Neuronal coupling in rod-signal pathways of the retina.
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Animals↗
Biomedical subjects
D I Vaney
Publications and source records attributed to D I Vaney.
The immunocytochemical detection of amino-acid neurotransmitters in paraformaldehyde-fixed tissues.
In this study, we show that specific antibodies can be raised against paraformaldehyde conjugates of amino acids, including the neurotransmitters glycine, gamma-amino-butyric acid and glutamate, and a non-neuroactive amino acid, glutamine. These antibodies against paraformaldehyde conjugates specifically detect the above amino acids in paraformaldehyde-fixed tissues. The penetration of antibodies into paraformaldehyde-fixed tissues is much superior to the penetration of antibodies into glutaraldehyde-fixed tissues; hence good labeling can be observed through the depth of the tissues. Unlike glutaraldehyde, fixation with paraformaldehyde does not give rise to high levels of tissue autofluorescence and, thus, these antibodies are very effective for immunofluorescence studies. Furthermore we suggest that the ability of these antibodies to detect amino acids in paraformaldehyde-fixed tissues will permit their use in situations where it is necessary to detect other other fixation-sensitive antigens, such as neurotransmitter receptors and transporters.
Amino Acids↗
pH-gated dopaminergic modulation of horizontal cell gap junctions in mammalian retina.
Horizontal cells mediate lateral inhibition in the outer retina, and this process is dependent on electrical coupling through gap junctions, giving rise to receptive fields that are much wider than the dendritic fields. This study on rabbit retina shows that the permeability of the gap junctions between A-type horizontal cells, as assessed by Lucifer yellow dye coupling, is modulated by dopamine through a D1 receptor linked to adenylate cyclase. Both exogenously applied dopamine and endogenously released dopamine uncoupled the horizontal cells, but the effect was pH-gated whereby it occurred only at an extracellular pH 7.2 +/- 0.05. The horizontal cells also uncoupled in acidic media (pH 7.0 or below) in the absence of dopamine. Our results show that horizontal cell coupling in the mammalian retina is regulated by both dopamine and pH. Given that the pH in the outer retina varies with the metabolic activity of the photoreceptors, these results suggest that ambient light conditions could gate the activity of neurotransmitters through pH-sensitive mechanisms.
Adenylyl Cyclases↗
Territorial organization of direction-selective ganglion cells in rabbit retina.
The On-Off direction-selective (DS) ganglion cells in the rabbit retina comprise four discrete subtypes that orthogonally code the direction of image motion. This study examined the dendritic relationships between different subtypes of On-Off DS cells, which were identified by their distinctive type 1 bistratified (BiS1) morphology following the intracellular injection of a biotinylated tracer or Lucifer yellow under direct microscopic control. The dendrites of BiS1 cells that had closely spaced somata, which presumably comprised subtypes of On-Off DS cells with different preferred directions, were not randomly superimposed but were fasciculated into loose bundles. By contrast, tracer coupling revealed that neighboring On-Off DS cells of one subtype were highly territorial, providing complete coverage of the retina with minimal overlap. This mirrors, on a larger scale, the territorial organization within the dendritic tree of individual DS cells, suggesting that similar interactions shape both the branching pattern and the spatial extent of these neurons. Moreover, the dendrites at the edge of the dendritic field often formed tip-to-shaft or tip-to-tip contacts with dendrites from coupled cells, thus appearing to form closed dendritic loops that may be equivalent to those found within the dendritic tree. Consequently, the dendrites of one subtype are distributed uniformly and economically across the retina. The resulting plexus forms a strikingly regular scaffold on which the presynaptic interneurons generate direction selectivity.
Animals↗
Unidirectional coupling of gap junctions between neuroglia.
Gap junctions permit the passage of ions and small molecules between cells, thereby providing a basis for direct intercellular communication. In the rabbit retina, the low molecular weight dyes Lucifer yellow and biocytin passed readily from astrocytes into adjacent astrocytes, oligodendrocytes, and Müller cells. However, the dyes rarely passed from either oligodendrocytes or Müller cells into astrocytes. Unidirectional passage of dye suggests the presence of an asymmetric barrier to the movement of molecules through heterologous gap junctions and indicates the potential for a hierarchy of command between interconnected cells.
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The coupling pattern of axon-bearing horizontal cells in the mammalian retina.
In most vertebrate retinae, horizontal cells of the same functional type are homologously coupled through gap junctions. However, ultrastructural, physiological and dye-coupling studies have shown that the axon terminals of horizontal cells in mammalian retinae are not connected by gap junctions. In this study, intracellular injection of the junction-permeant tracer, Neurobiotin, combined with photochromic intensification of the diaminobenzidine (DAB) reaction product, has revealed that the B-type horizontal cells in rabbit retina show strong tracer coupling, both between the dendritic trees and between the axonal arborizations. These findings establish that the coupling pattern of axon-bearing horizontal cells in a mammalian retina in qualitatively similar to the coupling patterns of axon-bearing horizontal cells in non-mammalian retinae.
