-accumulating processes in the outer half of the inner plexiform layer was discovered. In the inner half of that layer the branching pattern is diffuse. In the electron microscope the indoleamine-accumulating neurons were seen to have synapses of the conventional type. Their main synaptic contacts are reciprocal synapses with rod bipolar terminals in sublamina b of the inner plexiform layer. They also have synapses with flat cone bipolar terminals in sublamina a, and occasionally with amacrine cells and ganglion cells throughout the inner plexiform layer. Synapses with invaginating cone bipolar terminals, in sublamina b, appear to be rare. The synaptic arrangement with reciprocal synapses with rod bipolar terminals is similar to that of the recently reported AI amacrine cells. It is also similar to that of the indoleamine-accumulating neurons in the retinae of other mammals investigated earlier.     

The actions of serotonergic agonists and antagonists on the activity of brisk ganglion cells in the rabbit retina

Extracellular electrophysiological recordings were obtained from rabbit retinal ganglion cells in either a superfused eyecup or an in vivo preparation. Selective antagonists or agonists of serotonin at the 5-HT2 or 5-HT1A receptors were applied, and the changes in light-evoked and spontaneous activity were studied. Both 5-HT1A agonists and 5-HT2 antagonists reduced the ON-components of the light-evoked responses of all classes of brisk ganglion cell; spontaneous activity was reduced in these cells as well. These results suggest that the indoleamine-accumulating amacrine cells of the rabbit retina serve to facilitate the output of the depolarizing rod bipolar cell and thereby increase the efficacy of transmission between this and other cells in the rabbit retina, and that this process is mediated by 5-HT2 receptors. On the basis of the similarity of the actions of the 2 classes of drug studied, we hypothesize further that 5-HT1A receptors mediate an inhibitory process that serves to terminate the indoleamine-induced facilitation. This process may be located either in the bipolar terminal or presynaptic to it in the terminal of the putative indoleaminergic cells.     

Light- and electron-microscopic analysis of vasoactive intestinal polypeptide-immunoreactive amacrine cells in the guinea pig retina

Vasoactive intestinal polypeptide (VIP) is a neuroactive substance that is expressed in both nonmammalian and mammalian retinas. This study investigated the morphology and synaptic connections of VIP-containing neurons in the guinea pig retina by immunocytochemistry, by using antisera against VIP. Specific VIP immunoreactivity was localized to a population of wide-field and regularly spaced amacrine cells with processes ramifying mainly in strata 1 and 2 of the inner plexiform layer (IPL). Double-label immunohistochemistry demonstrated that all VIP-immunoreactive cells possessed gamma-aminobutyric acid immunoreactivity. The synaptic connectivity of VIP-immunoreactive amacrine cells was identified in the IPL by electron microscopy. The VIP-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in strata 1 to 3 of the IPL. The most frequent postsynaptic targets of VIP-immunoreactive amacrine cells were other amacrine cell processes in strata 1 to 3 of the IPL. Synaptic outputs to bipolar cells were also observed in strata 1 to 3 of the IPL. In addition, ganglion cell dendrites were also postsynaptic to VIP-immunoreactive neurons in the sublamina a of the IPL. These studies show that one type of VIP-immunoreactive amacrine cells make contact predominantly with other amacrine cell processes. This finding suggests that VIP-containing amacrine cells may influence inner retinal circuitry, thus mediating visual processing.     

