GABA-like immunoreactivity in the cat retina: light microscopy

Semithin sections of the cat retina were stained with antibodies against GABA conjugated to bovine serum albumin with glutaraldehyde. Labelled cells were visualized by means of the peroxidase-antiperoxidase method. In the outer plexiform layer both A- and B-type horizontal cells and the B-type axon terminal system expressed GABA-like immunoreactivity. Approximately 25-30% of all amacrine cells and the whole inner plexiform layer were heavily labelled. Two types of putative GABA-ergic interplexiform cells could be distinguished. One of them also expressed tyrosine-hydroxylase-like immunoreactivity. A few bipolar cells were also GABA-immunolabelled. GABA-like immunoreactivity and 3H-muscimol uptake were colocalized in 90% of the amacrine cells labelled. However, horizontal cells did not accumulate 3H-muscimol.     

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.     

Interaction between enkephalin and GABA in the chicken retina: further analyses of coexisting relationships

Previous studies have indicated an interactive relationship between enkephalin and γ-aminobutyric acid (GABA) in the vertebrate retina. Among these studies are those that have demonstrated the colocalization of enkephalin and GABA in retinal amacrine cells. In the present study, enkephalin immunocytochemistry was combined with either autoradiography of tritiated GABA high-affinity uptake or GABA immunocytochemistry to further investigate the coexistence of GABA in enkephalin-amacrine cells of the chicken retina. A regional analysis revealed that the percentage colocalization of GABA high-affinity uptake in enkephalin-amacrine cells did not vary appreciably throughout the retina. Overall, 15.2% of enkephalin-amacrine cells exhibited high-affinity GABA uptake. Double-label immunofluorescence histochemistry revealed that 15.1% of enkephalin-amacrine cells express endogenous GABA-like immunoreactivity. These double-labelled cells were observed throughout central and peripheral regions of the retina. In each of the double-label analyses, only less intensely labelled enkephalin-amacrine cells expressed markers of GABA activity. The two double-label analyses reveal almost identical percentages of coexistence of GABA markers in chicken enkephalin-amacrine cells and therefore, provide supportive evidence for the GABAergic nature of these cells. These results suggest a functional diversity in the population of chicken enkephalin-amacrine cells and imply the possibility of multiple signalling through amacrine cells which contain enkephalin and GABA.     

Amacrine cells in the ganglion cell layer of the cat retina

Following transection of the optic nerve, ganglion cells in the cat retina undergo retrograde degeneration. However, many small profiles (less than or equal to 10 micron) survive in the ganglion cell layer. Previously considered to be neuroglia, there is now substantial evidence that they are displaced amacrine cells. Their density increases from approximately 1,000 cells/mm2 in peripheral retina to 7,000 cells/mm2 in the central area. Their total number was found to be 850,000, which is five times the number of ganglion cells and also five times the number of astrocytes. Uptake of 3H-muscimol followed by autoradiography labelled 75% of the displaced amacrine cells; hence, the majority seem to be GABAergic. Immunocytochemistry with an antibody directed against choline-acetyl-transferase labelled approximately 10% of the displaced amacrines in the peripheral retina and 17% in the central area. Uptake of serotonin (5-HT) followed by immunocytochemistry was found in 25-30% of displaced amacrines. NADPH diaphorase histochemistry labelled approximately 5% of displaced amacrine cells. The sum of the various percentages make colocalization likely. Intracellular injection of Lucifer Yellow under microscopic control revealed that displaced amacrine cells constitute several morphological types.     

GABA-like immunoreactivity in the macaque monkey retina: a light and electron microscopic study

The distribution of GABA-like immunoreactivity in the macaque monkey retina was studied by using postembedding techniques on semithin and ultrathin sections. At the light microscopic level, both inner and outer plexiform layers showed strong GABA-like immunoreactivity in the central retina. All the horizontal cells, some bipolar cells, 30-40% of amacrine cells, occasional interplexiform cells, and practically all displaced amacrine cells were labeled. In the peripheral retina (beyond 5 mm eccentricity), the outer plexiform layer and the horizontal cells were not labeled, but all other cell types showed the same labeling pattern as in the central retina. Synapses of the inner plexiform layer involving a pre- or postsynaptic GABA-labeled process were studied electron microscopically. Synapses involving a GABA-labeled presynaptic amacrine cell process made up 80% of the synapses observed. These GABA-labeled amacrine processes synapsed onto amacrine, bipolar, and ganglion cell processes as well as onto amacrine and ganglion cell bodies. Synapses involving a postsynaptic GABA-labeled process made up 20% of the synapses studied. The GABA-like immunoreactive processes were postsynaptic to bipolar cells at the dyads and to amacrine cells at conventional synapses.     

