Search PubMed⌕ Search

Biomedical subjects

R H Masland

Publications and source records attributed to R H Masland.

At least 19 recordsLinked to original sources
PubMed1992

Direct visualization of the dendritic and receptive fields of directionally selective retinal ganglion cells.

Optical methods were used to locate the cell bodies of directionally selective ganglion cells in isolated rabbit retinas. These neurons detect the direction in which images move across the retinal surface and transmit that information to the brain. The receptive field of each identified cell was determined, after which the cell was injected with Lucifer yellow. An image of the receptive field border was then projected onto the fluorescent image of the dendrites, allowing precise comparison between them. The size of the receptive field matched closely the size of the dendritic arbor of that cell. This result restricts the types of convergence that can be postulated in modeling the mechanism of retinal directional selectivity.

Amidines↗
PubMed1992

Co-release of acetylcholine and GABA by the starburst amacrine cells.

Rabbit retinas were isolated from the eye and maintained in vitro. When they were incubated for 60 min in the presence of 3H-GABA, subsequent autoradiography showed radioactivity to be present primarily in amacrine cells. Under these conditions, most of the radioactivity contained in the retinas remained in the chemical form of GABA. Autoradiography and immunohistochemistry of alternate sections showed the amacrine cells that accumulate 3H-GABA to be the same cells that contain endogenous GABA immunoreactivity. These include the starburst cells, the indoleamine-accumulating cells, and other, as yet unidentified amacrine cells. The localization confirms previous immunohistochemical findings. When retinas containing 3H-GABA were expressed to elevated concentrations of K+, their content of 3H-GABA decreased. Autoradiography showed a reduced 3H-GABA content in all of the cells that contained 3H-GABA. Since those include the starburst cells, previously shown to be cholinergic, the finding demonstrates that the starburst cells release both ACh and GABA. Retinas simultaneously labeled with 14C-GABA and 3H-ACh were superfused, and the release of radioactive compounds from the retina was studied. Depolarization by elevated K+ caused an increased recovery of both ACh and GABA in the superfusate, but the predominant mechanisms of their release appeared to be different. The stimulated release of ACh was entirely Ca2+ dependent, while the release of radioactivity originating from GABA was much less so. A concentration-dependent counterflux (homoexchange) of intracellular GABA was demonstrated by raising the extracellular concentration of GABA (or nipecotic acid). These results suggest that a large outward flux of GABA occurs via the GABA transporter, probably by the potential-sensitive mechanism studied by Schwartz (1982, 1987). Stimulation of double-labeled retinas by flashing light or moving bars always increased the release of ACh, and the release was entirely dependent on the presence of extracellular Ca2+. Stimulation with light never caused a detectable release of GABA. This was unexpected, since the two neurotransmitters are present in the same amacrine cells: stimulation adequate to release one neurotransmitter should release both.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1989

Connections of indoleamine-accumulating cells in the rabbit retina.

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

5,7-Dihydroxytryptamine↗
PubMed1989

Shape and distribution of an unusual retinal neuron.

Rabbit retinas were exposed to exogenous indoleamines and fixed with mixed aldehydes. The indoleamines were accumulated by two types of amacrine cell and by an unusual cell (type 3) that branches widely in both plexiform layers. The type 3 cells were studied after immunohistochemistry, photooxidation of the fluorescent label, or injection with Lucifer Yellow. Their cell bodies are located at the scleral margin of the inner nuclear layer. The cells' arbors in the outer plexiform layer range from 800 to 1,500 microns in diameter. A descending process crosses the inner nuclear layer and branches in layer 5 of the inner plexiform layer. The arbor in the inner retina can exceed 500 microns in diameter. The distribution of type 3 cells was mapped in a series of retinal whole mounts. The number of type 3 cells ranged from 58 to 270 in different retinas. In two retinas from a single animal, however, it was virtually identical. Type 3 cells are concentrated in the region ventral to the visual streak, so that large areas of the retina are not covered by any type 3 cell. Because of their incomplete retinal coverage and variable number from animal to animal, the type 3 cells appear to be developmental anomalies. Paradoxically, their generation must be precisely controlled because of the numerical symmetry between an individual animal's two eyes.

Aldehydes↗
PubMed1989

Indoleamine accumulation by retinal neurons exposed to blood.