Dopaminergic and indoleamine-accumulating amacrine cells express GABA-like immunoreactivity in the cat retina

Colocalization of indoleamine uptake and GABA-like immunoreactivity was studied in the cat retina. Consecutive, semithin sections were incubated in antisera to either 5-HT (5-hydroxytryptamine) or GABA. More than 90% of all 5-HT-accumulating amacrine cells expressed GABA-like antigens. With the same approach, the colocalization of 5-HT uptake and GABA-like immunoreactivity was studied in rabbit and 75-80% of the 5-HT-accumulating amacrine cells expressed GABA-like immunoreactivity, thus confirming a previous study (Osborne and Beaton, 1986). Since, in both cat and rabbit, endogenous 5-HT could not be found by immunocytochemistry, one must consider the possibility that some GABAergic amacrine cells take up indoleamines. In the cat retina, antibodies against tyrosine hydroxylase (TH) label dopaminergic amacrine cells (Oyster et al., 1985). By incubating consecutive, semithin sections in antisera to either TH or GABA, it was found that 84% of the dopaminergic amacrine cells also expressed GABA-like immunoreactivity. GABA-like immunoreactivity and 3H-muscimol uptake were found to be colocalized in more than 90% of the amacrine cells labeled. However, dopaminergic amacrine cells did not accumulate 3H-muscimol. Evidence is presented from colocalization studies for 2 types of interplexiform cell in the cat retina. One is stained by GABA-like immunocytochemistry and by 3H-muscimol uptake. The other is the dopaminergic amacrine cell, which also expresses GABA-like immunoreactivity, but does not accumulate 3H-muscimol.     

Synaptic organization of the dopaminergic neurons in the rabbit retina.

A number of substances were tested for their ability to label amine-accumulating neurons in the rabbit retina after fixation with OsO₄ or glutaraldehyde and OsO₄. Useful results were obtained with 5,6-dihydroxytryptamine (5,6-DHT) and 6-hydroxydopamine (6-HDA). Labelled processes were characterized by small (40–50 mm) pleomorphic synaptic vesicles containing electron-dense cores, and at times by swelling of mitochondria and by increased electron density of membranes and cytoplasm. Fluorescence microscopy showed that 5,6-DHT labelled both dopaminergic and indoleamine-accumulating neurons. In most experiments, therefore, the indoleamine-accumulating neurons were removed with 5,7-dihydroxytryptamine. In such retinas the dopaminergic processes labelled by 5,6-DHT were found to make synapses of the conventional type, characterized by an accumulation of synaptic vesicles on the presumed presynaptic side and some aggregation of material on the cytoplasmic side of the synaptic membranes and within the synaptic cleft. The dopaminergic processes were found to contact each other and also non-dopaminergic amacrine cells and their processes. Conventional synapses onto dopaminergic processes were observed from both labelled and unlabelled amacrine processes. The input from labelled neurons was observed on varicose dopaminergic processes whereas input from non-labelled elements was found on the intervaricose parts of the dopaminergic processes. No Contacts of dopaminergic processes with bipolar or ganglion cells were observed. Injections of 6-HDA gave the same results, although this drug gave less distinct labelling which made the observations less decisive than with 5,6-DHT. In retinas treated with 5,6-DHT alone (i.e., in which the indoleamine-accumulating neurons remained) numerous processes were observed which were both pre- and postsynaptic to bipolar terminals. These observations suggest that the indoleamine-accumulating processes synapse with bipolar cells. The results show that the dopaminergic neurons form a network involving only amacrine cells, suggesting a regulatory function for them. By analogy with the dopaminergic interplexiform cells of the goldfish retina, it is suggested that the dopaminergic neurons in the rabbit may regulate lateral inhibitory effects mediated by amacrine cells. Furthermore, the finding that the dopaminergic and indoleamine-accumulating cells apparently have a different synaptic organization suggests that it is appropriate to categorize amacrine cells according to their transmitter content as well as their morphology.     

