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.     

Quantitative studies of enkephalin's coexistence with γ-aminobutyric acid, glycine and neurotensin in amacrine cells of the chicken retina

Previous double-label studies demonstrate that enkephalin coexists with γ-aminobutyric acid, glycine or neurotensin in amacrine cells of the chicken retina. The present study utilizes double- and triple-label paradigms to quantitatively analyze these coexisting relationships. Twenty-eight percent of enkephalin-like immunoreactive amacrine cells were found to exhibit high-affinity uptake of [³H]GABA, while 53% of enkephalin-amacrine cells specifically accumulate [³H]glycine. Moreover, the present study predicts that at least 26% of enkephalin-amacrine cells which accumulate [³H]glycine should also be immunoreactive for neurotensin.     

Interactions between enkephalin and gamma-aminobutyric acid in the larval tiger salamander retina

Both double-label and intracellular electrophysiological recording techniques were utilized to investigate the interactions between enkephalin and gamma-aminobutyric acid in the larval tiger salamander retina. Double-label studies revealed that the vast majority (greater than 96%) of enkephalin-immunostained amacrine cells also exhibit high affinity uptake of [3H]gamma-aminobutyric acid. Electrophysiological evidence demonstrated that morphine and gamma-aminobutyric acid exert opposite effects on a population of On-Off ganglion cells. gamma-Aminobutyric acid decreased the activity of these cells, while enkephalin increased their activity. These findings support the idea that opiate-mediated pathways inhibit GABAergic pathways in the vertebrate retina.     

Immunocytochemical and autoradiographic localization of GABAergic neurons in the goldfish retina

The putative neurotransmitter gamma-aminobutyric acid (GABA) was localized in goldfish retina by using an antiserum directed against GABA itself. The same types of cells were stained with this antibody as were labelled with an antiserum directed against the synthesizing enzyme for GABA, glutamic acid decarboxylase. Stained neurites of these cells were located throughout the inner plexiform layer (IPL) but staining was more intense in the proximal IPL. The GABA-immunoreactive staining could be reduced or completely abolished by preabsorbing the primary antibody with GABA. Uptake of [3H]-GABA or the GABA agonist [3H]-muscimol was localized in GABA-stained retinas using light microscope autoradiography. These experiments demonstrated that all types of GABA-immunoreactive amacrine cells had high-affinity uptake mechanisms for both [3H]-GABA and -muscimol. Thirty percent of proximal inner nuclear layer (INL) and some cells in the ganglion cell layer (GCL) were labelled by all three GABAergic markers. Most GABA-immunoreactive amacrine cells were lightly labelled due to [3H]-GABA uptake but a few amacrines (Ab) were heavily labelled. These findings demonstrate that the autoradiographic localization of [3H]-GABA or [3H]-muscimol uptake and the immunocytochemical localization of GAD or GABA are appropriate methods for localizing GABAergic neurons in the retina. Few GABA-immunoreactive amacrine cells accumulated the putative amino acid transmitter [3H]-glycine, verifying that the goldfish retina contains distinct subpopulations of glycinergic and GABAergic amacrine cells.     

Interactions between enkephalin and γ-aminobutyric acid in the larval tiger salamander retina

Both double-label and intracellular electrophysiological recording techniques were utilized to investigate the interactions between enkephalin and γ-aminobutyric acid in the larval tiger salamander retina.Double-label studies revealed that the vast majority (96%) 96%) of enkephalin-immunostained amacrine cells also exhibit high affinity uptake of [³H]γ-aminobutyric acid. Electrophysiological evidence demonstrated that morphine and γ-aminobutyric acid exert opposite effects on a population of On-Off ganglion cells. γ-Aminobutyric acid decreased the activity of these cells, while enkephalin increased their activity. These findings support the idea that opiate-mediated pathways inhibit GABAergic pathways in the vertebrate retina.     

GABAergic input to the synaptic terminals of mb1 bipolar cells in the goldfish retina

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.     

Two types of pyriform Ab amacrine cells in the goldfish retina: an EM analysis of [3H]GABA uptake and somatostatin-like immunoreactivity

Pyriform Ab amacrine cells in the goldfish retina take up [3H]GABA and show somatostatin-like immunoreactivity (SLIR), leading to the question of whether these two markers are labeling the same or different types of Ab amacrine cells. We used a double-label radio/immuno-technique at the electron microscopical level to visualize the comparative location of [3H]GABA uptake and SLIR in Ab amacrine cells and their processes. SLIR was restricted to large dense-cored vesicles in processes of Ab amacrine cells. In no case were processes labeled with SLIR observed to take up [3H]GABA. Thus, there are at least two types of pyriform Ab amacrine cells: one that takes up [3H]GABA and one that shows SLIR. These may correspond to the two types of Ab amacrine cells described by Cajal in his classic studies on the retina.     