3,3'-Diaminobenzidine↗
The spatial organization of tyrosine hydroxylase-immunoreactive amacrine cells in the chicken retina and the consequences of myopia.
We examined the spatial organization of the putative dopaminergic amacrine cells in the chicken retina and how this organization was affected by myopic eye enlargement. Myopia was produced by monocular lid suture for 4-7 months from hatching. Dopaminergic amacrine cells (TH-IR) were labelled by tyrosine hydroxylase immunohistochemistry. The somata of the TH-IR cells were usually located at the inner border of the inner nuclear layer; they gave rise to a dense plexus in stratum 1 (S1) of the inner plexiform layer, to a sparse plexus in stratum 3 (S3), and to short spiny dendrites at the border of strata 4 and 5 (S4/S5). The long thin processes in S1 and S3 could seldom be traced to their cell of origin, whereas the S4/S5 dendrites formed discrete fields that tiled the retina with little overlap. Lid suture resulted in retinal expansion of between 25-70%, but the total number of TH-IR amacrine cells was unaltered. Per retina, there were about 4700 TH-IR amacrine cells which showed a 3:1 density gradient from central to peripheral retina. The size of the S4/S5 dendritic fields increased proportionately in the expanded retinae, thus maintaining their coverage across the retina. The increase was achieved through scaled growth of the S4/S5 dendrites, involving both terminal and non-terminal dendrites. These findings suggest that the expansion of retinal neurons during myopia occurred through normal, albeit excessive, growth mechanisms.
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Photochromic intensification of diaminobenzidine reaction product in the presence of tetrazolium salts: applications for intracellular labelling and immunohistochemistry.
The diaminobenzidine (DAB) reaction product can be greatly intensified by incubating the reacted tissue in either nitro blue tetrazolium or tetranitro blue tetrazolium and then exposing the tissue to strong light. Epi-illumination through a microscope objective enables the photochromic intensification to be carried out under direct visual control, with optimal intensification taking only 10-30 s through a 20x objective. Alternatively, the whole preparation can be intensified in a few minutes by passing it back and forth under a fibre light guide. The method can be used to intensify cells that have been labelled either by immunoperoxidase techniques or with intracellular tracers such as horseradish peroxidase and neurobiotin.
Animals↗
Dopaminergic modulation of gap junction permeability between amacrine cells in mammalian retina.
In mammalian retina, the rod bipolar cells synapse on the AII amacrine cells, which are therefore the third-order neurons in the rod-signal pathway. The AII amacrine cells are connected by gap junctions, both to each other and to fourth-order, On-center cone bipolar cells. They also receive synaptic input from the dopaminergic amacrine cells, and in this study, we investigated whether dopamine modulates the permeability of the gap junctions between AII amacrine cells in the isolated rabbit retina. The small biotinylated tracer Neurobiotin was injected into nuclear yellow-labeled AII cells under direct microscopic control. The extent of tracer coupling to neighboring AII cells, 40-60 min after Neurobiotin injection (0.5 nA for 60 sec), provided a standard measure of the permeability of the homologous gap junctions. Under control conditions, individual AII amacrine cells were coupled to 73 +/- 15 neighboring cells, and this was unaffected by changes in pH from 6.6 to 7.8. Exogenous dopamine significantly reduced the tracer coupling at concentrations as low as 10 nM (26 +/- 16 cells), with the effect increasing with dopamine concentration up to 10 microM (6 +/- 4 cells). The uncoupling effect of dopamine was both blocked by the selective D1 antagonist SCH-23390 (10 microM) and mimicked by the specific D1 agonist SKF-38393 (500 microM). Moreover, the AII amacrine cells were also uncoupled when the retina was incubated in forskolin (60 microM) and isobutylmethylxanthine (200 microM). Taken together, these results indicated that the uncoupling was mediated by a D1-like receptor that stimulates cAMP production. Although the selective D1 antagonist on its own did not increase tracer coupling, suggesting that there was little release of endogenous dopamine in the superfused photo-bleached retina, veratridine-evoked release of endogenous transmitters did uncouple the AII amacrine cells, and this effect was blocked by the specific D1 antagonist.
1-Methyl-3-isobutylxanthine↗
Rod-signal interneurons in the rabbit retina: 1. Rod bipolar cells.
