Biomedical subjects

C J Barnstable

Publications and source records attributed to C J Barnstable.

At least 19 recordsLinked to original sources

PubMed2000

Dorsal retinal pigment epithelium differentiates as neural retina in the microphthalmia (mi/mi) mouse.

PURPOSE: Microphthalmia, a bHLH-zip transcription factor associated with the onset and maintenance of pigmentation, identifies the retinal pigment epithelial (RPE) compartment during optic vesicle and optic cup development. To determine a role for microphthalmia (mi) during eye development, the effects of an mi loss of function mutation on RPE and neural retinal were investigated in the mi/mi mouse. METHODS: A series of embryonic and postnatal mi/mi and wild-type eyes were sectioned and labeled with neural retina- and RPE cell type-specific antibodies. Photoreceptor loss was quantified by counting the number of photoreceptor nuclei spanning the outer nuclear layer throughout postnatal retinal development. RESULTS: Early neural retinal differentiation is not affected in the mi/mi mouse. The mi/mi ventral retinal pigment epithelial layer begins to develop normally, but does not pigment or attain a differentiated cuboidal morphology. The dorsal region of mi/mi retinal pigment epithelium expands and forms an ectopic retina, which develops all major retinal cell types along a similar time course as the wild type. After birth, mi/mi photoreceptors begin to form rosettes, outer segments fail to elongate, and over an extended time period, the retina degenerates. CONCLUSIONS: Together these results suggest that early retinal development can proceed normally in the mi/mi mutant, but later retinal histogenesis is dependent on the presence of a differentiated retinal pigment epithelium. Most importantly, loss of mi function permits a change in cell fate from RPE to retina in the dorsal eye.

Animals↗

PubMed2000

Impairment of rod cGMP-gated channel alpha-subunit expression leads to photoreceptor and bipolar cell degeneration.

PURPOSE: To determine whether alterations in rod cGMP-gated channel function mediate retinal degeneration, a transgenic approach in which the alpha subunit of the rod cGMP-gated channel is reduced by the expression of an antisense RNA was used. METHODS: A 890-bp fragment of the 5' mouse rod cGMP-gated channel cDNA was cloned in the antisense orientation under the control of the strong bacterial cytomegalovirus promoter. This antisense construct was used to generate transgenic mice in which the expression of the antisense transgene was measured by reverse transcription-polymerase chain reaction. Histologic, immunocytochemical, and TdT-dUTP terminal nick-end labeling (TUNEL) analyses were performed on control and transgenic retina sections to determine the effects of antisense expression on specific cell types. RESULTS: The antisense RNA was able to inhibit the translation of the rod channel protein in an in vitro assay. Three transgenic mouse lines expressing the antisense construct were obtained. Molecular analyses showed that the antisense is expressed in the eye of each transgenic mouse line, where it reduces rod cGMP-gated channel RNA expression. Histologic and immunocytochemical data showed that transgenic retinas have a reduced number of photoreceptors and bipolar cells. TUNEL staining confirmed that photoreceptor and bipolar cells degenerate along an apoptotic pathway. CONCLUSIONS: Impairment of rod cGMP-gated channel alpha subunit expression leads to photoreceptor and bipolar cell degeneration. These transgenic mice are the first model of retinal degeneration caused by an alteration in the expression of the rod cGMP-gated channel. This model system can be used to test therapies designed to slow or stalled retinal degenerations.

Animals↗

PubMed1999

Widespread expression of olfactory cyclic nucleotide-gated channel genes in rat brain: implications for neuronal signalling.