GABA-ergic pathways in the goldfish retina.

A high-affinity uptake mechanism for [3H]-gamma-aminobutyric acid (GABA) has been localized to type H1 cone horizontal cells and type Ab pyriform amacrine cells in the retina of the goldfish by light and electron microscopy autoradiography. By stimulating isolated retinas with colored lights during incubation we have been able to use [3H]-GABA uptake as a probe of light-evoked changes in membrane potential. All colors of lights increase and darkness decreases [3H]-GABA uptake by H1 cone horizontal cells. Our model of voltage dependence of GABA uptake predicts that all colors of light should hyperpolarize H1 cone horizontal cells and other investigators have shown by intracellular recording and dye-marking that type H1 cone horizontal cells hyperpolarize to all wavelengths of light. We have also obtained evidence that dark-induced depolarization of cone horizontal cells leads to release of GABA. Type Ab pyriform amacrine cells show maximal [3H]-GABA uptake in darkness and when exposed to green or blue lights, but red lights dramatically suppress uptake. We predict these neurons to be red-depolarizing, and recent intracellular recordings and dye-marking by Famiglietti et al. ('77) support our conclusions. Synaptic relations of apparently GABA-ergic neurons were investigated in the electron microscope. We propose type H1 cone horizontal cells to be both pre- and post-synaptic to red-sensitive cones and type Ab pyriform amacrine cells to be both pre- and post-synaptic to red-sensitive center-depolarizing bipolar cells.     

Two types of pyriform Ab amacrine cells in the goldfish retina: an EM analysis of [H]GABA uptake and somatostatin-like immunoreactivity

Pyriform Ab amacrine cells in the goldfish retina take up [³H]GABA and show somatostatin-like immunoreactivity (SLIR), leading to the question of whether these two markers are labeling the same or different types of Ab amacrine cells. We used a double-label radio/immuno-technique at the electron microscopical level to visualize the comparative location of [³H]GABA uptake and SLIR in Ab amacrine cells and their processes. SLIR was restricted to large dense-cored vesicles in processes of Ab amacrine cells. In no case were processes labeled with SLIR observed to take up [³H]GABA. Thus, there are at least two types of pyriform Ab amacrine cells: one that takes up [³H]GABA and one that shows SLIR. These may correspond to the two types of Ab amacrine cells described by Cajal in his classic studies on the retina.     

Identification and characterisation of cell types accumulating GABA in rat retinal cultures using cell type specific monoclonal antibodies

Monolayer and reaggregate cultures have been established from neonatal rat retina. After 7 days in culture, 60 nM [3H]gamma-aminobutyric acid (GABA) was used to identify cells with a high affinity uptake mechanism for GABA. Approximately 80% of the process-bearing cells were found to be labelled. These cells were identified as amacrine cells by double-labelling experiments combining [3H]GABA uptake with immunocytochemical labelling with monoclonal antibody HPC-1 which in retina is specific for amacrine cells. The ability of cultures to synthesize GABA from glutamate was investigated at various times. Little synthesis was observed during the first few days in culture. This lag was followed by an increase in the amount of synthesis until 3 weeks of culture. When clumps and reaggregate cultures of retinal cells were examined by [3H]GABA uptake, a time-dependent redistribution of labelled cells was observed. After 20 h in culture, GABA-positive cells were distributed over the whole cell mass. Over the next few days, the labelled cells became more common on the outer edge of the aggregates and less common in inner regions. By 7 days of culture, no labelled cell bodies were found on the inside of the aggregates, although such cells could be labelled by [3H]D-aspartate. The results provide positive identification of a subclass of retinal cells in culture, and show that at least one aspect of retinal histogenesis is not dependent upon extra-retinal tissues or the position imposed by the temporal order of retinal cell birth.     

Development of GABAergic function of dissociated hippocampal cultures from fetal mice

The GABA uptake mechanism has been characterized in hippocampal cell cultures prepared from fetal mice of 13-19 days gestational age [3H]GABA is accumulated selectively by neuronal cells by a high-affinity (Km = 3 micro M) mechanism that is an early property of the neurons. Autoradiography of [3H]GABA uptake revealed that approximately 30% of the neuronal cells had uptake. A unique small neuronal type did not have [3H]GABA uptake but was found to have [3H]glutamic acid uptake, suggesting that these neurons may be granule cells. The selective labeling of neurons with [3H]GABA and [3H]glutamic acid is consistent with the idea that high-affinity uptake of a transmitter may be unique to neurons that use that transmitter.     