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.     

Morphological and immunocytochemical identification of indoleamine-accumulating neurons in the cat retina

The type and distribution of indoleamine-accumulating neurons were studied in the cat retina. The neurons were characterized by their capacity to take up an exogenous indoleamine, 5,6-dihydroxytryptamine, which could then be visualized using formaldehyde-induced fluorescence. Under direct microscopic control, intracellular injection of Lucifer yellow into the labeled cells revealed that the indoleamine-accumulating neurons comprise 2 distinct morphological types of amacrine cell and 1 class of ganglion cell. The dendritic morphology and retinal distribution of these cells were studied in detail. The proportion of retinal cells that accumulate 5-HT was measured using 5-HT (5-hydroxytryptamine) preloading in vitro and immunocytochemistry with an anti-5-HT antibody. Only 1 type of amacrine cell showed uptake of 5-HT at a concentration of 10(-7) M. Cells containing endogenous 5-HT were not detected by immunocytochemistry.     

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

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

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.     

Dopaminergic innervation of A II amacrine cells in mammalian retina

Dopaminergic amacrine cells were stained in cat, rat, and rabbit retina using an antibody against tyrosine hydroxylase (TH). Following intraocular injection of DAPI (4,6,diamidino-2-phenylindole), subsequent retinal whole-mount preparations revealed that the dopaminergic fiber plexus formed rings around amacrine cell bodies. Intracellular injection of Lucifer yellow (LY) into A II amacrine cells confirmed that this rod-related, bistratified interneuron has its cell body within the dopaminergic rings. Using a photooxidation process, LY was transformed into an electron-dense reaction product, enabling ultrastructural examination of LY-injected A II amacrine cells. In retinae counterstained with an antibody against TH, it was possible to show synapses from TH-positive fibers onto these cells. The dopaminergic plexus was further investigated by injecting single dopaminergic cells with LY and thus revealing their branching pattern. The present results emphasize the role of dopamine in modulating the rod pathway in mammalian retina.     

Localization of 3H-GABA, -muscimol, and -glycine in goldfish retinas stained for glutamate decarboxylase

Glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotransmitter GABA, has been localized in goldfish retina using a new antiserum. We observed at least six types of GAD-immunoreactive amacrine cells, one of which was large and pyriform (Ab type). In addition, immunoreactive synaptic terminals were located throughout the inner plexiform layer (IPL). Amacrine cells that were GAD-immunoreactive also had high-affinity uptake mechanisms for both 3H-GABA and 3H-muscimol that were detectable autoradiographically. Type Ab pyriform amacrine cells were heavily labeled because of 3H-GABA uptake and were GAD-immunoreactive. Other types of GAD-immunoreactive amacrine cells, including a subpopulation of Ab amacrines, were lightly labeled because of 3H-GABA uptake. Because the same neurons that were GAD-immunoreactive also accumulated 3H-GABA and 3H-muscimol, these three are appropriate markers for GABAergic cells in the goldfish retina. However, the uptake of 3H-muscimol by many non-GAD-immunoreactive cells, detectable at longer autoradiographic exposure times, indicates that this label must be used with caution. Thirty percent of goldfish retinal amacrine cells are GABAergic, and their processes are distributed throughout all levels of the IPL. Few GAD-immunoreactive amacrine cells accumulated 3H-glycine, so the goldfish retina contains distinct populations of glycinergic and GABAergic amacrine cells.     

GABA and tyrosine hydroxylase immunocytochemistry reveal different patterns of colocalization in retinal neurons of various vertebrates

Colocalization of GABA- and tyrosine hydroxylase-like immunoreactivity was studied in the retinae of various vertebrate species in order to ascertain whether the presumed coexistence of GABA and dopamine, reported earlier for mammals (Kosaka et al.: Exp. Brain Res. 66:191-210, '87: W?ssle and Chun: J. Neurosci. 8:3383-3394,'88) is a common phenomenon. GABA-immunopositive cells constituted a separate population from tyrosine hydroxylase-positive cells in fish and amphibians, whilst in higher--i.e., amniote--vertebrates, such as reptiles, birds, and mammals, all dopaminergic cells contained GABA-like immunoreactivity. No clear correlation was found between the type of dopaminergic cell (amacrine/interplexiform) and the presence or absence of colocalization.     