Fluorescence and electron microscopical observations on the amine-accumulating neurons of the cebus monkey retina

The organization of the Cebus monkey regina was analysed after the intraocular injection of 5,6-dihydroxytryptamine. This amine was taken up not only by the previously known dopaminergic neurons, but also by a set of indoleamine-accumulating neurons, whose processes are confined to the inner plexiform layer. The synaptic contacts of the dopaminergic neurons were analysed in the electron microscope after the processes of the indoleamine-accumulating neurons were destroyed by the intravitreal injection of the neurotoxic indoleamine, 5,7-dihydroxytryptamine. The subsequent injection of 5,6-dihydroxytryptamine induces certain changes in the dopaminergic neurons which accumulate the substance: electron-dense cores appear in the synaptic vesicles, and increased electron-density of mitochodrial and cellular membranes is often observed. The dopaminergic neurons were found to be presynaptic to amacrine cell perikarya and processes in the inner plexiform layer. In the outer plexiform layer they were presynaptic to both bipolar and horizontal cells, but they did not contact photoreceptors. The dopaminergic neurons received synapses only in the inner plexiform layer, from amacrine cell processes. It is inferred that in Cebus most dopaminergic neurons belong to a special class of retinal neuron, the interplexiform cells, which appear to transmit information centrifugally within the retina, from the inner to the outer plexiform layers. There are considerable similarities between the synaptology of the dopaminergic interplexiform neurons in the Cebus monkey and the goldfish retina, and the function of interplexiform neurons may therefore be similar in these two species.     

5-HT2A receptors are differentially expressed in bullfrog and rat retinas: a comparative study

Expression of 5-hydroxytryptamine (5-HT) 2A receptor (5-HT2A) was studied in bullfrog and rat retinas by immunocytochemistry. In the bullfrog retina, 5-HT2A-immunoreactivity was observed in both the outer and inner plexiform layers (OPL and IPL). Double labeling experiments further showed that 5-HT2A was expressed in Müller cells stained by GFAP. Labeling for 5-HT2A was strong in the somata and endfeet and relatively weak in the major processes and fine branchets of Müller cells. In contrast, 5-HT2A immunoreactivity was hardly detected in the rat retina, and no rat Müller cells were labeled. Furthermore, immunocytochemical assay demonstrated that labeling for 5-HT was present in amacrine cells and displaced amacrine cells in the inner retina of bullfrog, but not in the rat retina. These results suggest that 5-HT may modulate retinal information processing via activating 5-HT2A expressed in neuronal and glial elements in bullfrog, but that such modulation is unlikely to occur in rat.     

Differential regulation of ciliary neurotrophic factor receptor-α expression in all major neuronal cell classes during development of the chick retina

Ciliary neurotrophic factor (CNTF) exerts a multiplicity of effects on a broad spectrum of target cells, including retinal neurons. To investigate how this functional complexity relates to the regulation of CNTF receptor α (CNTFRα) expression, we have studied the developmental expression of the receptor protein in chick retina by using immunocytochemistry. During the course of development, the receptor is expressed in all retinal layers, but three levels of specificity can be observed. First, the expression is regulated temporally with immunoreactivity observed in ganglion cells (embryonic day 8 [E8] to adult), photoreceptor precursors (E8–E12), amacrine cells (E10 to adult), bipolar cells (E12–E18), differentiated rods (E18 to adult), and horizontal cells (adult). Second, expression is restricted to distinct subpopulations of principal retinal neurons: preferentially, large ganglion cells; subpopulations of amacrine cells, including a particular type of cholinergic neuron; a distinctly located type of bipolar cell; and rod photoreceptors. Third, expression exhibits subcellular restriction: it is confined largely to dendrites in mature amacrine cells and is restricted entirely to outer segments in mature rods. These data correlate with CNTF effects on the survival of ganglion cells and mature photoreceptors, the in vitro differentiation of photoreceptor precursors and cholinergic amacrine cells, and the number of bipolar cells in culture described here or in previous studies. Thus, our results demonstrate an exceptional degree of complexity with respect to the regulation of neuronal CNTFRα expression in a defined model system. This suggests that the same signaling pathway is used to mediate a variety of regulatory influences, depending on the developmental stage and cell type. J. Comp. Neurol. 400:244–254, 1998. © 1998 Wiley-Liss, Inc.     