Glutamate receptor agonists release [3H]GABA preferentially from horizontal cells

A total of 5-6 different cell types in vertebrate retinas accumulate [3H]gamma-aminobutyric acid (GABA). In frog retina, specific populations of cells in the horizontal, amacrine and ganglion cell layers are labeled autoradiographically after a 15-min in vitro incubation with [3H]GABA. Cells which may be bipolar or interplexiform cells are also labeled. Similar autoradiographic patterns are observed in chick retina except for the absence of labeled bipolar or interplexiform cells. In rat retinas, [3H]GABA uptake is limited primarily to Muller and amacrine cells. Depolarizing glutamate receptor agonists (glutamate, aspartate and kainic acid) applied in an in vitro perfusion system, stimulated massive release of [3H]GABA from frog and chick retina but not from rat retina. Under these conditions, autoradiographic labeling of horizontal cells was virtually depleted, while labeling of other cell types remained robust. In contrast, potassium caused release of the label from all 3 types of retina, and loss of autoradiographic labeling occurred uniformly in all cell types. We conclude that [3H]GABA-accumulating horizontal cells possess depolarizing glutamate receptors and that activation of these receptors leads to a release of GABA stores. On the other hand, Muller cells and the various subclasses of [3H]GABA-accumulating amacrine, bipolar and/or interplexiform cells, do not release GABA in response to glutamate receptor stimulation and thus appear to be relatively insensitive to excitatory amino acids.     

Autoradiographic localization of [H]muscimol in the cat retina

In the cat retina, [³H]muscimol is localized in 5 morphologically distinct sub-populations of neurons with cell bodies in the amacrine layer and in other neurons located in the ganglion cell layer. Mu¨ller cells are unlabeled. The labeled subpopulations in the amacrine layer correspond to the subpopulations which also exhibit preferential uptake of [³H]GABA. The [³H]muscimol-labeled cells include interplexiform cells and type-AI reciprocal amacrine cells.     

Interaction between enkephalin and γ-aminobutyric acid in the chicken retina: A double-label immunoelectron microscopic analysis

In the present study, double-label immunoelectron microscopy was used to examine the synaptic relationships between amacrine cell populations in the chicken retina that contain either enkephalin or γ-aminobutyric acid (GABA) or both enkephalin and GABA. The objectives of the present study were twofold. First, the ultrastructural features and synaptic organization of enkephalin and enkephalin/GABA amacrine cells were compared. Second, the synaptic interactions between these populations and the population of GABA amacrine cells were examined. A total of 475 synaptic arrangements were observed to involve enkephalin or enkephalin/GABA amacrine cell processes. The synaptic relationships of enkephalin and enkephalin/GABA amacrine cells were quite similar. Each population was pre- and postsynaptic to amacrine cells, postsynaptic to bipolar cells, and presynaptic to processes possibly originating from ganglion cells. A substantial percentage of each population's pre- and postsynaptic relationships were with the processes of GABAergic amacrine cells. Moreover, when enkephalin and enkephalin/ GABA amacrine cell processes were postsynaptic to bipolar cells, their dyadic partner was observed frequently to be a GABA amacrine cell process. The present study suggests a diversity in the population of chicken enkephalin amacrine cells with respect to their expression of the classical inhibitory transmitter GABA. Moreover, a functional relationship between enkephalinergic and GABAergic pathways is indicated by studies showing that both enkephalin and enkephalin/GABA amacrine cells exhibit substantial synaptic interaction with GABA amacrine cells. © 1994 Wiley-Liss, Inc.     

Carrier-mediated release of GABA from retinal horizontal cells

H1 horizontal cells in goldfish retina are probably GABAergic and receive excitatory synaptic input from red cones. This input should affect the synaptic release of GABA from H1 cells. We studied the uptake and release of [3H]GABA from the isolated goldfish retina by use of autoradiography. When retinas were incubated in the light for 15 min in 0.72 microM [3H]GABA, heavy label was found over the somata (HS) and axon terminals (HAT) of H1 horizontal cells, and over pyriform amacrine cell bodies and their processes in sublamina b of the IPL. Postincubation of retinas, preloaded with [3H]GABA, in 0.5-10 mM L-glutamate or 0.1-10 mM L-aspartate, resulted in a dose-dependent and selective loss of [3H]GABA from HS and very little loss from HAT. This loss was not due to an efflux of metabolites of [3H]GABA or to any calcium-dependent vesicular release of [3H]GABA from HS. The glutamate-evoked release of [3H]GABA by H1 cells was sodium dependent, sensitive to substitution of lithium for sodium, and inhibited by nipecotic acid. In addition, [3H]GABA was released from HS by 0.1 mM ouabain but not by 50 mM potassium chloride. Our results suggest that the chemically evoked release of [3H]GABA from HS is mediated by a sodium-dependent transport carrier which may be responsible for the high affinity uptake of [3H]GABA by H1 cells as well. Since synaptic vesicles are not found at presumed synaptic release sites in H1 cells, we suggest that the GABA which is released synaptically from H1 cells may derive from a cytoplasmic pool of GABA and is released by means of a transport carrier. This carrier appears to depend primarily on the sodium concentration gradient across the H1 cell membrane rather than on the membrane potential of the H1 cell for its action. The relevance of the carrier-mediated release of GABA from HS in regard to the synaptic function of H1 cells is discussed.     