The cyclic nucleotides cAMP and cGMP are important intracellular messengers involved in a wide variety of signal transduction events in the nervous system. It has been proposed that cAMP/cGMP elicit some of their effects through direct gating of a novel class of Ca2+ -permeable ion channels that are termed cyclic nucleotide-gated (CNG) channels. Previous studies have identified the expression of a gene encoding one major CNG channel subtype, the olfactory receptor neuron alpha subunit, in the brain [El-Husseini et al. (1995) NeuroReport 6:1331-1335; Kingston et al. (1996a) Proc. Natl. Acad. Sci. U.S.A. 93:10440-10445; Bradley et al. (1997) J. Neurosci. 17:1993-2085]. We, therefore, proposed that the actions of cAMP/cGMP on neurons in the brain might occur through the activation of these CNG channels. To determine how widespread such a function might be, the regional and cellular distribution of the olfactory CNG channel alpha subunit has been examined in detail. Primers for multiple portions of the olfactory CNG channel were used in polymerase chain reaction (PCR) to amplify cDNA reverse-transcribed from several brain regions. The identities of PCR products were confirmed with Southern blots and by sequencing. In situ hybridization experiments demonstrated localization of CNG channel mRNA in discrete neuronal populations throughout the brain. In agreement with previous work, relatively strong hybridization signals are present in neuronal cell bodies of the cerebellum, olfactory bulb, cerebral cortex, and brainstem. Additionally, somewhat lesser signals are found in thalamus, hypothalamus, midbrain, and spinal cord while no hybridization signal was detectable in the caudate nucleus. This surprisingly wide distribution throughout the rat brain strengthens the hypothesis that CNG channels may influence numerous processes as downstream effectors of cyclic nucleotide cascades. Interestingly, the distribution of CNG channels is very similar to that of the nitric oxide/cGMP system, suggesting that one function of CNG channels in the brain could be to link diffusible messengers to elevated Ca2+ entry into neurons.

Animals↗

PubMed1999

Temporal and spatial pattern of expression of cyclic nucleotide-gated channels in developing rat visual cortex.

Cyclic nucleotide-gated channels, ligand-gated and highly permeable to calcium, are good candidates for transducing signals received by migrating cells, growth cones and developing synapses. The level of calcium in growth cones is important for axon guidance. Further, cyclic nucleotides, whose levels can be altered by nitric oxide and other transmitters, are known to alter growth cone motility. We use rat visual cortex as a model in our semi-quantitative RT-PCR and in situ hybridization studies to determine the developmental time course and localization of all three CNG family members (rod, olfactory and cone/testis). We demonstrate that in the cortex, the three channel subtypes are each expressed in a distinct temporal and spatial pattern in only sensorimotor and occipital regions of the cortex. Specifically, the rod and olfactory subtypes are present at the time of migration and rapid dendritic outgrowth, and the cone/testis subtype is highly expressed after eye opening. These results suggest CNG channels may play a role in both early and late events in visual cortical development.

Animals↗

PubMed1998

Erx, a novel retina-specific homeodomain transcription factor, can interact with Ret 1/PCEI sites.

Our previous studies on the transcriptional regulation of rod opsin gene expression had defined a strikingly conserved element, Ret 1/PCEI, present in the upstream regulatory regions of opsin and other photoreceptor-specific genes. This element interacts with a 40 kDa, developmentally regulated, retina-specific protein. In this study we report the cloning of the novel retina-specific homeodomain protein Erx. Erx contains a homeodomain that is 79% homologous to that of Drosophila empty spiracles. This 40 kDa protein can interact with the Ret 1 element in electrophoretic mobility shift assays. Mutation of key residues in Ret 1 eliminates all Erx binding. Transient transfection of Y79 retinobalstoma cells with Erx leads to significant transcriptional activation of a reporter gene via Ret 1 elements. We conclude that Erx is the Ret 1 binding activity. This is the first example of a Q50 homeodomain protein expressed in retinal photoreceptors.

Amino Acid Sequence↗

PubMed1998

Substituted cGMP analogs can act as selective agonists of the rod photoreceptor cGMP-gated cation channel.

Cyclic nucleotide-gated (CNG) channels are expressed in many cell types in both the nervous system and nonexcitable tissues. In order to understand the roles of cGMP-gated channels, and to distinguish actions of cGMP mediated through CNG channels from those through cGMP-dependent protein kinase (G-kinase), several new cGMP analogs were tested for potency as CNG channel agonists. Using Xenopus oocytes expressing the rat rod cGMP-gated ion channel alpha-subunit, we showed that an analog containing a pCPT group at the 8-position, 8-pCPT-cGMP, was 80 times more potent than cGMP and 14 times more potent than 8-Br-cGMP. 8-pCPT-cGMP is the most potent CNG channel agonist so far described and also has the advantages of much better membrane permeability as well as much higher resistance to PDE-hydrolysis, as compared with 8-Br-cGMP. Modification of both 8-Br-cGMP and 8-pCPT-cGMP by introduction of a sulphur atom into the cyclic phosphate group gave smaller changes in agonist efficiency. Both Sp-8-Br-cGMPS and Sp-8-pCPT-cGMPS acted as agonists of CNG channels and are also G-kinase activators. In contrast, Rp-8-Br-cGMPS was a channel agonist, with an EC50 of 173.5 microM, but a G-kinase antagonist with a Ki of 4 microM. Finally, Rp-8-pCPT-cGMPS was a channel agonist and showed additional noncompetitive antagonist activity at higher concentrations. The results suggest that 8-pCPT-cGMPS is a highly potent photoreceptor CNG channel agonist with high membrane permeability and PDE-resistance and furthermore Rp-8-Br-cGMPS can be used to test whether the actions of cGMP are selectively mediated by CNG channels.

