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Sample records for aii amacrine cell

  1. The AII amacrine cell connectome: a dense network hub

    PubMed Central

    Marc, Robert E.; Anderson, James R.; Jones, Bryan W.; Sigulinsky, Crystal L.; Lauritzen, James S.

    2014-01-01

    The mammalian AII retinal amacrine cell is a narrow-field, multistratified glycinergic neuron best known for its role in collecting scotopic signals from rod bipolar cells and distributing them to ON and OFF cone pathways in a crossover network via a combination of inhibitory synapses and heterocellular AII::ON cone bipolar cell gap junctions. Long considered a simple cell, a full connectomics analysis shows that AII cells possess the most complex interaction repertoire of any known vertebrate neuron, contacting at least 28 different cell classes, including every class of retinal bipolar cell. Beyond its basic role in distributing rod signals to cone pathways, the AII cell may also mediate narrow-field feedback and feedforward inhibition for the photopic OFF channel, photopic ON-OFF inhibitory crossover signaling, and serves as a nexus for a collection of inhibitory networks arising from cone pathways that likely negotiate fast switching between cone and rod vision. Further analysis of the complete synaptic counts for five AII cells shows that (1) synaptic sampling is normalized for anatomic target encounter rates; (2) qualitative targeting is specific and apparently errorless; and (3) that AII cells strongly differentiate partner cohorts by synaptic and/or coupling weights. The AII network is a dense hub connecting all primary retinal excitatory channels via precisely weighted drive and specific polarities. Homologs of AII amacrine cells have yet to be identified in non-mammalians, but we propose that such homologs should be narrow-field glycinergic amacrine cells driving photopic ON-OFF crossover via heterocellular coupling with ON cone bipolar cells and glycinergic synapses on OFF cone bipolar cells. The specific evolutionary event creating the mammalian AII scotopic-photopic hub would then simply be the emergence of large numbers of pure rod bipolar cells. PMID:25237297

  2. The AII amacrine cell connectome: a dense network hub.

    PubMed

    Marc, Robert E; Anderson, James R; Jones, Bryan W; Sigulinsky, Crystal L; Lauritzen, James S

    2014-01-01

    The mammalian AII retinal amacrine cell is a narrow-field, multistratified glycinergic neuron best known for its role in collecting scotopic signals from rod bipolar cells and distributing them to ON and OFF cone pathways in a crossover network via a combination of inhibitory synapses and heterocellular AII::ON cone bipolar cell gap junctions. Long considered a simple cell, a full connectomics analysis shows that AII cells possess the most complex interaction repertoire of any known vertebrate neuron, contacting at least 28 different cell classes, including every class of retinal bipolar cell. Beyond its basic role in distributing rod signals to cone pathways, the AII cell may also mediate narrow-field feedback and feedforward inhibition for the photopic OFF channel, photopic ON-OFF inhibitory crossover signaling, and serves as a nexus for a collection of inhibitory networks arising from cone pathways that likely negotiate fast switching between cone and rod vision. Further analysis of the complete synaptic counts for five AII cells shows that (1) synaptic sampling is normalized for anatomic target encounter rates; (2) qualitative targeting is specific and apparently errorless; and (3) that AII cells strongly differentiate partner cohorts by synaptic and/or coupling weights. The AII network is a dense hub connecting all primary retinal excitatory channels via precisely weighted drive and specific polarities. Homologs of AII amacrine cells have yet to be identified in non-mammalians, but we propose that such homologs should be narrow-field glycinergic amacrine cells driving photopic ON-OFF crossover via heterocellular coupling with ON cone bipolar cells and glycinergic synapses on OFF cone bipolar cells. The specific evolutionary event creating the mammalian AII scotopic-photopic hub would then simply be the emergence of large numbers of pure rod bipolar cells.