Sequential course of uptake of intravitreal 5,7-dihydroxytryptamine by carp retinal cells

The sequential course of uptake by retinal cells of intravitreally injected 5,7-dihydroxytryptamine (5,7-DHT) together with dopamine (DA) was investigated in juvenile carp retinas, which were removed at various intervals (1-24 h) after injection. The cells taken up 5,7-DHT were visualized immunohistochemically with anti-serotonin (5-HT) antibody and FITC-conjugated IgG. After a mixture of 5,7-DHT and DA (2.5, 10 or 20 micrograms each) was given, large-sized indoleamine (IA) amacrine cells first (1-4 h), and then small-sized indoleamine-accumulating amacrine amacrine (IAA) cells (4-12 h), bipolar cells (8-12 h) and in some cases photoreceptor cells (12-24 h) were sequentially observed, and finally the immunoreactive structures almost disappeared around 24 h after injection. When the mixture of 5,7-DHT and DA (10 micrograms each) was injected into the eyes of reserpinized fish, the same sequential uptake of 5,7-DHT was seen in a faster time course, but additionally various classes of retinal cells (horizontal, ganglion and Müller cells) became visible as irregular clusters. However, DA cells were never visualized at any stages of all the experiments, indicating that DA cells do not take up 5,7-DHT in the carp retina, which was further confirmed by double labeling of 5-HT- and tyrosine hydroxylase-like immunoreactive cells. Double labeling also revealed that 5,7-DHT-accumulating bipolar cells appear to represent a subclass different from that of protein kinase C-like immunoreactive bipolar cells.     

Serotonergic and serotonin-accumulating neurons in the goldfish retina

Autoradiography of goldfish retinas incubated in micromolar levels of 3H-serotonin displayed 3 kinds of labeled somas in the inner nuclear layer: S1 amacrine cells with heavy labeling, large somas, and a sparse distribution (approximately 93/mm2); S2 amacrine cells with moderate labeling, smaller somas, and a denser distribution (approximately 500/mm2); and a subset of bipolar cells with light labeling, small somas, and a very dense distribution (approximately 4000/mm2). Serotonin-like immunoreactivity was observed only in S1 amacrine cells and their synaptic terminals. Radiolabeled terminals in the inner plexiform layer formed 4 strata that were differentially assigned to the 3 cell types. S1 amacrine cells arborized in sublayers 1 and 5, received inputs from type a1 bipolar cells and amacrine cells, and made synapses on other amacrine cells, type a1 bipolar cells and unidentified processes. Thus, S1 amacrine cells seem to receive significant input from "off-center" pathways. S2 amacrine cells arborized in sublayer 3 and made synapses onto amacrine cells. Labeled bipolar cell terminals were exclusively located in sublayer 2 and were identified as type a2 mixed rod-cone bipolar cells. We conclude that the S1 amacrine cell is truly serotonergic and that radiolabeling of S2 amacrine cells and type a2 bipolar cells is due to cross-specificity for another carrier or processes unrelated to their neurochemical identities. These observations partially reconcile many previous observations on the types, numbers, and synaptologies of teleost retinal neurons identified by different markers for indoleaminergic transmission.     

Development of catecholaminergic, indoleamine-accumulating and NADPH-diaphorase amacrine cells in rabbit retinae.

