Amacrine Processes Research Articles

Developmental Regulation and Activity-Dependent Maintenance of GABAergic Presynaptic Inhibition onto Rod Bipolar Cell Axonal Terminals

Presynaptic inhibition onto axons regulates neuronal output, but how such inhibitory synapses develop and are maintained invivo remains unclear. Axon terminals of glutamatergic retinal rod bipolar cells (RBCs) receive GABAA and GABAC receptor-mediated synaptic inhibition. We found that perturbing GABAergic or glutamatergic neurotransmission does not prevent GABAergic synaptogenesis onto RBC axons. But, GABA release is necessary for maintaining axonal GABA receptors. This activity-dependent process is receptor subtype specific: GABAC receptors are maintained, whereas GABAA receptors containing α1, but not α3, subunits decrease over time in mice with deficient GABA synthesis. GABAA receptor distribution on RBC axons is unaffected in GABAC receptor knockout mice. Thus, GABAA and GABAC receptor maintenance are regulated separately. Although immature RBCs elevate their glutamate release when GABA synthesis is impaired, homeostatic mechanisms ensure that the RBC output operates within its normal range after eye opening, perhaps to regain proper visual processing within the scotopic pathway.

Differential distribution of glycine transporters in Müller cells and neurons in amphibian retinas

Amphibian retinas are commonly used for electrophysiological studies on neural function and transduction because they share the same general properties as higher vertebrate retinas. Glycinergic synapses have been well described in amphibian retinas. However, the role of glycine transporters in the synapses is largely unknown. We studied the distribution and function of glycine transporters in the retinas from tiger salamanders, mudpuppies, and leopard frogs by immunofluorescence labeling and whole-cell recording methods. Our results indicated that GlyT1- and GlyT2-like transporters were present in Müller cells and neurons, respectively. GlyT1 labeling was present in Müller glial cells and co-localized with Glial fibrillary acidic protein (GFAP), a Müller cell marker, whereas the GlyT2 immunoreactivity was present in the somas of amacrine cells (ACs) and processes in the inner plexiform layer (IPL) and the outer plexiform layer (OPL). Because the axon processes of glycinergic interplexiform cells (IPCs) are the only source of glycine input in the OPL, GlyT2 staining revealed a spatial pattern of the axon processes of IPCs in the OPL. The function of GlyT2 in the IPCs was studied in tiger salamander retinal horizontal cells (HCs) by whole-cell gramicidin perforated recording. The results demonstrated that inhibition of GlyT2 by a specific inhibitor, amoxapine, increased a tonic glycine input to HCs. Thus, the GlyT2 transporter is responsible for uptake of synaptic glycine in the outer retina. We also compared the distribution of glycine transporters in other amphibian species: salamander, mudpuppy, and frog. The results are consistent with the general pattern that GlyT1-like transporters are present in Müller cells and GlyT2-like transporters in neurons in amphibian retinas.

Sign-conserving amacrine neurons in the fly's external plexiform layer

Amacrine cells in the external plexiform layer of the fly's lamina have been intracellulary recorded and dye-filled for the first time. The recordings demonstrate that like the lamina's short photoreceptors R1-R6, type 1 lamina amacrine neurons exhibit nonspiking, "sign-conserving" sustained depolarizations in response to illumination. This contrasts with the sign-inverting responses that typify first-order retinotopic relay neurons: monopolar cells L1-L5 and the T1 efferent neuron. The contrast frequency tuning of amacrine neurons is similar to that of photoreceptors and large lamina monopolar cells. Initial observations indicate that lamina amacrine receptive fields are also photoreceptor-like, suggesting either that their inputs originate from a small number of neighboring visual sampling units (VSUs), or that locally generated potentials decay rapidly with displacement. Lamina amacrines also respond to motion, and in one recording these responses were selective for the orientation of moving edges. This functional organization corresponds to the anatomy of amacrine cells, in which postsynaptic inputs from several neighboring photoreceptor endings are linked by a network of very thin distal processes. In this way, each VSU can receive convergent inputs from a surround of amacrine processes. This arrangement is well suited for relaying responses to local intensity fluctuations from neighboring VSUs to a central VSU where amacrines are known to be presynaptic to the dendrites of the T1 efferent. The T1 terminal converges at a deeper level with that of the L2 monopolar cell relaying from the same optic cartridge. Thus, the localized spatial responses and receptor-like temporal response properties of amacrines are consistent with possible roles in lateral inhibition, motion processing, or orientation processing.

