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V Sarthy

Publications and source records attributed to V Sarthy.

11 recordsLinked to original sources
PubMed2006

Characterization of green fluorescent protein-expressing retinal cells in CD 44-transgenic mice.

Sensory information in the retina is transferred from rod and cone photoreceptors to higher visual centers via numerous parallel circuits that sample the photoreceptor mosaic independently. Each circuit consists of a unique combination of ganglion cell, bipolar and amacrine cell types. The morphology and physiological responses of many amacrine cells have been characterized. However, the synaptic connections and retinal circuits in which they participate are only rarely understood. A major problem that has prevented fuller characterization of retinal circuitry is the need for specific cellular markers for the more than 50 inner retinal cell types. One potential strategy for labeling cells is to use transgenic expression of a reporter gene in a specific cell type. In a recent study of cluster of differentiation 44 (CD44)-enhanced green fluorescent protein (EGFP) transgenic mice, we observed that the green fluorescent protein (GFP) was expressed in a population of amacrine and ganglion cells in the inner nuclear layer (INL) and the GCL. To characterize the morphology of the GFP-labeled cells, whole mount preparations of the retina were used for targeted iontophoretic injections of Lucifer Yellow and Neurobiotin. Furthermore, immunocytochemistry was used to characterize the antigenic properties of the cells. We found that many GFP-expressing cells were GABAergic and also expressed calretinin. In addition to the somatic staining, there was a strong GFP(+)-band located about 50-60% depth in the inner plexiform layer (IPL). Double labeling with an antibody to choline acetyltransferase (ChAT) revealed that the GFP-band was located at strata 3 inner retina. The best-labeled GFP-expressing cell type in the INL was a wide-field amacrine cell that ramified in stratum 3. The GFP-expressing cells in the GCL resemble the type B1, or possibly A2 ganglion cells. The CD44-EGFP mice should provide a valuable resource for electrophysiological and connectivity studies of amacrine cells in the mouse retina.

Amacrine Cells↗
PubMed1997

Molecular characterization and in situ localization of a mouse retinal taurine transporter.

Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high-affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na+- and Cl(-)-dependent [3H]taurine transport activity in Xenopus laevis oocytes that saturated with an average Km of 13.2 microM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl- was varied, a single saturation process with an average apparent equilibrium constant (K(Cl-)) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na+ was varied (apparent equilibrium constant, K(Na+) approximately 54.8 mM). Analyses of the Na+- and Cl(-)-concentration dependence data suggest that at least two Na+ and one Cl- are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > beta-alanine > L-diaminopropionic acid > guanidinoethane sulfonate > beta-guanidinopropionic acid > chloroquine > gamma-aminobutyric acid > 3-amino-1-propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4alpha-phorbol 12,13-didecanoate but was inhibited significantly by the active phorbol ester phorbol 12-myristate 13-acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.

Amino Acid Sequence↗
PubMed1996

Immunocytochemical localization of retinoid-binding protein in the dogfish retina.

PURPOSE: The cellular localization of retinoid-binding proteins has been extensively studied in the mammalian retina. Less is known about the distribution and identity of retinoid-binding proteins in lower vertebrate retinas. To understand their cellular functions, we have examined the distribution of specific retinoid-binding proteins in the neural retina and RPE of an elasmobranch, the dogfish. METHODS: Retinoid-binding proteins were localized in cryostat sections of dogfish eyes by the indirect immunofluorescence technique. We used well-characterized antibodies that have been previously employed for immunostaining. Immunoblotting was carried out to identify the antigen. RESULTS: The studies show that the cellular retinaldehyde-binding protein is present in the RPE and retinal Müller cells, and the interphotoreceptor retinoid-binding protein is found in the interstitial space between pigment epithelium and retina. CONCLUSIONS: The distribution of retinoid-binding proteins in dogfish retina is similar to that found in the mammalian retina which suggests that the sites of action of the retinoid-binding proteins are highly conserved in vertebrates.

Animals↗
PubMed1995

Transient induction of the glial intermediate filament protein gene in Müller cells in the mouse retina.

The glial intermediate filament protein (GFAP) gene is not normally expressed by retinal Müller cells but it is transcriptionally activated following photoreceptor degeneration. In the present study, we have examined the relationship between progressive photoreceptor loss and changes in GFAP gene activity in Müller cells. In albino mice with light-induced photoreceptor degeneration, GFAP level was strongly elevated after 2 weeks. GFAP level remained high even after 3 months in light. In situ hybridization studies showed that GFAP transcripts were quite sparse in the first week but increased dramatically after 2 weeks of light exposure. After 4 weeks in constant light, however, little GFAP mRNA was detected in Müller cells. RNA blotting also showed that there was an approximately 20-fold increase in GFAP mRNA content at 2 weeks; but at 4 weeks, the RNA content fell to about four-fold higher than the basal level. These results show that GFAP level remains high long after its synthesis, probably as a consequence of low GFAP turnover in the Müller cell cytoskeleton, while GFAP mRNA level rises and declines rapidly due to transient activation of the GFAP gene in Müller cells.

Animals↗
PubMed1995

Differential regulation of a glial fibrillary acidic protein-LacZ transgene in retinal astrocytes and Müller cells.