We have investigated the ontogeny of four classes of amacrine cells in the rabbit retina. In particular, the distribution, number, soma diameter, dendritic field diameter, and pattern of dendritic stratification were studied in catecholaminergic (CA) and indoleamine-accumulating (IA) amacrines and in two classes of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase amacrine cells. The first CA and IA cells are observed on the 27th postconceptional day (27PCD) and the first NADPH-diaphorase cells on 28PCD. These first cells are concentrated in the central part of the visual streak, and at subsequent ages, cells in this part of the streak have larger somata and more mature dendritic fields than those elsewhere, supporting the notion that the peak density region is a developmentally advanced part of the retina. Throughout development, amacrine cells of all classes are concentrated in the visual streak, with their density reaching minima at the superior and inferior retinal margins. As their total number increases, the difference in cell density between the streak and the periphery decreases, presumably because proportionately more cells are added at the periphery. Their total number peaks around 42PCD, followed by a decline of 12-31% to adult values. Once the peak number of cells has been reached, the difference in cell density between the streak and periphery begins to increase. The rate of this increase is closely correlated with the increase in retinal area. This redistribution of amacrine cells, as well as a greater expansion of their dendritic fields in peripheral retina, is almost certainly the product of nonuniform retinal expansion.     

Connections of indoleamine-accumulating cells in the rabbit retina

To study the connections of the neurons of the rabbit retina that accumulate indoleamines, we injected 5,7-dihydroxytryptamine into the vitreous body. It accumulated within a subset of amacrine cells and could be visualized there by aldehyde-induced fluorescence. The fluorescent labeling was photo-converted to an insoluble, osmiophilic product by irradiation in the presence of diaminobenzidine, and the tissue was examined by electron microscopy. Preservation of the structure of the tissue after photoconversion was satisfactory and the dendrites of the indoleamine-accumulating cells could easily be identified. They form a dense plexus near the junction of the inner plexiform and ganglion cell layers, where they exhibit large synaptic endings that occupy a substantial fraction of the surface of rod bipolar terminals. The dendrites of the indoleamine-accumulating cells receive input from rod bipolars at dyad synapses, where the other postsynaptic partner is a dendrite of a narrow-field, bistratified amacrine cell; in addition, they receive amacrine cell input throughout the inner plexiform layer. The only outputs we observed are reciprocal synapses onto the rod bipolar endings. Thus, these amacrine cells appear to exert an important effect on the transmission of scotopic information through the retina.     

A system of indoleamine-accumulating neurons in the rabbit retina

The indoleamine-accumulating neurons of the rabbit retina were labeled by intraocular injection of 5,7-dihydroxytryptamine (5,7-DHT). The retinas were fixed with 2.5% paraformaldehyde and 0.2% glutaraldehyde and inspected by fluorescence microscopy. Five kinds of cell accumulated the indoleamine. They were labeled to essentially the same brightness and remained so despite variations in the concentration at which 5,7-DHT had been applied or the duration of its application. Experiments in which 5,7-DHT was applied to retinas incubated in vitro gave identical results. To see the whole shape of the cells, we visually guided micropipettes to the fluorescent cell bodies and injected the cells with Lucifer yellow CH. To study the cells as a population, we used a new method in which the fluorescence of 5,7-DHT is photochemically converted to an insoluble diaminobenzidine product. The dendrites of all of the indoleamine-accumulating cells were then simultaneously visible. Used together, these techniques revealed an interrelated system of indoleamine-accumulating neurons. All of the cells contribute processes to a dendritic plexus that lies at the inner margin of the inner plexiform layer. The plexus is roughly 4 micron thick. It is pierced by the stalks of the Müller cells and is occasionally interrupted by ganglion cell bodies, where they extend above the average margin of the ganglion cell layer. Otherwise it fills much of the space at the junction of the plexiform and ganglion cell layers. The type 1 and type 2 cells are amacrine cells with cell bodies at the inner margin of the inner nuclear layer. They have 5-8 radially branching primary dendrites which extend horizontally across the inner plexiform layer before descending to join the dendritic plexus. They differ from each other in cell body shape, dendritic morphology, and the course of their dendrites within the inner plexiform layer. Each has a "displaced" counterpart, with a morphology similar to the type 1 or type 2 cell but with a cell body located in the ganglion cell layer. The displaced cells are separate functional elements because, in contrast to the type 1 and type 2 cells, they have no dendrites (and hence can have no synaptic connections) in the outer part of the inner plexiform layer. The fifth kind of cell (type 3) appears not to have been described before. Its cell body is located at the outer margin of the inner nuclear layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Serotoninergic axonal contacts on identified cat spinal dorsal horn neurons and their correlation with nucleus raphe magnus stimulation