Identification and characterization of an aquaporin 1 immunoreactive amacrine-type cell of the mouse retina

Using immunocytochemistry, a type of amacrine cell that is immunoreactive for aquaporin 1 was identified in the mouse retina. AQP1 immunoreactivity was found in photoreceptor cells of the outer nuclear layer (ONL) and in a distinct type of amacrine cells of the inner nuclear layer (INL). AQP1-immunoreactive (IR) amacrine cell somata were located in the INL and their processes extended through strata 3 and 4 of the inner plexiform layer (IPL) with thin varicosities. The density of the AQP1-IR amacrine cells increased from 100/mm(2) in the peripheral retina to 350/mm(2) in the central retina. The AQP1-IR amacrine cells comprise 0.5% of the total amacrine cells. The AQP1-IR amacrine cell bodies formed a regular mosaic, which suggested that they represent a single type of amacrine cell. Double labeling with AQP1 and glycine, gamma-aminobutyric acid (GABA) or GAD(65) antiserum demonstrated that the AQP1-IR amacrine cells expressed GABA or GAD(65) but not glycine. Their synaptic input was primarily from other amacrine cell processes. They also received synaptic inputs from a few cone bipolar cells. The primary synaptic targets were ganglion cells, followed by other amacrine cells and cone bipolar cells. In addition, gap junctions between an AQP1-IR amacrine process and another amacrine process were rarely observed. In summary, a GABAergic amacrine cell type labeled by an antibody against AQP1 was identified in the mouse retina and was found to play a possible role in transferring a certain type of visual information from other amacrine or a few cone bipolar cells primarily to ganglion cells.

Localization of metabotropic glutamate receptors mGluR1alpha and mGluR2/3 in the cat retina.

The distribution of metabotropic glutamate receptors 1alpha (mGluR1alpha) and mGluR2/3 in the cat retina was studied through the use of preembedding immunocytochemistry for light and electron microscopy. Staining for mGluR1alpha in the outer plexiform layer was seen in numerous punctate structures that were identified as rod spherules. Cone pedicles remained unlabeled. A number of amacrine and ganglion cell somata also were stained with processes ramifying throughout the inner plexiform layer. These processes were postsynaptic to cone bipolar cells in both sublaminae, where they comprised one but not both of the postsynaptic elements at dyad contacts. Immunostaining for mGluR2/3 was observed in horizontal cells as well as in numerous amacrine and displaced amacrine cells. Labeled amacrine processes were postsynaptic to cone bipolar cells in both sublaminae but, similar to mGluR1alpha, comprised only one of the postsynaptic elements. Staining for mGluR2/3 also was seen in amacrine processes postsynaptic to rod bipolar terminals; these processes were identified as belonging to type A17 amacrine cells. The distribution patterns indicate that both mGluR1alpha and mGluR2/3 are positioned for postsynaptic function, whereas mGluR1alpha also may contribute to the presynaptic regulation of glutamate release from rod photoreceptors.

Synaptic connections involving immunoreactive glycine receptors in the turtle retina.

