Genetic and molecular identification of a Drosophila histidine decarboxylase gene required in photoreceptor transmitter synthesis.

Drosophila mutants of a single complementation group with defective on-/off-transients of the electroretinogram (ERG) were found to be deficient in synthesis of the photoreceptor transmitter, histamine, in a gene-dosage dependent manner, suggesting that the gene identified by the mutants (hdc) might be the structural gene for Drosophila histidine decarboxylase (HDC). A rat HDC cDNA was used to isolate a Drosophila homolog which shows approximately 60% sequence identity with mammalian HDCs over a region of 476 amino acids. In RNA blots, the Drosophila homolog detects four transcripts that are expressed primarily in the eye and are severely reduced in hdc mutants.

Developmental expression of the glial fibrillary acidic protein (GFAP) gene in the mouse retina.

1. In the nervous system, Glial fibrillary acidic protein (GFAP) is a well-known, cell type-specific marker for astrocytes. 2.

Histamine: a neurotransmitter candidate for Drosophila photoreceptors.

Recent experimental evidence suggests that histamine might be the synaptic transmitter used by invertebrate photoreceptors. In the present study, we have examined whether histamine is a transmitter candidate for Drosophila photoreceptors. Our findings are as follows: (a) Large amounts of histamine are synthesized by wild-type heads, whereas heads from the eye-deficient mutants, eyes absent and sine oculis, show reduced histamine synthesis.

Intermediate filaments in the inner ear of normal and experimentally damaged guinea pigs.

The hypothesis that proteins known to occur in glial cells in the central nervous system may be present in inner-ear supporting cells was investigated. Immunocytochemical techniques were used to look for the existence of two classes of intermediate filaments, vimentin and glial fibrillary acidic protein (GFAP), in cellular elements of the inner-ear epithelium in normal and experimentally damaged guinea-pig cochleas. Vimentin is present in two types of supporting cells in the normal organ of Corti: Deiters' cells and inner pillar cells.

Localization of laminin B1 mRNA in retinal ganglion cells by in situ hybridization.

In the nervous system, neuronal migration and axonal growth are dependent on specific interactions with extracellular matrix proteins. During development of the vertebrate retina, ganglion cell axons extend along the internal limiting (basement) membrane and form the optic nerve. Laminin, a major component of basement membranes, is known to be present in the internal limiting membrane, and might be involved in the growth of ganglion cell axons.

Localization of L-glutamic acid decarboxylase mRNA in cat retinal horizontal cells by in situ hybridization.

Retinal horizontal cells receive synaptic input from photoreceptors and provide a pathway for lateral interactions in the vertebrate retina. In nonmammalian retinas, the H1 horizontal cells appear to use gamma-amino butyric acid (GABA) as their neurotransmitter. The transmitter used by mammalian horizontal cells, however, remains to be identified.

Localization of L-glutamic acid decarboxylase mRNA in monkey and human retina by in situ hybridization.

Immunocytochemical studies with gamma-aminobutyric acid (GABA) antibodies and glutamic acid decarboxylase antibodies have shown that the primate retina contains GABAergic amacrine, interplexiform, and displaced amacrine cells. In addition, subpopulations of photoreceptors and horizontal cells have also been suggested to be GABAergic in this retina. In the present study, we have used in situ hybridization to localize GABAergic neurons in human and monkey retinas.

Subcellular localization of an intermediate filament protein and its mRNA in glial cells.

Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell.

A monoclonal antibody marker for the paraboloid region of cone photoreceptors in turtle retina.

Monoclonal antibodies that specifically label one or more cell types in retina have been produced; however, only a few antibodies that, in addition, recognize distinct subcellular structures in these cells have been reported. During a search for monoclonal antibodies that bind to specific cell types in the turtle (Pseudemys scripta elegans) retina, we obtained an antibody (20 93; an IgG) that labels the inner segment of cone photoreceptors. Ultrastructural immunocytochemistry using immunogold and avidin/biotin-peroxidase techniques showed that 20 93 antigen is localized to the paraboloid, a region specifically involved in glycogen metabolism in cones.