The cellular morphology and topographic distribution of the rod bipolar cells in the rabbit retina have been investigated by selective labelling with protein kinase C-immunohistochemistry (Negishi et al., Neurosci, Lett. 94:247-252, 1988) and by Lucifer Yellow injection of microscopically identified cells in a superfused retinal preparation. The distribution of the rod bipolar cells parallels that of their input neurons, the rod photoreceptors, in that the rod bipolars reach maximum densities of 5,000-7,000 cells/mm2 on the inferior and superior flanks of the visual streak, dropping to slightly lower densities at the peak visual streak. The centre-to-periphery density gradient of the rod bipolars is about 2.5:1, and the density ratio of rods to rod bipolars shows little variation across the retina, ranging from 43:1 in superior retina to 58:1 in inferior retina. The dendritic field area of the rod bipolar cells increases from 600 microns2 on the visual streak to 1,200 microns2 in the far-superior retina, with each point on the retina overlapped by 2.5-3.5 dendritic fields. The axonal field area of the rod bipolar cells increases from about 100 microns2 at the peak visual streak to about 250 microns2 at the retina edge, and the axonal field coverage ranges from 0.55 in the visual streak to about 0.8 in peripheral retina. Although there appear to be gaps in the local array of rod bipolar somata, these areas are covered by the axonal arbours of neighbouring rod bipolar cells.
Animals↗
Rod-signal interneurons in the rabbit retina: 2. AII amacrine cells.
AII amacrine cells, which are the third-order neurons in the rod pathway, can be differentially labelled in rabbit retina by injecting Nuclear Yellow into the posterior chamber. Under ultraviolet excitation, the labelled retina appears strongly metachromatic, with the AII nuclei fluorescing silvery-yellow and the nuclei of other amacrine cells fluorescing blue. Labelled AII cells were injected with Lucifer Yellow under direct microscopic control in a superfused retinal preparation, and the dye was later photoconverted to an opaque reaction product. Rabbit AII amacrines, which number about 525,000 cells, reach a maximum density of 2,500-3,000 cells/mm2 on the peak visual streak, dropping to 400-500 cells/mm2 at the superior margin. These narrow-field amacrines have a bistratified dendritic morphology, with distinctive "lobular appendages" in sublamina a of the inner plexiform layer and wider ranging "arboreal dendrites" in sublamina b. Although the lobular field area increases 10-fold from the visual streak to the far periphery, the lobular field coverage is almost uniform across the retina, averaging 1.0 in inferior retina and 0.8 in superior retina. The dendritic field area of the arboreal dendrites also increases with eccentricity from the visual streak, but there are pronounced differences between inferior and superior retina. The arboreal fields are 2 to 3 times larger than the lobular fields throughout the inferior retina but up to 15 times larger in the superior retina. The arboreal field overlap is only 1.8 at the peak visual streak, increasing slightly to about 2.4 over most of the inferior retina; the overlap increases sharply in the superior retina, however, reaching values of 10 or more in the far periphery. Both the lobular and arboreal fields of AII cells are spaced more regularly than the somata, thus covering apparent gaps in the somatic array. An analysis of the potential convergence and divergence between rod bipolar cells and AII amacrine cells in the rabbit retina indicates that the neuronal architecture of the rod circuit is not organized in a uniform module that is simply scaled-up from central to peripheral retina. Moreover, peripheral fields in the superior and inferior retina that have equivalent densities of interneurons show markedly different rod bipolar----AII amacrine convergence ratios, with the result that many more rod photoreceptors converge on an AII amacrine cell in the superior retina than in the inferior retina.(ABSTRACT TRUNCATED AT 400 WORDS)
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Many diverse types of retinal neurons show tracer coupling when injected with biocytin or Neurobiotin.
This study demonstrates that the junctional connections between rod-signal interneurons in mammalian retina can be visualized by tracer coupling, following intracellular injection of the biotinylated compounds, biocytin and Neurobiotin. In addition, many other types of retinal neurons -including B-type horizontal cells and several types of retinal ganglion cells-show specific patterns of tracer coupling, usually to cells of the same neuronal type but occasionally to cells of other neuronal classes. These findings suggest that electronic transmission occurs commonly throughout the retina and, consequently, diverse types of retinal neurons may form functional networks of coupled cells.
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The retinae of Prototherian mammals possess neuronal types that are characteristic of non-mammalian retinae.
This study has shown that the retinae of Prototherian (egg-laying) mammals possess two neuronal types that are present in non-mammalian retinae, but absent or morphologically different in the retinae of Eutherian (placental) mammals. First, endogenous serotonin-like immunoreactivity has been localized in a population of presumptive amacrine cells in the platypus retina, the first such report in a mammalian retina. Second, the protein kinase C-immunoreactive (PKC-IR) bipolar cells in the echidna retina appear similar to the PKC-IR bipolars in the chicken retina, in that their dendrites give rise to a Landolt's club and their axons are multistratified. By contrast, the PKC-IR rod bipolar cells in the rabbit and in the brushtail possum, a Metatherian (marsupial) mammal, have no Landolt's clubs and their axons form terminal lobes in the innermost stratum of the inner plexiform layer.