The distribution of glycine receptors in the turtle retina was studied with the aid of a monoclonal antibody that detects the 93-kD protein associated with the strychnine-sensitive glycine receptor. Light microscopically, receptors were found in the inner plexiform layer and, more sparsely, in the innermost parts of the inner nuclear layer. No receptors were seen to be associated with photoreceptor cells, horizontal cells, or any other structures in the distal inner nuclear layer or outer plexiform layer. Ultrastructurally, glycine receptors were found on the inner face of postsynaptic membranes of processes from amacrine and presumed ganglion cells and always involved amacrine cell processes as the presynaptic element. Such glycine receptor immunoreactive synapses onto amacrine cell processes were distributed throughout the inner plexiform layer with a peak density near the middle. On the other hand, output synapses onto ganglion cell processes displaying immunoreactive glycine receptor sites showed a bimodal distribution in the inner plexiform layer. Glycine receptor immunoreactivity was not detected on bipolar cells, but presumed glycine-utilizing processes (i.e. those presynaptic to immunoreactive glycine receptors) were occasionally found to be postsynaptic in bipolar cell dyads. The majority of the synaptic input to the presumed glycine-utilizing amacrine cell processes was from other amacrine processes, some of which were themselves glycine utilizing. The observations suggest that glycinergic synapses in the turtle retina are, to a large extent, engaged in processing interamacrine signals.

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.

Distribution of GABA-immunoreactive amacrine cell synapses in the inner plexiform layer of macaque monkey retina

The distribution patterns of GABA immunoreactive (+) and immunonegative (-) amacrine cell synapses and profiles in the inner plexiform layer (IPL) were analyzed in three macaque monkey retinas using postembedding electron-microscopic (EM) immunogold cytochemistry. Synapses and profiles were counted at 5% intervals throughout the IPL depth in three EM montages (total area = 6509 microns 2), with 0% depth at the inner nuclear layer/IPL border. Nearly 70% of all amacrine synapses were GABA+, and they contacted all major classes of neurons that arborize in the IPL: bipolars (45%), ganglion cells (25%), and GABA+ (20%) and GABA- (10%) amacrines. A major relationship was seen between GABA+ amacrine processes and bipolar terminals: 76% of all amacrine-to-bipolar synapses were GABA+, and 82% of bipolar output dyads contained at least one GABA+ amacrine. GABA+ amacrine profiles (N = 2455) were concentrated in three wide bands at IPL depths of 0-25%, 40-60%, and 75-100%, corresponding to the dense bands seen with light-microscopic immunocytochemistry. In contrast, GABA+ amacrine synapses (N = 1081) were distributed evenly throughout the IPL depth, rather than being confined to the three dense bands. GABA- amacrine synapses (N = 516) were concentrated at 40% and 60% depths. Each category of amacrine output synapses had a characteristic pattern of stratification in the IPL. GABA+ amacrine-to-bipolar synapses occurred throughout the IPL but were most frequent at 20% and 80% IPL depths, where the dendrites of midget cone bipolars arborize (Polyak, 1941). In contrast, GABA+ amacrine-to-ganglion cell synapses were concentrated at 30% and 70% IPL depths, near the dendritic arborizations of parasol ganglion cells (Watanabe & Rodieck, 1989). GABA+ synapses onto bipolars and amacrines were also concentrated at the level of rod bipolar terminals. GABA+ amacrines must play significant but different roles in ON and OFF midget and parasol pathways as well as the rod pathway.

Structure of the starburst amacrine network in the cat retina and its association with alpha ganglion cells

To investigate indirect pathways to ganglion cells we studied the starburst amacrine cell network and its relationship to the alpha ganglion cell. Starburst cells were identified by an antiserum to choline acetyltransferase and alpha cells by injection of Lucifer yellow. The density of on and off starburst cells peaks at the area centralis and decreases with eccentricity by a factor of seven. The fine amacrine processes, interrupted by distinct varicosities, arborize in a planar fashion in the inner plexiform layer. The on network, at the junction of strata 3 and 4, and the off network, in stratum 2, have a similar appearance. Neighboring starburst processes run in intimate association to form a network of bundles. As bundles cross each other, loops of irregular size and shape are formed. The loops are smallest in the area centralis and increase by a factor of three towards the periphery; correspondingly, bundle length per unit area decreases with eccentricity. However, the number of varicosities/bundle length stays constant with eccentricity as does the number of processes per bundle. Segments of the starburst network associate over fairly long distances with dendrites of alpha ganglion cells. About 26% of the alpha ganglion dendritic tree shows such association, and this is significantly greater than would be expected if the alpha and starburst processes were independent. We conclude that the functional unit of the starburst cell is a linear bundle of processes and that the starburst network may connect synaptically to the alpha cell.