Animals↗

PubMed1998

Yoav Citri.

Explore the source record for details and available documents.

Animals↗

PubMed1998

Molecular and pharmacological analysis of cyclic nucleotide-gated channel function in the central nervous system.

Most functional studies of cyclic nucleotide-gated (CNG) channels have been confined to photoreceptors and olfactory epithelium, in which CNG channels are abundant and easy to study. The widespread distribution of CNG channels in tissues throughout the body has only recently been recognized and the functions of this channel family in many of these tissues remain largely unknown. The molecular biological and pharmacological properties of the CNG channel family are summarized in order to put in context studies aimed at probing CNG channel functions in these tissues using pharmacological and genetic methods. Compounds have now been identified that are useful in distinguishing CNG channel activated pathways from cAMP/cGMP dependent-protein kinases or other pathways. The ways in which these interact with CNG channels are understood and this knowledge is leading to the identification of more potent and more specific CNG channel subtype-specific agonists or antagonists. Recent molecular and genetic analyses have identified novel roles of CNG channels in neuronal development and plasticity in both invertebrates and vertebrates. Targeting CNG channels via specific drugs and genetic manipulation (such as knockout mice) will permit better understanding of the role of CNG channels in both basic and higher orders of brain function.

Animals↗

PubMed1998

Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells.

PURPOSE: To determine whether low concentrations of glutamate induce cell death in purified rat retinal ganglion cells (RGCs). METHODS: Rat retinal ganglion cells were purified from dissociated retinal cells by a modified two-step panning method and were cultured in serum-free medium containing neurotrophic factors and forskolin. Survival of RGCs after exposure to glutamate, with or without glutamate receptor antagonists, was measured by calcein-acetoxymethyl ester staining after 3 days in culture. To visualize calcium signals, RGCs were loaded with the calcium indicator dye, fluo-3 acetoxymethyl ester, and fluorescence was measured by laser scanning confocal microscope. Electrophysiological properties of RGCs were examined by using the whole-cell, patch-clamp technique. RESULTS: The application of increasing concentrations (5-500 microM) of glutamate caused a dose-dependent increase in RGC death after 3 days in culture. Neurotoxic effects of low doses of glutamate were totally blocked by a specific alpha-amino-3-dihydro-5-methyl-isoxazol-4-propionic acid-kainate (AMPA-KA) receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but not by a specific N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonovalerate (APV). In addition, calcium imaging and patch-clamp recordings showed that intracellular calcium accumulation and glutamate-evoked inward currents were completely blocked by DNQX but not by APV. CONCLUSIONS: Low doses of glutamate can activate AMPA-KA receptors in RGCs, which causes increases in intracellular calcium and decreases in cell survival. This is the first report to show the functional role of calcium-permeable AMPA-KA receptors in cultured RGCs.

2-Amino-5-phosphonovalerate↗

PubMed1998

HPC-7: a novel oligodendrocyte lineage protein which appears prior to galactocerebroside.

The identification of cell type-specific molecules expressed at different developmental stages can help to elucidate the regulatory mechanisms governing the survival, differentiation, and development of cells in the central nervous system (CNS). A cell surface protein, HPC-7, was detected on rat oligodendrocytes (OL) in culture by a monoclonal antibody generated against adult rat hippocampal membranes. Adult rat brain and sciatic nerve sections showed selective labeling of white matter and other myelinated fibers in both the CNS and peripheral nervous system (PNS). Double-labeling of secondary cultures of OL, O-2A, and type-2 astrocytes and primary cultures of type-1 astrocytes with independent cell type-specific antibodies confirmed that HPC-7 was expressed only by the OL lineage. By using a series of OL stage-specific antigenic markers (A2B5, 04, OL-1, galactocerebroside, myelin basic protein) HPC-7 was found to appear at the time when OL precursors became A2B5 negative and began their terminal differentiation in OL. On immunoblots, anti-HPC-7 antibody recognized a single 66 kDa band in rat OL and a single band at 100 kDa in adult myelin. N-glycosidase treatment showed that the HPC-7 protein did not contain substantial amounts of N-linked carbohydrate. Thus, HPC-7 appears to be a cell surface protein of the OL lineage that marks the important transition from proliferative precursor to postmitotic OL.