Transcriptional activation of an intermediate filament protein gene in mice with retinal dystrophy.

We are interested in understanding neuronal interactions that regulate expression of specific genes in glial cells in the nervous system. In the normal mouse retina, the glial intermediate filament protein (GFAP) is not detectable in Müller cells, the predominant glial cells in the retina. Photoreceptor degeneration resulting from retinal degeneration (rd) mutation or environmental light damage, however, leads to the appearance of GFAP in Müller cells.

Distribution of Müller cells in the turtle retina: an immunocytochemical study.

Müller cells are the major type of glial cell in the vertebrate retina, and appear to participate in important structural and metabolic functions. Although the morphological features of Müller cells have been extensively studied, their topographic distribution across the retina has not been previously reported. We have used a Müller cell-specific monoclonal antibody, 19-33, to study the distribution of Müller cells in turtle retina.

Localization of glycine-containing neurons in the Macaca monkey retina.

Autoradiography following 3H-glycine (Gly) uptake and immunocytochemistry with a Gly-specific antiserum were used to identify neurons in Macaca monkey retina that contain a high level of this neurotransmitter. High-affinity uptake of Gly was shown to be sodium dependent whereas release of both endogenous and accumulated Gly was calcium dependent. Neurons labeling for Gly included 40-46% of the amacrine cells and nearly 40% of the bipolars.

A monoclonal antibody that binds to photoreceptors in the turtle retina.

We have raised monoclonal antibodies to photoreceptor cells in the retina of the turtle (Pseudemys scripta elegans). One of these antibodies, 15-18 (an IgG1), was studied by immunoelectron microscopy using colloidal gold, and found to bind to the outer segments of all rods and some single cones, but did not stain turtle double cones. Immunoblotting and immunoprecipitation show that antibody 15-18 binds to an antigen of apparent Mr approximately 34,5000 which is probably turtle opsin.

L-glutamate: a neurotransmitter candidate for cone photoreceptors in the monkey retina.

In order to examine whether L-aspartate and L-glutamate are photoreceptor transmitters in monkey retina, we have carried out two different studies: an autoradiographic localization of the high-affinity uptake sites for aspartate (Asp) and glutamate (Glu), and an immunocytochemical localization of the biosynthetic enzyme, L-aspartate aminotransferase (AAT). Our results show that L-Glu is taken up by a sodium-dependent, high-affinity uptake system with a Km = 8 +/- 3.5 X 10(-6) M and a Vmax = 48 +/- 14 X 10(-12) mol/min/mg protein; 3H-L-Glu or 3H-L- and D-Asp are taken up and accumulated by rod somata and inner segments in retinas incubated at 37 degrees C or at ambient temperature; cones accumulate 3H-L-Glu at ambient temperature but not at 37 degrees C.

Developmental expression of a synaptic vesicle-specific protein in the rat retina.

In order to examine the appearance of synaptic vesicles and to correlate it with the formation of the synaptic layers, we have determined the staining pattern of a murine monoclonal antibody (SV 48) to a synaptic vesicle-associated protein in developing rat retina. The antigen was detected by the indirect immunofluorescence technique using cryostat sections of paraformaldehyde-fixed retinas. In the adult retina, the antibody stained both the outer plexiform (OPL) and the inner plexiform layers (IPL).

Establishment of Muller cell cultures from adult rat retina.

During a recent study of the effects of photoreceptor degeneration on Muller cells, we observed that unlike glia from normal adult retinas, glia from degenerating rat retinas could be cultured and maintained in vitro for several weeks. Using a variety of cell type specific antisera, we show that at least 70% of cells in these cultures are derived from Muller cells.

Ionic and electrophysiological properties of retinal Müller (glial) cells of the turtle.