The ON-alpha ganglion cell of the cat retina and its presynaptic cell types

Anatomical circuits converging onto the ON-alpha (Y) ganglion cell were studied by computer-assisted reconstruction of substantial portions of 2 alpha cells from electron micrographs of serial sections. The alpha cells in the area centralis were labeled by a Golgi-like retrograde filling with horseradish peroxidase, and certain presynaptic amacrine processes were labeled by uptake of 3H-glycine. About 4400 synapses contacted the alpha cell. Eighty-six percent were from amacrine cells; the rest were from bipolar cells. About one-quarter of the amacrine synapses were specifically labeled by 3H-glycine and probably belong to the A4 amacrine. The bipolar inputs were provided by several types: cone bipolar CBb1 (85%), cone bipolar CBb5 (2%), the rod bipolar (5%), and some unidentified cone bipolars (11%). Contacts from each type occurred in specific strata, with the consequence that they tended to form spots or annulli over the alpha dendritic field. The CBb1 bipolars formed a moderately dense array (8000/mm2), with a nearest-neighbor distance of 8.6 +/- 1.3 microns. Most members of the array (84%) contacted the alpha cell, providing 1-7 synapses (average, 2.7 +/- 1.6). The placement of contacts from an individual CBb1 followed certain rules: they were restricted to a parent branch of the alpha arbor or to 2 daughter branches, but almost never crossed a branch of the alpha arbor. The synaptic territory of an individual CBb1 was not shared with other b1s (or cone bipolars of any sort), although it was shared with amacrine contacts. Rod bipolar cells also formed a very dense array (54,500/mm2) in the alpha dendritic field, but only a few of these (3%) contacted the alpha cell. The concentric receptive field of the CBb1, combined with the spatial organization of its array, is used to predict the CBb1 contribution to the alpha cell receptive field; this contribution resembles the spatial and temporal organization of the alpha receptive field itself.

Synaptic organization of dopaminergic interplexiform cells in the goldfish retina.

The synaptic organization of dopaminergic interplexiform cells (DA-IPC) in the goldfish retina was studied by a combined double-label electron-microscopical (EM) immunocytochemical/autoradiographical study. DA-IPCs were labeled with antisera against tyrosine hydroxylase. The possibility of synaptic contact with GABAergic amacrine cells in the proximal inner plexiform layer (IPL) was studied by using 3H-GABA uptake. Most synaptic input and output from DA-IPC processes involved amacrine cell processes. In addition, synaptic interactions were observed between DA-IPC processes and bipolar cell terminals, other DA-IPC processes, very small dendrites in the IPL, ganglion cell and optic fiber layers (OFL), and cell bodies in the ganglion cell layer (GCL). Input and output synapses with GABAergic amacrine processes also were observed. Two-thirds of the DA-IPC boutons in the proximal IPL were involved in "junctional appositions," that is, the junctions appeared to be specialized but they were different than classical chemical synapses. The synaptic organization of DA-IPCs in the goldfish IPL appears to be far more complex than previously thought. Although earlier studies have attempted to explain the action of dopamine in terms of interaction only with amacrine cells, the present study shows that effects involving bipolar cells, other DA-IPCs, unidentified processes and cell bodies in the GCL and OFL must be considered as well.

Rod bipolar array in the cat retina: pattern of input from rods and GABA-accumulating amacrine cells.

