P. Vijay Sarthy

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). The nuclear layers and the nerve fiber layer (NFL) were devoid of any staining. In prenatal and early postnatal (P) retinas, the antibody stained two bands which corresponded to the respective locations of the NFL and IPL. Staining in the NFL increased until P-4 and began to decline subsequently, and by P-8 little staining was left in this layer. In contrast, in the IPL, the intensity of staining increased gradually and leveled off by P-10. In the outer retina, a band of fluorescence corresponding to the OPL was first observed at P-5 and increased in intensity up to P-10. Immunoblotting studies showed that the major immunoreactive material from adult and embryonic retinas had a Mr approximately 65,000-67,000. As expected from its developmental pattern, all bands appeared initially in the central retina and subsequently in the peripheral retina. Our results show that the synaptic vesicle-protein is present in the nerve fiber layer before synaptogenesis in the central nervous system. Subsequently, the protein is lost from the NFL, possibly as a consequence of synapse formation.

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

Summary1.In the nervous system, Glial fibrillary acidic protein (GFAP) is a well-known, cell type-specific marker for astrocytes.2.In the mammalian retina, Muller cells, the major class of retinal glia, do not express GFAP or contain only low amounts of this protein. In retinas with photoreceptor degeneration, however, high levels of GFAP are found. It is possible that GFAP synthesis in these retinas could result from “dedifferentiation” of Muller cells as a consequence of disruption of normal neuron-glia interactions.3.We have carried out immunocytochemical andin situ hybridization studies to examine whether GFAP or its mRNA is expressed by retinal cells early in embryonic development.4.Our results show that GFAP-containing cells, which are probably astrocytes, are found only in the ganglion cell and nerve fiber layers and that these cells appear after postnatal day-1 (P-1) and continue to form until P-10.5.Astrocyte formation starts from the optic disc and moves toward the periphery of the retina at a rate of ~ 160–200µm per day.6.An unexpected result from these studies is that GFAP mRNA levels are high in the first week of birth and decline rapidly as the animal develops.7.Finally, we did not find either GFAP or GFAP mRNA in retinal cells other than astrocytes during normal development.

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.

The uptake of [3h] γ-aminobutyric acid by isolated glial (müller) cells from the mouse retina

Abstract We have examined the uptake of [ 3 H]GABA by isolated glial (Muller) cells using light microscope autoradiography. The aim of these studies was to determine whether Muller cells dissociated from papain-treated mouse retina have the capacity to accumulate exogenous GABA and to further characterize the uptake process. Our results show that isolated Muller cells retain the capacity to accumulate exogenous [ 3 H]GABA and this uptake is inhibited by excess GABA, DABA and β-alanine. This communication also describes a new method for performing light microscope autoradiography.

Localization of the Na-K pump in turtle retina

SummaryThe kinetics of ouabain binding to Na-K pump and the distribution of pump sites were examined in the retina ofPseudemys scripta elegans. Binding to retinal slices followed bimolecular kinetics characterized by aKD of 1.5 × 10−6m and a maximum binding capacity of 11.2 × 10−8 mol g−1 of protein. Quantitative autoradiography of slices revealed a high concentration of bound ouabain in the inner segment, outer plexiform, inner plexiform and optic nerve layers, and correspondingly, a low level of binding in layers containing cell bodies. In the few instances that outer segments remained attached to cones, little or no binding to outer segments was observed. The membrane density of inner segment binding sites was measured by combining autoradiographic measurements of pump site concentration with stereological measurements of membrane concentration. The densities were 6.3 and 3.7 × 103 sites μm−2 of cone-ellipsoid and cone-fin cell membrane, respectively. The density of Müller cell microvilli was measured similarly but in enzymatically isolated cells and found to be 600 sites μm−2 of membrane. Measurements of cone-ellipsoid pump site density in enzymatically isolated cones were not different from measurements in slices. Calculation of Na-K pump site turnover number for the cone inner segment from pump site densities and published dark current measurements yielded a value of 30 Hz.

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. Antibody 15-18 binds visual pigments from several species, including bovine opsin. In order to determine the antigenic site bound by 15-18 in bovine opsin, synthetic peptides were used as competitors in an enzyme-linked immunoassay (ELISA). The antigenic site is located in the surface loop connecting rhodopsin helices IV-V, in the sequence 190-197. Antibody 15-18 binds to the external surface of rod cell outer segments, thus providing direct evidence for the predicted orientation of rhodopsin in the plasma membrane.

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. Our experimental results show that: (1) the major peaks of DNA, RNA, and protein synthesis occur at different times during regeneration and that the synthesis of RNA precedes that of DNA which is followed by the protein; (2) synthesis and accumulation of ACh and GABA is lost by the fifth day following ocular devascularization and it reappears by day 23-24. ACh and GABA syntheses continue to increase until they attain plateau around day 35; (3) the appearance of choline acetyltransferase and glutamate decarboxylase activities parallel ACh and GABA synthesis; and (4) transmitter synthesis precedes development of the ERG. The first light evoked response is seen around day 29 by which time the transmitter synthesis is well advanced.

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

SummaryMüller 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. The antibody was obtained during a search for cell type-specific monoclonal antibodies in the rat retina. Immunoblotting studies show that 19–33 reacts with a 58 KDa protein that is present in Müller cells. Immunocytochemical studies withen face sections of turtle retina show that the density of Müller cells is fairly uniform across the retina although there are small regional differences. We estimate that the mean Müller cell density is about 1600 cells mm−2 of turtle retina and that each turtle retina contains about 54 000 Müller cells.

Retrograde labeling and dissociation of mouse retinal ganglion cells

Mouse retinal ganglion cells were labeled by retrograde axonal transport of the fluorescent dyes, DAPI and Primulin. The labeled retinae were dispersed by papain treatment and mechanical dissociation. Among the cells in the suspension, the ganglion cells could be unequivocally identified by their intense DAPI/Primulin fluorescence. These results establish that it is possible to label specific neurons in the central nervous system by retrograde transport of fluorescent dyes and to identify the labeled cells after proteolytic dissociation.

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. Although little uptake is seen before postnatal day 5, between days 6 and 9, the dye selectively stains putative horizontal cell bodies and their process. Later, other cell bodies located deeper in the INL begin to show staining until day 21, when the uptake pattern becomes analogous to that seen in adult retina. By nearest neighbor analysis of labeled cell bodies in wholemounts of D-9 retina, we have shown that the labeled cells are distributed in a quasi-regular mosaic in the retina. Upon proteolytic dissociation of labeled retina, individual cells with Lucifer yellow fluorescence could be readily identified in the cell suspensions.