The ionic and electrophysiological properties of Müller cells, the principal glial element of the vertebrate retina, were investigated. The membrane potential of enzymatically dissociated and in situ Müller cells was about -80 mV and depended on external K+ concentration in a manner that was described by the Goldman-Hodgkin-Katz equation with a Na+-K+ permeability ratio of 0.037.

Localization of the Na-K pump in turtle retina.

The kinetics of ouabain binding to Na-K pump and the distribution of pump sites were examined in the retina of Pseudemys scripta elegans. Binding to retinal slices followed bimolecular kinetics characterized by a KD of 1.5 X 10(-6) M and a maximum binding capacity of 11.

Müller cell expression of glial fibrillary acidic protein after genetic and experimental photoreceptor degeneration in the rat retina.

Glial fibrillary acidic protein (GFAP) is normally found in astrocytes. In the normal rat retina at all ages, only astrocytes stain for GFAP. This staining pattern is also found in RCS rats with inherited retinal dystrophy younger than 38 days.

Release of [3H]gamma-aminobutyric acid from glial (Müller) cells of the rat retina: effects of K+, veratridine, and ethylenediamine.

In several neural systems, glial cells appear to take up and release gamma-aminobutyric acid (GABA) upon depolarization. We have studied the release of [3H]GABA from Müller (glial) cells in the rat retina by a double isotope-labeling technique in which Müller cells are preloaded with 3H-GABA while a population of neurons is prelabeled with [14C]glycine. By autoradiography, we have confirmed that [3H]GABA is taken up by the radially oriented Müller cells, whereas [3H]glycine is accumulated by a subset of amacrine cells (neurons).

Lucifer yellow uptake in developing rat retina: selective staining of horizontal cells.

We have examined the uptake of Lucifer yellow CH by developing rat retina. We find that selective dye uptake occurs in retina incubated in vitro in the absence of external Ca2+. The pattern of uptake appears to depend on the developmental stage examined.

Retrograde labeling, enrichment, and characterization of retinal ganglion cells from the neonatal rat.

We have developed a method for labeling retinal ganglion cells in neonatal rats by retrograde transport of the fluorescent dye, True Blue (TB), injected into the optic chiasm. Following proteolytic dissociation of labeled retinas into single cells, the labeled cells could be enriched 50- to 100-fold by centrifugation in a 5%/10% metrizamide gradient. When plated in Ham's F-10 medium in the presence of fetal calf serum and chick optic tectum-conditioned medium, the labeled cells could be maintained in vitro up to 48 hr.

Retinal regeneration in the adult newt, Notophthalmus viridescens: appearance of neurotransmitter synthesis and the electroretinogram.

The removal or destruction of retina in the adult newt, Notophthalmus viridescens, leads to the formation of a new functional retina by metaplasia of the pigment epithelium. In order to understand the physiological and biochemical changes that occur during regeneration, we have examined the time-course of appearance and maturation of the electroretinogram (ERG) and correlated it with the synthesis of the neurotransmitters, acetylcholine (ACh) and gamma-aminobutyric acid (GABA). In addition, we have also determined the pattern of DNA, RNA, and protein synthesis in the regenerating retina.

Selective uptake of lucifer yellow by retinal cells.

When turtle retinae were incubated with the fluorescent dye, lucifer yellow, in the absence of Ca2+, the dye was selectively accumulated by cell bodies located in the inner nuclear layer (INL). The morphological features of the labeled cells suggested that they were bipolar cells. Other fluorescent dyes, Procion yellow and Primulin, were also taken up by somata in the INL, in the absence of external Ca2+, although the identity of the labeled cells was uncertain.

The ultrastructure of isolated glial (Müller) cells from the turtle retina.

Sarthy and Lam (1978) described a method for isolation of glial (Müller) cells from turtle retina which involves papain treatment, mechanical dissociation of the retinae, and subsequent separation of the dispersed cells by velocity sedimentation at unit gravity. In order to establish the cytologic integrity and the extent of contamination, we have carried out ultrastructural studies on the isolated cells using scanning and transmission electron microscopy. During the course of these studies we have also examined the effect of papain on the morphology of the retina.