PURPOSE: Glial fibrillary acidic protein (GFAP) is normally expressed in astrocytes, but not in Müller cells, in the mouse retina. In response to retinal injury or photoreceptor degeneration, however, GFAP gene transcription is strongly activated in the Müller cell. To identify the genetic elements involved in GFAP gene induction, the authors have studied gene expression in transgenic mice in which beta-galactosidase (beta-gal) expression is under control of GFAP regulatory sequences. METHODS: Histochemical methods were used to study gene expression. The transgene expression was followed by x-gal staining, whereas GFAP expression was monitored by immunostaining with GFAP antibody. RESULTS: In GFAP-LacZ transgenic mice, beta-gal activity and GFAP immunostaining were found in retinal astrocytes. Transgene expression showed the same developmental pattern as that of endogenous GFAP in retinal astrocytes. In addition, beta-gal staining also was observed in lens epithelial cells. Neither GFAP nor beta-gal expression was seen in Müller cells in the adult or developing retina. When focal retinal lesions were introduced into the retina, strong GFAP immunostaining was observed in Müller cells throughout the retina. No beta-gal staining was seen in Müller cells in these retinas. In astrocytes, however, beta-gal and GFAP both were present. CONCLUSIONS: The observations suggest that cis elements responsible for GFAP expression in retinal astrocytes are present in 5' flanking region of the GFAP gene, whereas the regulatory elements involved in GFAP induction in Müller cells are located elsewhere.

Animals↗
PubMed1994

Cloning, expression, and localization of a mouse retinal gamma-aminobutyric acid transporter.

PURPOSE: To isolate a cDNA clone encoding a high-affinity gamma-aminobutyric acid (GABA) transporter from mouse retina, to examine its biochemical and pharmacologic properties, and to determine the sites of its mRNA expression in retinal cells. METHODS: A mouse retinal cDNA library was screened using a fragment of a rat brain GABA transporter (GAT-1) cDNA as a probe. One homologous clone, mouse retinal GAT-1, was chosen for further characterization. RNA transcribed from mouse retinal GAT-1 was microinjected into Xenopus oocytes, and pharmacologic properties of the expressed transporter were determined. Sites of mouse retinal GAT-1 mRNA expression were examined by in situ hybridization. RESULTS: The protein sequence deduced from the DNA sequence of mouse retinal GAT-1 cDNA was virtually identical to that of the rat and the mouse brain GAT-1. RNA transcribed from this clone induced a [3H]-GABA uptake activity in microinjected Xenopus oocytes that was both sodium and chloride dependent. The apparent Km and Vmax for the GABA uptake were 8.3 microM and 40.0 pmol/egg per hour, respectively. The mouse retinal GAT-1 induced GABA uptake was inhibited by L-diaminobutyric acid, guvacine, cis-4-hydroxynipecotic acid, nipecotic acid, and 4,5,6,7-tetrahydroisoxazolo [4,5c]-pyridin-3-ol with IC50 values of 320, 79, 71, 7.1, and 200 microM, respectively. However, beta-alanine was unable to inhibit the induced GABA uptake significantly (IC50 approximately 2,500 microM). In situ hybridization studies showed that mouse retinal GAT-1 mRNA was present in a subpopulation of amacrine, interplexiform, and displaced amacrine cells. Hybridization signal in the Müller cells was significantly lower, and GAT-1 transcripts were not detected in the bipolar, horizontal, or photoreceptor cells of mouse retina. CONCLUSIONS: The mouse retinal GAT-1 cDNA encodes a Na(+)-dependent, high-affinity GABA transporter that is mainly expressed in a subset of mouse retinal inter neurons.

Animals↗
PubMed1993

Collagen IV mRNA expression during development of the mouse retina: an in situ hybridization study.

PURPOSE: During development of the nervous system, neuronal migration and axonal growth depend on specific interactions with molecules in the extracellular matrix. In a recent study of laminin expression, it was shown that laminin B1 mRNA was expressed by both nonneural cells and the retinal ganglion cells during development of the mouse retina. Because collagen IV is associated with laminin in basement membranes, this report examined whether collagen IV mRNA also is expressed by neurons and nonneural cells during retinal development. METHODS: Collagen IV was localized by immunocytochemistry, whereas the sites of collagen IV mRNA synthesis were identified by in situ hybridization. RESULTS: Collagen IV immunostaining was detected at embryonic day 12 (E-12), the earliest stage examined. At E-12 and E-15, collagen was found in the lens, the embryonic (hyaloid) blood vessels, and the internal limiting membrane (ILM) of the retina. At E-17, immunostaining was reduced in the ILM, whereas the lens and hyaloid were strongly stained. Collagen was barely detected in the ILM in postnatal retinas. In the in situ hybridization experiments, at E-12, collagen IV mRNA was found in the lens and the hyaloid vessels. Only sparsely labeled cells were present in the retina. After E-17, the density of labeling in these structures decreased dramatically. Collagen IV mRNA was not found in the retina at any stage in development or in the adult. Northern blot analysis showed that a 6 Kb collagen IV transcript was present in the eye. CONCLUSIONS: These findings establish that high levels of collagen IV are present at the ILM only during early development (E-12 to E-17), when most axonal growth occurs. Retinal collagen IV is possibly derived from nonretinal sources, such as the lens, or more likely from the hyaloid vessels.

Animals↗
PubMed1986

gamma-Aminobutyric acid (GABA) uptake by Xenopus oocytes injected with rat brain mRNA.

A major pathway for inactivation of biogenic amine and amino acid neurotransmitters is by uptake into neurons or glia cells through Na+-dependent, high-affinity uptake systems. Here, we show that Xenopus oocytes, microinjected with poly(A)+ RNA from developing rat brain, express a Na+-dependent GABA uptake system, which is similar in its properties to the high-affinity GABA uptake system, present in rat brain. These results suggest that the oocyte expression system may be useful in the isolation of mRNAs and subsequent cloning of the genes encoding the polypeptides involved in GABA transport in the nervous system.

Animals↗