GABA uptake by purified avian Müller glia cells in culture

GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na(+)/Cl (-) dependent transporters (GATs). It has been reported in the past that, in the avian retina, [(3)H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [(3)H]GABA in a Na(+) and Cl(+) dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [(3)H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K[(+) or veratridine stimulation, Müller glial cells did not release [(3)H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.     

Double-label analyses demonstrating the non-coexistence of enkephalin and glycine in amacrine cells of the larval tiger salamander retina

Enkephalin immunocytochemistry was combined with either glycine immunocytochemistry or autoradiography of high-affinity glycine uptake to examine for colocalization of enkephalin and glycine in amacrine cells of the larval tiger salamander retina. A total of 995 enkephalin-immunoreactive amacrine cells were visualized in double-label preparations. None of the enkephalin-labelled cells was observed to co-label for markers of glycinergic activity.     

Interaction between enkephalin and dopamine in the avian retina

Biochemical and pharmacological techniques were utilized to investigate the interaction between the enkephalinergic and dopaminergic systems in the chicken retina. Exogenously applied enkephalin and its analogues were observed to inhibit the release of preloaded dopamine from the retina. This inhibition was concentration-dependent and was suppressed by the opiate antagonist, naloxone. The relationship between enkephalinergic and dopaminergic amacrine cells was studied in retinas which were subjected to 6-hydroxydopamine (6-OHDA) treatments. 6-OHDA degenerated approximately 80-90% of those cells which exhibit high affinity uptake of [3H]dopamine. In 6-OHDA-treated retinas, the capacity of 3H-labelled [D-Ala2]methionine enkephalinamide to bind specifically to opiate receptors was substantially reduced (only 70-75% of the control). Scatchard analyses and ligand displacement studies indicated that this decrease in binding was due to a reduction in the number of opiate receptors. Taken together, these observations strongly indicate that a fraction of the opiate receptors in the chicken retina (25-30%) are closely associated with the population of dopaminergic amacrine cells.     

Quantitative analyses of the coexistence of gamma-aminobutyric acid in substance P-amacrine cells of the larval tiger salamander retina

The present study was performed as part of a systematic examination of gamma-aminobutyric acid's (GABA) coexistence with other classical transmitter and neuropeptides in neuronal populations of the larval tiger salamander retina. Substance P immunocytochemistry was combined with either GABA immunocytochemistry or autoradiography of high-affinity GABA uptake to examine for the presence of GABA in substance P-amacrine cells of the larval tiger salamander retina. Double-label analyses revealed two populations of substance P-amacrine cells that express both markers of GABA activity. One population was situated in the innermost cell row of the inner nuclear layer, while the other population was located in the ganglion cell layer. In both cases, these double-labelled cells accounted for approximately 10% of substance P-amacrine cells in their respective layers. The present study demonstrates, therefore, that substance P-amacrine cells in the larval tiger salamander retina can be categorized on the basis of their coexisting/non-coexisting relationships with GABA and suggests a possible functional diversity in the population of substance P-amacrine cells.     

Effects of gamma-aminobutyric acid on cones and bipolar cells of the tiger salamander retina

The gamma-aminobutyric acid (GABA) system in the tiger salamander retina was studied using autoradiographic and electrophysiological techniques. A high-affinity uptake mechanism for GABA has been localized in about 60% of the horizontal cells and about 30% of the amacrine cells. Effects of exogeneously applied GABA on the membrane conductance of cones and hyperpolarizing bipolar cells (HBC) were examined using the two electrode current-clamp technique in the living retinal slices. In both cell types, 1 mM of GABA caused a conductance increase. In perfused eyecups, 2 mM of GABA selectivity abolished the surround response of the HBC and left the center response unchanged. These results are consistent with the notion that a population of horizontal cells and a population of amacrine cells in the salamander retina may use GABA as their neurotransmitter.