Animals↗

PubMed1997

Direct blockade of both cloned rat rod photoreceptor cyclic nucleotide-gated non-selective cation (CNG) channel alpha-subunit and native CNG channels from Xenopus rod outer segments by H-8, a non-specific cyclic nucleotide-dependent protein kinase inhibitor.

Using excised inside-out patch techniques, a non-specific cyclic nucleotide-dependent protein kinase inhibitor, H-8 (N-2-(methylamino)ethyl-5-isoquinolinesulfonamide), has been shown to suppress directly the activities of both rat rod photoreceptor cyclic GMP-gated channel alpha-subunits expressed in Xenopus oocytes and native cGMP-gated channels from Xenopus rod outer segments. When co-applied with 100 microM cGMP on the cytoplasmic side of the patches, current suppression by H-8 increased with membrane depolarization. Blockade by H-8 was not relieved by supersaturating concentrations of cGMP (1 mM). In addition, blockade by H-8 showed significant inhibition of channel activity at negative holding potentials when acting from the extracellular side of the channel. The results were consistent with the conclusion that H-8 can act as an open channel blocker from either side of the channel. Even though H-8 is a much more effective kinase inhibitor, it is frequently used at concentrations that cause inhibition of cGMP-gated channels and this effect needs to be taken into account when evaluating results obtained with this compound.

Animals↗

PubMed1997

Differentiation and transdifferentiation of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies between the retina and the choroid of the eye and plays a vital role in ocular metabolism. The RPE develops from the same sheet of neuroepithelium as the retina and the two derivatives become distinguished by different expression patterns of a number of transcription factors during embryonic development. As the RPE layer differentiates it expresses a set of unique molecules, many of which are restricted to certain regions of the cell. PRE cells undergo both a loss of polarity and a loss of expression of many of these cell type-specific molecules when placed in monolayer culture. The RPE of many species, including mammals, can be induced to transdifferentiate by growth factors such as basic fibroblast growth factor. Under the influence of such factors the RPE is triggered to alter expression of a wide array of molecules and to take on a retinal epithelium fate, from which differentiated retinal cell types including rod photoreceptors can be produced.

Animals↗

PubMed1997

Cyclic nucleotide gated channels as regulators of CNS development and plasticity.

Cyclic nucleotide gated (CNG) cation channels are critical for signal transduction in vertebrate visual and olfactory systems. Members of the CNG channel gene family have now been cloned from a number of species, from Caenorhabditis elegans to humans. An important advance has been the discovery that CNG channels are present in many neurons of the mammalian brain. CNG channels act as molecular links between G-protein-coupled cascades, Ca2+-signalling systems, and gaseous messenger pathways. Perhaps most striking are recent data implicating CNG channels in both developmental and synaptic plasticity.

Animals↗

PubMed1997

Experimental preretinal neovascularization by laser-induced venous thrombosis in rats.

PURPOSE: Retinal ischemia and neovascularization (NV) are important components of many retinal disorders. To facilitate further investigation of retinal ischemia and neovascularization, we sought to develop a reproducible in vivo experimental model of venous occlusion by photodynamic thrombosis in rats. METHODS: After anesthesia, 27 eyes of pigmented rats received an intraperitoneal injection of 0.2 ml of, 10% sodium fluorescein 15 minutes prior to laser treatment. With a blue-green argon laser, selected venous sites next to the optic nerve head were photocoagulated indirectly with a 78 diopter lens. Venous occlusion was accomplished using laser parameters of 1.0 second, 50 microns, and 50-100 mW. For a control group, 10 eyes were coagulated on the retina between major vessels using the same parameters after fluorescein injection. For a second control group, 1% sodium hyaluronate was injected into the subretinal space to make a long-standing retinal detachment in 5 eyes. RESULTS: With 1-8 laser impulses, each venous occlusion was obtained and was associated with extreme venous constriction and tortuousity. Retinal edema became evident 10-30 minutes after treatment in the sectors associated with the occluded veins. This edema became a bullous retinal detachment (RD) within 12 hours and intra-retinal hemorrhage was observed. The retinal edema continued for 3-10 days and the retinas reattached spontaneously. Prior to or after retinal reattachment 70% (19/27) of eyes developed retinal NV and tractional RD. Of these, 11 developed NV of the optic disc (NVD), 6 developed NV elsewhere (NVE), and 2 developed NVD and NVE. In 30% (8/27) of the eyes, retinal edema resolved without evidence of NV. In control groups no eyes showed either circulatory disorders or evidence of NV. CONCLUSIONS: This is a new model of retinal ischemia and associated neovascularization established by venous thrombosis that is easily reproducible. Many aspects of rat retinal physiology are known and this model has promise as an avenue for further investigation.