The potential and actual connections between rod and rod bipolar arrays in the area centralis of the cat retina were studied by electron microscopy of serial ultrathin sections. In the region studied there were about 378,000 rods/mm2 and 36,000-47,000 rod bipolars/mm2. The tangential spread of rod bipolar dendrites was 11.2 microns in diameter, and the "coverage factor" for the rod bipolar cell was 3.5-4.6. We estimate that about 37 rods potentially converge on a rod bipolar cell and that one rod potentially diverges to about four rod bipolar cells. The actual connections, however, are less than this by about half: 16-20 rods actually converge on a bipolar cell and one rod actually diverges to slightly less than two rod bipolar cells. The degree of convergence appears to reflect a compromise between the need to signal graded stimulus intensities (requiring wide convergence) and the need to maintain a good signal/noise ratio (requiring narrow convergence). Amacrine varicosities that provide reciprocal contact at the rod bipolar dyad were studied in serial electron microscopic autoradiograms following intraocular administration of 3H-GABA or 3H-glycine. More that 90% of the reciprocal amacrine processes accumulated GABA in a specific fashion. This information, in conjunction with Nelson's recordings from the rod bipolar and amacrine cells postsynaptic at the dyad (Nelson et al: Invest. Ophthalmol. 15:946-953, '76; Kolb and Nelson: Vision Res. 23:301-312, '83), suggests that feedback at the rod bipolar output might be positive.

Morphological changes induced in turtle retinal neurons by exposure to 6-hydroxydopamine and 5,6-dihydroxytryptamine.

Following intraocular injection of the dopamine neurotoxin 6-hydroxydopamine (10-50 micrograms on two successive days in a Ringer vehicle containing ascorbate and pargyline) and an incubation period of 1 to 18 days, degeneration was noted in presumptive amacrine cells in the retina of the turtle, Pseudemys scripta elegans. Injection of vehicle alone produced no effect. Affected perikarya initially showed swollen mitochondria, lysosomes and distended cisternae. At later stages the cells took on a darkened appearance. In contrast, affected amacrine processes in the inner plexiform layer became markedly distended and lost their cytoplasmic contents, resulting in empty, very swollen profiles. No degeneration was noted distal to the affected cell bodies, i.e. the affected cells were not interplexiform neurons. Cells lesioned by 6-hydroxydopamine were shown to accumulate [3H]dopamine. Intraocular administration of 5,6-dihydroxytryptamine (a single dose of 10-40 micrograms in the same vehicle) followed by 4-6 days incubation resulted in a marked darkening of certain bipolar cell axon terminals, cell bodies and Landolt's clubs. The toxic effects of 5,6-dihydroxytryptamine were blocked by zimelidine, a serotonin uptake blocker. Thus, these two neurotoxins have different targets in the turtle retina. At the highest dose tested, however, 6-hydroxydopamine did produce degenerative changes in the presumed serotonergic bipolar cell.

Molecular specificity of defined types of amacrine synapse in cat retina

The inner plexiform layer of cat retina contains synaptic structures belonging to 50 or more types of "identified" neurons. To learn whether there are antigens confined to subsets of these synaptic structures, we raised monoclonal antibodies to homogenates of neural retina. Binding patterns of these antibodies were visualized by the peroxidase-antiperoxidase method and studied in serial, ultrathin sections by electron microscopy. Four antibodies stained the synaptic varicosities of certain amacrine cells. Many of the stained varicosities formed reciprocal synapses with a rod bipolar axon terminal, but only about half of the reciprocal synapses associated with a rod bipolar were stained. Other stained varicosities formed synapses with cone bipolar axons, ganglion cell dendrites, and unstained amacrine processes. The patterns were essentially the same for each antibody and were not altered by staining with the antibodies two at a time; therefore, it is likely that all four antibodies stain the same subset of synaptic structures. These patterns would be accounted for if there were staining of all the synaptic varicosities of three of the four types of identified amacrine reciprocally connected to the rod bipolar (A6, A8, A13). This localization suggests that the antigen responsible for the binding pattern is not associated with synaptic transmission. Staining is present in the inner plexiform layer during the period of synaptogenesis and consequently the antibodies are serving as markers for following the development of identified synapses in an identified neural circuit.

Aspartate aminotransferase-like immunoreactivity in the guinea pig and monkey retinas.