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.     

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.     

Postnatal development of neurons containing both catecholaminergic and GABAergic traits in the rat main olfactory bulb

Postnatal development of catecholaminergic and gamma-aminobutyric acid (GABA)-ergic neurons in the periglomerular region of the rat main olfactory bulb was studied immunohistochemically using antisera against tyrosine hydroxylase (TH), glutamic acid decarboxylase and GABA. TH-like immunoreactive neurons almost always contained GABA-like immunoreactivity in the first postnatal week, but about 10% of them did not contain GABA-like immunoreactivity in older animals.     

Double-label analyses of somatostatin's coexistence with enkephalin and gamma-aminobutyric acid in amacrine cells of the chicken retina

Double-label analyses were performed to investigate somatostatin's coexistence with either enkephalin or gamma-aminobutyric acid (GABA) in amacrine cells of the chicken retina. Double-label immunocytochemistry revealed that although some amacrine cells labelled only for somatostatin or enkephalin, approx. 81% and 85% of somatostatin-immunopositive cells in the center and periphery of the retina, respectively, were also enkephalin-immunoreactive. Somatostatin-immunocytochemistry combined with autoradiography of high-affinity [3H]GABA uptake revealed that approx. 18% of somatostatin-immunoreactive amacrine cells exhibit high-affinity uptake of [3H]GABA.     

Colocalization of enkephalin-, glucagon-, and corticotropin-releasing factor-like immunoreactivity in GABAergic amacrine cells in turtle retina.

The large number of amacrine cells which contain gamma-aminobutyric acid (GABA) in the turtle retina makes it difficult to examine specific GABAergic cell types. In order to selectively label subpopulations of GABAergic neurons, we have used fluorescent double-labeling immunocytochemical techniques to examine the localization of GABA-like immunoreactivity (LI) in amacrine cells which contain antigens resembling the neuropeptides glucagon (GLUC), corticotropin-releasing factor (CRF) or enkephalin (ENK). GABA-LI was found in 41% of the cells with GLUC-, 100% of the cells with CRF-, and 69% of the cells with ENK-LI. There were regional differences in the presence of GABA-LI in amacrine cell populations with ENK-LI. GABA-LI was present in about 80% of the cells with ENK-LI outside of the visual streak, while only 37% of the cells within the streak had GABA-LI. Based on the distinct morphologies and regional distributions of these peptidergic amacrine cells, we conclude that they represent different subpopulations of GABAergic amacrine cells in the turtle retina. Future studies can now utilize existing information regarding the synaptic connectivity of these peptidergic amacrine cells to help delineate the functions of GABAergic amacrine cells in the turtle retina.     

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.     

Presence of glutamate, glycine, and gamma-aminobutyric acid in the retina of the larval sea lamprey: comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, gamma-aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter-synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin-immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin-immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA-immunolabeled perikarya were observed. GABA-immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate-immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter-expressing cells of the larval retina appear to participate only in the vertical processing pathway.     

Neuronal subpopulations in cat retina which accumulate the GABA agonist, (3H)muscimol: a combined Golgi and autoradiographic study

Golgi impregnation techniques were combined with electron microscopic autoradiography of (3H-muscimol in order to provide morphological identification of labeled neurons in the cat retina. This gamma-aminobutyric acid (GABA) agonist has been shown to label the same neurons which accumulate (3H)GABA. Selected cells were photographed and drawn by the aid of a camera lucida drawing tube prior to being thin sectioned for autoradiography. The (3H)muscimol-accumulating neurons which were identified include an interplexiform cell, five classes of conventional amacrine cell, and another cell with its soma located in the ganglion cell layer which is either a ganglion cell or a displaced amacrine. The conventional amacrine cells were compared with the recent morphological classification of Kolb et al. (Vision Res. 21: 1081-1114, '81) and identified as A2, A10, A13, A17, and A19 amacrine cells. These cells exhibit a widespread distribution providing input to all five strata of the inner plexiform layer.