Exposing rabbit retinas for one minute to an incubation medium containing 10 microliters of blood diluted in 20 ml of medium was sufficient to produce serotonin-like immunoreactivity in some of the retinal indoleamine-accumulating neurons. Retinas from rabbits that had been perfused before the eyes were removed had no detectable immunoreactivity. Our results support the conjecture that the serotonin sometimes detected in the retina originates in the blood. Why the cells have a carrier for a molecule that they do not normally contain remains unclear.

Amines↗
PubMed1989

Co-release of acetylcholine and gamma-aminobutyric acid by a retinal neuron.

Rabbit retinas were vitally stained with 4',6-diamidino-2-phenylindole (DAPI), a fluorescent compound that selectively accumulates within the cholinergic amacrine cells. The retinas were then incubated in vitro in the presence of radioactive gamma-aminobutyric acid (GABA) and autoradiographed. The cells that accumulated DAPI were found to accumulate GABA, confirming immunohistochemical evidence that the cholinergic amacrine cells contain GABA. Incubation of retinas in the presence of elevated concentrations of K+ caused them to release acetylcholine and GABA, and autoradiography showed depletion of radioactive GABA from the cholinergic amacrine cells. This indicates that the cholinergic amacrine cells can secrete acetylcholine and GABA. Retinas were double-labeled with [14C]GABA and [3H]acetylcholine, allowing simultaneous measurement of their release. The release of [14C]GABA was found to be independent of extracellular Ca2+. Radioactive GABA synthesized endogenously from [14C]glutamate behaved the same way as radioactive GABA accumulated from the medium. In the same experiments the simultaneously measured release of [3H]acetylcholine was strongly Ca2+-dependent, indicating that the releases of acetylcholine and GABA are controlled by different mechanisms. Synaptic vesicles immunologically isolated from double-labeled retinas contained much [3H]acetylcholine and little or no [14C]GABA. These results suggest that the cholinergic amacrine cells release acetylcholine primarily by vesicle exocytosis and release GABA primarily by means of a carrier.

Acetylcholine↗
PubMed1988

Amacrine cells.

Explore the source record for details and available documents.

Animals↗
PubMed1988

Photoconversion of some fluorescent markers to a diaminobenzidine product.

Retinal whole mounts, brain sections, and astrocyte cultures were labeled with various fluorescent markers. Tissues or cells were then irradiated by light in the presence of diaminobenzidine. Irradiation initiated a reaction in which specific fluorescent labeling was replaced by an insoluble diaminobenzidine product. The diaminobenzidine product is more stable than the original fluorescent labeling and can be processed for electron microscopy. In some cases, the reaction product reveals cellular detail that cannot be resolved in the fluorescent labeling. The 10 fluorescent markers tested have widely differing structures, span a broad range of wavelengths, and label several different cellular elements. The photoconversion reaction was successful with all markers and tissues tested.

Animals↗
PubMed1987

The resting release of acetylcholine by a retinal neuron.

The cholinergic amacrine cells of the rabbit retina secrete acetylcholine by two mechanisms. One is activated by stimulation of the retina by light or depolarization of the amacrine cells by K+ ions. It requires the presence of extracellular Ca2+. The second is independent of extracellular Ca2+ and is unaffected by large depolarizations of the cells. It bears some similarity to the acetylcholine 'leakage' described at the neuromuscular junction. Although the Ca2+-independent mechanism accounts for about two thirds of the total acetylcholine release in the dark, the amount of acetylcholine released in this way is small compared with the release of acetylcholine triggered by stimulation of the retina with light. Its biological significance is unclear.

Acetylcholine↗
PubMed1986

A system of indoleamine-accumulating neurons in the rabbit retina.

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

5,7-Dihydroxytryptamine↗
PubMed1985

Local order among the dendrites of an amacrine cell population.

The cholinergic amacrine cells of the rabbit retina branch within a narrow stratum of the retina's inner synaptic layer, and their dendritic fields overlap as much as 70-fold. Because each cell's dendrites have many branches, the overlap must create a dense meshwork of cholinergic dendrites. To learn how the overlapping dendrites are positioned with respect to each other, we filled the dendrites of groups of neighboring cells with Lucifer Yellow CH. The cholinergic amacrine cells were selectively stained by intraocular injection of the fluorescent molecule 4,6-diamidino-2-phenylindole. The retinas were then fixed with 2% paraformaldehyde and 0.01% glutaraldehyde. The stained cells were penetrated under visual control by Lucifer Yellow-filled micropipettes. A systematic arrangement of the dendrites was observed. When a pair of cells was injected, their dendrites were often seen to lie alongside each other. In the terminal dendritic region, there are virtually no dendrites that do not end in apposition to a dendrite of a neighboring cholinergic amacrine cell. When small clusters of nearby cells were injected, an ordered microstructure appeared. The dendrites of the cells join together to form curving bundles, which enclose spaces that rarely contain any cholinergic dendrites: the appearance of the dendritic mosaic is that of a lattice with a repeating unit roughly 10 microns in diameter. The significance of this ordering is not certain, but it is possible that the repeating structural unit participates in a modular functional arrangement.