Animals↗

PubMed1996

Identification of competitive antagonists of the rod photoreceptor cGMP-gated cation channel: beta-phenyl-1,N2-etheno-substituted cGMP analogues as probes of the cGMP-binding site.

cGMP is the natural activator of the cyclic nucleotide-gated channel originally isolated from rod photoreceptors but now known to be expressed in a wide variety of neural and non-neural cells. To identify antagonists of cGMP action and to better understand the interaction between cGMP and the channel protein, experimental studies were undertaken using four synthetic cGMP analogues, PET-cGMP, 8-Br-PET-cGMP, Rp-8-Br-PET-cGMPS, and Sp-8-Br-PET-cGMPS. With excised patches from either Xenopus oocytes expressing a cloned rat rod channel alpha-subunit or from native Xenopus rod photoreceptors, Rp-8-Br-PET-cGMPS competitively suppressed the cGMP-induced current with an IC50 of 25 microM and Sp-8-Br-PET-cGMPS inhibited this current with an IC50 of 105 microM. On the expressed rat rod channel, 8-Br-PET-cGMP behaved as a very weak partial agonist at high concentrations and an antagonist (IC50 = 64 microM) at lower concentrations when coapplied with cGMP. PET-cGMP did not activate channel currents alone but showed a synergism when coapplied with subsaturating concentrations of cGMP. Because Sp-8-Br-PET-cGMPS is a potent activator of type I cGMP-dependent protein kinase, but a competitive antagonist of channel activation, it will be a useful reagent for discriminating between those effects of cGMP that are mediated by a protein kinase and those mediated by channel activation. Because the PET derivatives all contain a phenyl-substituted 5-membered ring system fused to the amino group in position 2 and the nitrogen in position 1 of the guanine ring, the results support the idea that N1 and N2 are important for channel activation. They also suggest a minor role for the cyclic phosphate group in binding or activation.

Animals↗

PubMed1996

cGMP-mediated effects on the physiology of bovine and human retinal Müller (glial) cells.

1. Whole-cell currents of freshly dissociated or cultured Müller cells from human and bovine retinas were studied using the perforated-patch and standard whole-cell recording techniques. 2. We found that internal perfusion of cGMP or external exposure to 8-bromo-cGMP activated a calcium permeable, non-selective cation current in Müller cells, the principal glial cells of the retina. In addition, the activity of calcium-activated potassium channels increased markedly. These currents were minimally affected by cAMP. 3. Molecular studies using the reverse transcription-polymerase chain reaction demonstrated that human müller cells in culture contain transcripts closely related to the rod cyclic nucleotide-gated (CNG) channel. 4. Since guanylate cyclase is a known target for nitric oxide (NO), we tested the effect of NO donors on Müller cell currents. These agents induced currents that were qualitatively similar to those activated by cGMP. 5. Our experiments support the idea that the NO-cGMP pathway regulates the physiology of Müller cells and may play a role in integrating neuron-glia interactions in the retina.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

Animals↗

PubMed1996

Differential effects of bFGF on development of the rat retina.

A variety of growth factors can influence the expression of differentiated properties by cell types of the developing retina. One unresolved question has been whether these factors can direct the differentiation pathway of uncommitted precursors or whether they act to help the expression of properties by already committed cells. To address this question we have studied the effects of basic fibroblast growth factor (bFGF) on the differentiation of ganglion cells and rod photoreceptors in explant cultures of embryonic rat retinas. Incubation of retinas in the presence of bFGF accelerated the appearance of differentiated ganglion cells and incubation in the presence of anti-bFGF antibodies delayed the appearance. bFGF had no effect on the appearance of differentiated rod photoreceptors as judged by expression of opsin, although all-trans-retinoic acid did increase the number of cells expressing opsin. bFGF inhibited the formation of rod photoreceptor rosettes suggesting that it does influence some properties of rods or the adjacent Müller glial cells. The results suggest that bFGF can alter the timing of differentiation of retinal ganglion cells but not direct their production from retinal precursors.

Animals↗