The excitatory amino acids, aspartate and glutamate, have been proposed as retinal neurotransmitters. Aspartate aminotransferase (AAT) is an enzyme which is involved in the routine metabolism of these amino acids and may be involved in the specific synthesis of glutamate and/or aspartate for use as a neurotransmitter. On the basis of the hypothesis that increased levels of aspartate aminotransferase may reflect a transmitter role for aspartate and/or glutamate, we have localized aspartate aminotransferase in the guinea pig and cynamolgus monkey retinas with light and electron microscopic immunohistochemical techniques. AAT-like immunoreactivity is localized to the cones of guinea pig retina and to monkey rods. Both species contain a subpopulation of immunoreactive amacrine cells as well as a subpopulation of immunoreactive cells in the ganglion cell layer. Immunostaining is seen in bipolar cells and terminals in the monkey but not in the guinea pig retina. We have performed quantitative analysis of the immunoreactive staining in the outer plexiform layer and described the synaptic organization of immunoreactive processes in the inner plexiform layer (IPL). Labeled amacrine processes in both species form synaptic contacts predominantly to and from bipolar terminals in the inner third of the IPL and to and from other amacrine and small unidentified processes in the outer portion of the IPL. The majority of labeled bipolar terminals in the monkey retina are seen in the inner third of the IPL where they synapse exclusively onto amacrine processes. Labeled bipolar terminals in the outer third of the IPL occasionally synapse onto ganglion processes.

Intracellular control of axial shape in non-uniform neurites: a serial electron microscopic analysis of organelles and microtubules in AI and AII retinal amacrine neurites.

AI and AII cat retinal amacrine cells have highly varicose non-uniform, neuritic processes. Processes of both types were reconstructed via a computer system using serial electron micrographs. These reconstructions were analyzed for (a) varicosity volume, surface area, and length, (b) "neck" volume, surface area, and length, (c) number of microtubules within the varicosity, (d) number of microtubules within the "neck," and (e) volume and surface area of mitochondria and smooth endoplasmic reticulum and large smooth vesicular bodies within the processes. Correlation of these parameters revealed a linear relationship between the number of microtubules in the necks and mean neck cross-sectional area (rs = 0.780, P less than 0.001), while microtubule number within the varicosities showed no correlation with varicosity volume (rs = 0.239, P greater than 0.2). Varicosity volume did, however, correlate strongly with the summed volume of mitochondria and smooth vesicular bodies contained within the varicosity for both cell types examined. The ratio between membranous organelle volume and varicosity volume for AI amacrine processes of 1:6.97 (rs = 0.927), differed from the ratio of 1:1.80 for the AII amacrine processes (rs = 0.987). Similar relationships were observed in other nonvaricose neurites such as optic tract axons. Membranous organelles appear to contribute an additional obligatory volume to the cytosol that can be as much as seven times the organelles' direct volume. These observations suggest that both the cytoskeletal components, and the membrane organelles play a direct role in determining neurite shape.

Localization of synaptic and nonsynaptic nicotinic-acetylcholine receptors in the goldfish retina.

The localization of nicotinic-cholinergic receptors in the inner plexiform layer (IPL) of goldfish retina was studied by electron microscopic analysis of the binding pattern of a conjugate or horseradish peroxidase and alpha bungarotoxin (HRP-alpha BTx). Specific HRP reaction product (blockade by 1mM curare) was found at both synaptic and nonsynaptic sites. Synaptic binding sites for HRP-alpha BTx, which accounted for only 16% of the total specific reaction product sites, always involved an amacrine process as the presynaptic element, whereas amacrine, ganglion, and bipolar cells could be post-synaptic elements at labeled synapses. Only 17.5% of the total number of amacrine synapses were labeled by HRP-alpha BTx. Labeled synapses showed the same distribution in the IPL as unlabeled synapses: bimodal for amacrine-to-bipolar synapses with peak concentrations at the 20% and 80% layers and unimodal for amacrine-to-nonbipolar synapses with a peak concentration at the 60% layer. Nonsynaptic binding sites for HRP-alpha BTx (84% of total) were seen on the dendrites of ganglion, amacrine, and bipolar cells. The distribution of the nonsynaptic sites in the IPL largely accounts for the trilaminar binding pattern of 125I-alpha BTx as observed in light microscopic autoradiographs. If, as appears likely, the distribution of synapses is the relevant variable in determining the sites of neuronal interaction for a given transmitter system, then this study further illustrates the importance of distinguishing synaptic from nonsynaptic binding when using receptor-ligand probes to localize sites of chemical synaptic transmission.