This study examined the distribution of serotoninergic (5-HT) immunoreactive axonal contacts on spinal laminae I and II neurons by combining the intracellular horseradish peroxidase (HRP) method with immunocytochemistry. In addition, the 5-HT distribution was correlated with effects produced by electrical stimulation within the nucleus raphe magnus (NRM). Responses of lamina I neurons and lamina II stalked cells to noxious stimulation were markedly suppressed during NRM stimulation. In contrast, responses of nociceptive lamina IIa islet or non-nociceptive lamina IIb islet cells remained unchanged during nucleus raphe magnus stimulation. These inhibitory influences were positively correlated with the distribution of 5-HT immunoreactive contacts on these neurons. Nociceptive lamina I neurons and lamina II stalked cells received a significantly greater number of contacts (average of 74 and 63, respectively) than either nociceptive lamina IIa islet or non-nociceptive lamina IIb islet cells (average of 25 and eight contacts, respectively). Irrespective of cell type, most 5-HT contacts occurred on dendritic shafts rather than spines. These data reveal a differential distribution of 5-HT contacts on neurons in spinal laminae I and II, and indicate that at least a portion of the NRM modulation of dorsal horn neuronal activity is serotoninergic and concentrated on the dendritic shafts of nociceptive lamina I neurons and lamina II stalked cells.     

Calretinin in neurochemically well-defined cell populations of rabbit retina

In the rabbit retina, parvalbumin has been localized selectively to AII amacrine cells, while 28 kDa calbindin could be detected in horizontal cells, in one type of depolarizing cone bipolar cell and a population of wide-field amacrine cells. The distribution of the third neuronal calcium binding protein, calretinin, however, has not been studied to date in detail in the rabbit retina. Therefore in this study we aimed to describe the overall distribution of calretinin in the different retinal layers and the possible colocalization pattern with other neurochemical marker molecules. A few cone photoreceptor cells were found to be labeled, whereas the outer plexiform layer was free from immunoreactive elements. In the most proximal row of the inner nuclear layer amacrine cells were labeled, while more distally a few cells emitted beaded axon-like processes, toward the outer retina. There were large (18–28 μm in diameter) cells labeled in the ganglion cell layer, of which many apparently had their axon stained. Some of the calretinin immunoreactive amacrine cells (the AII neurons) also contained parvalbumin. Colocalization of calretinin and 28 kDa calbindin could not be ascertained in the same amacrine cell populations, nor was tyrosine hydroxylase present in calretinin-containing cells. There was partial colocalization of calretinin in the γ-aminobutyric acid-positive amacrine cell population. Parvalbumin containing ganglion cells were also positive for calretinin; however, the calretinin-positive ganglion cells were more numerous. γ-Aminobutyric acid could be colocalized in some calretinin-positive neurons of the ganglion cell layer.     

Presence of thyrotropin-releasing-hormone-immunoreactive (TRHir) amacrine cells in the retina of anuran and urodele amphibians