Animals↗
PubMed1984

Acetylcholine-synthesizing amacrine cells: identification and selective staining by using radioautography and fluorescent markers.

The fluorescent DNA stain 4,6,diamidino-2-phenylindole (DAPI) was applied to the cut axons of the rabbit optic tract, from which it was retrogradely transported to the retinal ganglion cell bodies. The labelled retinas were isolated from the eye and maintained in vitro in the presence of [3H]choline. They were then quick-frozen, freeze-dried, vacuum-embedded, and radioautographed on dry emulsion for identification of the acetylcholine-synthesizing cells. Inspection of the radioautographs by fluorescence microscopy showed the two labels not to co-exist: the cells that contained the transported fluorescence did not contain radioactive acetylcholine. In other animals the optic nerve was sectioned, causing retrograde degeneration of a large fraction of the ganglion cells. A population of small, round neurons in the ganglion cell layer was spared. These retinas synthesized [3H]acetylcholine at the same rate as control tissues; and radioautography showed an identical distribution of the acetylcholine-synthesizing cells. We conclude that the acetylcholine-synthesizing neurons of the ganglion cell layer are displaced amacrine cells. When DAPI was injected intraocularly instead of being applied to the optic tract, a regular mosaic of neurons in the ganglion cell layer was selectively stained, and two bands of fluorescence were observed in the inner plexiform layer, at the level where two bands of radioactive acetylcholine were observed in radioautographs. Quantitative analysis showed that the DAPI-stained cells were the same size as those that survive optic nerve section. Like the acetylcholine-synthesizing cells, they appear to be displaced amacrines; when wheatgerm agglutinin labelled by Evans blue was applied to the optic tract and DAPI was injected intraocularly, the red fluorescence of Evans blue and the blue fluorescence of DAPI accumulated in different cells. When DAPI was injected intraocularly and radioautography for acetylcholine was carried out, the cells brightly labelled by DAPI were found to have synthesized acetylcholine. We conclude that topically applied DAPI selectively labels the acetylcholine-synthesizing neurons of the ganglion cell layer. The distribution of the acetylcholine-synthesizing cells was established by counting the DAPI-labelled cells in whole-mounts.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1984

The shape and arrangement of the cholinergic neurons in the rabbit retina.

The acetylcholine-synthesizing neurons of the rabbit retina were selectively stained by intraocular injection of the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI). Retinas were then isolated from the eye, fixed for 10-30 min with 4% paraformaldehyde, and mounted flat on the stage of a fluorescence microscope. The acetylcholine-synthesizing cells were penetrated under visual control by microelectrodes filled with lucifer yellow CH. When the dye was electrophoretically injected into the cells, complete filling of their dendrites often occurred. Cells were successfully injected as long as one month after fixation of the tissue. Complete or nearly complete filling of 281 cells was accomplished, at retinal locations systematically covering the retinal surface. The cells stained with DAPI were found to form a single morphological population. They have two to seven primary dendrites, which branch repeatedly within a narrow plane and form a round or slightly oval dendritic tree. The branching becomes very fine for the distal one third of the dendritic tree, and the dendrites there are studded with small swellings. The distal dendritic tree lies mainly within one of the two thin strata of the inner plexiform layer where acetylcholine is present. The shape and size of the dendritic tree are continuously graded across the retina, the dendritic tree is narrower and the branching denser in the central retina, wider and sparser in the periphery. From knowledge of the population density and the shape of the neurons, one can reconstruct the array of dendrites that exists within the inner plexiform layer. The overlap of the dendritic fields is an order of magnitude greater than of any other retinal neuron previously described. Because the cells not only overlap widely but branch quite profusely, a very dense plexus of cholinergic dendrites is created.

Acetylcholine↗
PubMed1984

The functions of acetylcholine in the rabbit retina.