GABAergic amacrine cells in rat retina: immunocytochemical identification and synaptic connectivity.

GABAergic neurons have been identified in light and electron microscopic preparations of rat retina by an immunocytochemical localization of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD). GAD-positive neuronal somata are found only in the inner and middle parts of the inner nuclear layer, and GAD-positive neuronal terminals are observed exclusively within the inner plexiform layer (IPL) and the outermost part of the ganglion cell layer. Dense aggregations of GAD-positive terminals alternate with less dense zones to form a lamination of the IPL. GAD-positive terminals contain pleomorphic synaptic vesicles and are the presynaptic elements of conventional synapses onto bipolar and amacrine cell processes, as well as onto the somata and dendrites of ganglion cells. In addition, GAD-positive terminals are postsynaptic to unstained bipolar terminals and are components of synaptic dyads where they occasionally appear to form reciprocal synapses with the bipolar terminals, and serial with unstained amacrine processes. Probable synaptic contacts between adjacent GAD-positive terminals also have been observed. Most of the synaptic input to GAD-positive terminals comes from bipolar cells, while the small remaining input mainly comes from other GAD-positive terminals. The synaptic output to GAD-positive terminals is greatest to bipolar cells, followed in decreasing order by GAD-negative amacrine cells, ganglion cells, and other GAD-positive cells. The total synaptic output of GAD-positive cells appears to be more than twice as great as the total input of these cells. The location of GAD-positive somata, the distribution of GAD-positive terminals, and the synaptic relationships formed by these terminals all indicate that amacrine cells are the only GABAergic neurons in rat retina. Our observations also indicate that not all amacrines are GABAergic and suggest that GABAergic neurons may be limited to a narrow field subclass of amacrine cell. The findings concerning the synaptic connections of GABAergic amacrines suggest that such cells are the first link in several divergent pathways from bipolar to ganglion cells and that they probably serve more than one function since they feed synaptic activity forward directly upon ganglion cells as well as back upon bipolar cells.

Synaptic organization of the dopaminergic neurons in the rabbit retina

AbstractA 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.

Synaptic orgnization of the inner plexiform layer in the retina of the tiger salamander.

The synaptic morphology and organization of the inner plexiform layer of the salamander retina was examined in serial sections with the electron microscope. Processes were traced, whenever possible, from their cells of origin, and synapses were classified as ribbon or regular (non-ribbon, conventional). Amacrine processes always make regular synapses, while bipolar processes make both ribbon and regular synapses. For this reason, new and decisive criteria based on the differential morphology of synaptic vesicles and junctional membranes were sought to distinguish between the amacrine and bipolar processes in single sections. Amacrine processes contain a relatively uniform population of small round vesicles and they make regular symmetrical synapses. Bipolar processes contain vesicles that are generally larger and more pleomorphic than those of the amacrine processes, and they make ribbon synapses (monads, dyads and triads) and regular synapses of the asymmetrical type. Amacrine processes synapse upon other amacrine processes, bipolar axons, ganglion cell dendrites, and the perikarya of these three types of cells. Amacrine cells also give rise to somatodendritic synapses. Bipolar processes synapse upon amacrine processes, ganglion cell dendrites and other bipolar processes, but they have not been seen to synapse upon the somata of any cell. Both amacrine and bipolar processes engage in serial synapses, and these two groups often make reciprocal synapses with each other. Gap junctions have been found between two bipolar processes, between two amacrine processes, and less frequently, between a bipolar and an amacrine process.