The presence of thyrotropin-releasing-hormone-immunoreactive (TRH-ir) amacrine cells in the retina of amphibians is reported for the first time. The anuran and urodele retinas studied exhibit major differences in the distribution of TRH-ir cells. In the two urodele species investigated, most TRH-ir amacrine cells were located in the ganglion cell layer (GCL). These pear-shaped cells originate a dense TRH-ir dendritic plexus in strata 4-5 of the inner plexiform layer (IPL). A small number of TRH-ir amacrine cells were observed in the inner nuclear layer (INL). Most of these INL TRH-ir cells were multipolar neurons with radiating dendrites that originate a loose plexus in the IPL stratum 1. In the three anuran species investigated, most TRH-ir amacrine cells were located in the INL. Distribution of TRH-ir processes in the IPL of anurans was not so clearly layered as in urodeles, dendrites being observed throughout strata 1-5. In the toad retina THR-ir material was also observed in the outer plexiform layer, which suggests that toads may have some TRH-ir interplexiform neurons. In the frog and toad, TRH-ir fibers were also observed in the optic nerve, although their origin could not be ascertained. The number of TRH-ir amacrine cells per whole retina was higher in anurans than in urodeles, though urodeles have higher cell densities. The marked differences in distribution of TRH-ir amacrine cells observed between anurans and urodeles, and among the three anuran species, suggest different functions of TRH in retinal processing, perhaps related to the different specializations of the visual systems of these species.     

Neurotransmitter localization in the skate retina

The retina of the skate (Raja clavata, R. radiata and R. oscellata) was studied by autoradiography following intraocular injections or incubations with [3H]GABA, [3H]isoguvacine, [3H]glycine, [3H]dopamine or [3H]5-hydroxytryptamine. Fluorescence immunohistochemistry was also used to demonstrate the endogenous content, accumulation, and retention of 5-hydroxytryptamine. The [3H]GABA was taken up by glia, and [3H]isoguvacine failed to appreciably label any neurons. [3H]Glycine was accumulated by amacrine cells, possibly of two subtypes. The [3H]dopamine was taken up by a few rare cells in the inner nuclear layer, which sent processes into the inner plexiform layer. Both autoradiography and immunohistochemistry showed 5-hydroxytryptamine to be efficiently accumulated by two types of cells in the inner nuclear layer: a bipolar cell type and an amacrine cell type. The morphology of the bipolar cells suggests they are of the ON depolarizing type. Immunohistochemistry also demonstrated the retention of accumulated 5-hydroxytryptamine by these two cell types, and that the bipolar cells contained far less endogenous 5-hydroxytryptamine than the amacrine cells did. The latter cell type can be presumed to use 5-hydroxytryptamine as its neurotransmitter. The results show the distribution of presumed glycinergic, dopaminergic and indoleaminergic neurons. They also show that there are two fundamentally distinct types of indoleamine neurons, a bipolar cell type with a low and an amacrine cell type with a high content of 5-hydroxytryptamine.     

High vulnerability of GABA-immunoreactive neurons to kainate in rat retinal cultures: correlation with the kainate-stimulated cobalt uptake

Like other areas of the central nervous system, the retina is highly vulnerable to ischemia. In particular, neurons in the inner nuclear layer, including gamma-amino butyric acid (GABA)-ergic amacrine neurons, are highly vulnerable. Since excitotoxicity is likely a major mechanism of ischemic retinal injury, using rat retinal cell culture, we examined whether GABAergic retinal neurons are differentially vulnerable to particular excitotoxins. The neuronal population as a whole, identified by anti-microtubule associated protein-2 (MAP-2) immunocytochemistry, was equally vulnerable to kainate, but more resistant to N-methyl-d-aspartate (NMDA) than cultured cortical neurons. Compared to Thy-1 immunoreactive neurons, GABA immunoreactive neurons were more vulnerable to kainate, but more resistant to NMDA neurotoxicity. Double staining of cultures with anti-GABA immunocytochemistry and the kainate-stimulated cobalt uptake method, revealed a close correlation between the two. However, unlike in other neuronal cells, there was no clear correlation between GluR2 immunoreactivity and the cobalt staining. The heightened vulnerability of GABAergic neurons to kainate, as compared to the general neuronal population, may be due to the calcium-permeable AMPA/kainate receptors they have, as identified functionally by the kainate-stimulated cobalt uptake staining. Since these neurons are preferentially injured in ischemia, AMPA/kainate receptor-mediated neurotoxicity may contribute significantly to ischemic retinal injury.