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The morphology and topographic distribution of substance-P-like immunoreactive amacrine cells in the cat retina.
In cat retinal wholemounts, substance-P-like immunoreactivity (SP-IR) was localized in a distinct population of amacrines whose cell bodies were normally placed in the ganglion cell layer. Although displaced amacrines accounted for 80-95% of the SP-IR amacrines in peripheral retina, this proportion decreased considerably within the area centralis, accounting for 50-80% of the labelled cells at maximum density. The SP-IR cells in both the inner nuclear and ganglion cell layers gave rise to well-defined varicose dendrites of uniform appearance that stratified around 60% depth (S3/S4) of the inner plexiform layer. In addition, sparse fine dendrites in stratum 1 (S1) could sometimes be traced to inner nuclear cells and occasionally to displaced amacrines. The combined SP-IR cell density ranged from less than 50 cells mm-2 in the far periphery to more than 500 cells mm-2 in the area centralis; the maximum density showed little individual variation despite wide differences in the proportion of displaced cells. The 39,000 SP-IR amacrines in a mapped retina had a triangular topographic distribution, with intermediate isodensity lines extending vertically in superior retina and horizontally along both arms of the visual streak. Colocalization experiments established that all SP-IR cells in cat retina showed GABA-like immunoreactivity, and that the SP-IR amacrines were quite distinct from the cholinergic amacrines identified by choline acetyltransferase immunohistochemistry.
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Neurofibrillar long-range amacrine cells in mammalian retinae.
A distinct population of wide-field, unistratified amacrine cells are shown to be selectively stained by using neurofibrillar methods in rabbit and cat retinae. Their cell bodies may be located in the inner nuclear, inner plexiform or ganglion cell layers and they branch predominantly in stratum 2 of the inner plexiform layer. Characteristically, each cell has two or more long-range distal processes which extend for 2-3 mm beyond a more symmetrical, proximal dendritic field of 0.6-0.8 mm diameter. Although the neurofibrillar long-range amacrines account for less than 1 amacrine in 500, they achieve effective coverage of the retina by both the proximal and distal dendrites.
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GABA-like immunoreactivity in NADPH-diaphorase amacrine cells of the rabbit retina.
NADPH-diaphorase histochemistry selectively stains discrete populations of retinal interneurons in diverse mammals, including two amacrine types in the rabbit retina. In this study, we have demonstrated that most of these neurons show GABA-like immunoreactivity by combining indirect immunofluorescence and diaphorase histochemistry on frozen retinal sections. The NADPH-diaphorase amacrines account for only a small proportion of the GABA-positive cells in rabbit retina, thus reinforcing the emerging consensus that GABAergic amacrines are remarkably diverse in their morphology and function.
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GABA-like immunoreactivity in cholinergic amacrine cells of the rabbit retina.
In the ganglion cell layer of the rabbit retina, the inhibitory transmitter gamma-aminobutyric acid (GABA) and its analogues are accumulated by neurons that appear to match in size and number the population of displaced amacrine cells that synthesize the excitatory transmitter acetylcholine. In this double-label study, we have established directly that the cholinergic amacrine cells, selectively stained with diamidino-phenylindole, are strongly immunoreactive with GABA antisera. The coexistence of two classical transmitters, one excitatory and the other inhibitory, in this defined neuronal population, suggests that stimulation of the cholinergic amacrines may give rise to complex responses in their target neurons.
Acetylcholine↗
Morphological identification of serotonin-accumulating neurons in the living retina.
Neurons that accumulate the transmitter serotonin have been identified in the living retina by being labeled with 5,7-dihydroxytryptamine (5,7-HT), an autofluorescent serotonin analog. Iontophoretic injection of Lucifer yellow into the labeled cells under microscopic control revealed that the serotonin-accumulating neurons in rabbit retina constitute two morphological types of amacrine cells, termed S1 and S2, whose distal dendrites are stratified at the inner margin of the inner plexiform layer. The dendritic overlap of the S1 type is extraordinarily large: each point on the retina is covered by the fields of 550 to 900 S1 amacrines, and 6 to 8 meters of their dendrites are packed into each square millimeter of retina. Such a pervasive neuropil may provide an effective substrate for diffuse transmitter release, as proposed for serotonergic fibers elsewhere in the central nervous system.
5,7-Dihydroxytryptamine↗