Rabbit retinas were incubated in vitro under conditions known to maintain their physiological function. The acetylcholine stores of the cholinergic amacrine cells were labelled by incubation in the presence of [3H]choline. The tissue was then mounted in a fast-flow superfusion chamber, and the release of [3H]acetylcholine under various conditions was measured by liquid cation exchange or high-voltage electrophoresis. When the retina was stimulated by flashing light, the rate of appearance of radioactive acetylcholine in the superfusate increased, with a latency shorter than the resolution of the system. The rate of release of acetylcholine remained elevated as long as the light was flashing, and returned rapidly to baseline when the light was extinguished. A one minute stimulation with steady light caused a burst of acetylcholine release following stimulus onset and a second, smaller, burst following stimulus cessation. In the presence of 2-amino-4-phosphonobutyrate (APB), an agent known to eliminate selectively the transmission of ON responses to the proximal retina, steady light caused acetylcholine release only at stimulus cessation. Other retinas were labelled with [3H]choline, then incubated for 10-80 min in the presence of flashing light (to promote acetylcholine release) and either control medium or medium containing 100 micron APB (to prevent release from cells activated by stimulus onset). These retinas were quick-frozen, freeze-dried and radioautographed on dry emulsion. In retinas incubated under control conditions [3H]acetylcholine was initially present within two bands within the inner plexiform layer. The two bands became fainter together as the tissue's [3H]acetylcholine was released. APB selectively retarded the depletion of [3H]acetylcholine from the band nearest the ganglion cell layer. We conclude that the displaced cholinergic amacrine cells release acetylcholine at the transient when light appears, and the conventionally placed cholinergic amacrine cells release acetylcholine at the transient when light is extinguished. The retinal ganglion cells that receive a light-driven cholinergic input are distinguished from those that do not by a great sensitivity to slow stimulus motion. It is proposed that the dense plexus of cholinergic dendrites and the transient nature of acetylcholine release combine to create the local subunit that enables detection of motion within regions smaller than those ganglion cells' receptive fields.

Acetylcholine↗
PubMed1984

Monoclonal antibody to Thy-1 enhances regeneration of processes by rat retinal ganglion cells in culture.

Ganglion cells were dissociated from postnatal rat retinas, identified by specific fluorescent labels, and maintained in culture on a variety of substrates. Regeneration of processes by retinal ganglion cells was enhanced when the cells were plated on glass coated with a monoclonal antibody against the Thy-1 determinant. Plain glass and glass coated with polylysine, collagen, fibronectin, or other monoclonal antibodies supported the growth of neural processes, but were less effective than antibody to Thy-1.

Animals↗
PubMed1984

Biochemical interruption of membrane phospholipid renewal in retinal photoreceptor cells.

The rabbit retina's synthesis of new phosphatidylcholine from extracellular choline was interrupted by an intravitreal injection of the choline analogue hemicholinium-3. This disrupted the process by which new membrane is added to the rod photoreceptor outer segments and eventually caused outer segment degeneration. During the first 2 days after hemicholinium-3 was injected, rows of vesicles replaced the newly formed membrane discs at the outer segment's base. The region of vesicles then expanded, and the distal outer segment detached and was quickly phagocytosed by the pigment epithelium. Two weeks after hemicholinium-3 injection, all of the retina's outer segments had been lost and the inner segments were reduced in length. The threshold concentration of hemicholinium-3 was approximately 20 microM intraocularly. At this dose, the cell bodies, intracellular organelles, and synapses of the rod cells survived. A small group of amacrine cells, possibly those that synthesize acetylcholine, became pyknotic; but the other retinal neurons remained normal to both light and electron microscopy even upon exposure to intraocular concentrations as high as 1 mM. Biochemical experiments indicated that at 20 microM hemicholinium-3, the perturbation of choline metabolism is partial and transient. That it has major selective consequences for the outer segments probably reflects the large amount of new phospholipid required for renewal of their membranes. The selectivity of the lesion was also evidenced by electrophysiological activity recorded from hemicholinium-3-treated retinas. Hemicholinium-3 was injected in vivo, and at various times retinas were isolated and incubated in vitro. The normal components of the electroretinogram were observed, but its amplitude rapidly declined; 12 days after injection, no response to light could be detected. Spontaneous firing of single ganglion cells was observed at all times following hemicholinium-3 injection. As the outer segments degenerated and the threshold of the electroretinogram rose, the thresholds of the ganglion cells also rose, but normal ganglion cell receptive fields could sometimes be plotted. Fourteen days after injection, when all of the outer segments were gone, ganglion cell responses to bright light could still be recorded; their thresholds were about 3.5 log units above normal. This finding is consistent with a previous report of light-evoked responses in mice after outer segment degeneration.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