  3. Surround inhibition of mammalian AII amacrine cells is generated in the proximal retina

    PubMed Central

    Bloomfield, Stewart A; Xin, Daiyan

    2000-01-01

    Intracellular recordings were obtained from neurons in the superfused retina-eyecup preparation of the rabbit under dark-adapted conditions. Neurotransmitter agonists and antagonists were applied exogenously via the superfusate to dissect the synaptic pathways pharmacologically and thereby determine those pathways responsible for the generation of the on-centre/off-surround receptive fields of AII amacrine cells. Application of the metabotropic glutamate receptor agonist, APB, reversibly blocked both the on-centre and off-surround responses of AII cells. These data were consistent with the idea that both the centre- and surround-mediated responses are derived from inputs from the presynaptic rod bipolar cells. Whereas rod bipolar cells showed on-receptive fields ≈100 μm across, we found no evidence for an antagonistic off-surround response using light stimuli which effectively elicited the off-surrounds of AII amacrine cells. These results indicated that the surrounds of AII cells are not derived from rod bipolar cell inputs. Application of the ionotropic glutamate receptor antagonists CNQX or DNQX enhanced the on-centre responses of AII cells but attenuated the off-surround responses. These data indicated that the centre- and surround-mediated responses could not both be derived from signals crossing the rod bipolar-to-AII cell synapse. Application of the glycine antagonist, strychnine, had only minor and variable effects on AII cell responses. However, the GABA antagonists picrotoxin and bicuculline enhanced the on-centre response but attenuated or completely blocked the off-surround response of AII cells. The GABA antagonists had no effect on the responses of horizontal cells indicating that their effects on AII cell responses reflected actions on inner retinal circuitry rather than feedback circuitry in the outer plexiform layer. Application of the voltage-gated sodium channel blocker TTX enhanced the on-centre responses of AII cells but attenuated or

  4. AII amacrine cells discriminate between heterocellular and homocellular locations when assembling connexin36-containing gap junctions.

    PubMed

    Meyer, Arndt; Hilgen, Gerrit; Dorgau, Birthe; Sammler, Esther M; Weiler, Reto; Monyer, Hannah; Dedek, Karin; Hormuzdi, Sheriar G

    2014-03-15

    Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (Cx36) is the most abundant isoform; however, the mechanisms underlying formation of Cx36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native Cx36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-Cx36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of Cx36-EGFP clusters on an AII cell in the KO-Cx36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced - as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling Cx36. We suggest that employing different gap-junction-forming mechanisms could provide the means for a cell to regulate its gap junctions in a target-cell-specific manner, even if these junctions contain the same connexin.

  5. AII amacrine cells discriminate between heterocellular and homocellular locations when assembling connexin36-containing gap junctions

    PubMed Central

    Meyer, Arndt; Hilgen, Gerrit; Dorgau, Birthe; Sammler, Esther M.; Weiler, Reto; Monyer, Hannah; Dedek, Karin; Hormuzdi, Sheriar G.

    2014-01-01

    ABSTRACT Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (Cx36) is the most abundant isoform; however, the mechanisms underlying formation of Cx36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native Cx36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-Cx36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of Cx36–EGFP clusters on an AII cell in the KO-Cx36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced – as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling Cx36. We suggest that employing different gap-junction-forming mechanisms could provide the means for a cell to regulate its gap junctions in a target-cell-specific manner, even if these junctions contain the same connexin. PMID:24463820

  6. Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina

    PubMed Central

    Choi, Hannah; Zhang, Lei; Cembrowski, Mark S.; Sabottke, Carl F.; Markowitz, Alexander L.; Butts, Daniel A.; Kath, William L.; Singer, Joshua H.

    2014-01-01

    In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell. PMID:25008417

  7. Elucidating the role of AII amacrine cells in glutamatergic retinal waves.

    PubMed

    Firl, Alana; Ke, Jiang-Bin; Zhang, Lei; Fuerst, Peter G; Singer, Joshua H; Feller, Marla B

    2015-01-28

    Spontaneous retinal activity mediated by glutamatergic neurotransmission-so-called "Stage 3" retinal waves-drives anti-correlated spiking in ON and OFF RGCs during the second week of postnatal development of the mouse. In the mature retina, the activity of a retinal interneuron called the AII amacrine cell is responsible for anti-correlated spiking in ON and OFF α-RGCs. In mature AIIs, membrane hyperpolarization elicits bursting behavior. Here, we postulated that bursting in AIIs underlies the initiation of glutamatergic retinal waves. We tested this hypothesis by using two-photon calcium imaging of spontaneous activity in populations of retinal neurons and by making whole-cell recordings from individual AIIs and α-RGCs in in vitro preparations of mouse retina. We found that AIIs participated in retinal waves, and that their activity was correlated with that of ON α-RGCs and anti-correlated with that of OFF α-RGCs. Though immature AIIs lacked the complement of membrane conductances necessary to generate bursting, pharmacological activation of the M-current, a conductance that modulates bursting in mature AIIs, blocked retinal wave generation. Interestingly, blockade of the pacemaker conductance Ih, a conductance absent in AIIs but present in both ON and OFF cone bipolar cells, caused a dramatic loss of spatial coherence of spontaneous activity. We conclude that during glutamatergic waves, AIIs act to coordinate and propagate activity generated by BCs rather than to initiate spontaneous activity.

  8. Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling

    PubMed Central

    Kothmann, W. Wade; Massey, Stephen C.; O’Brien, John

    2010-01-01

    Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the central nervous system and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D1-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell type-specific responses dependent on the identity of the signaling complexes assembled. PMID:19940186

  9. Feedback inhibition in the inner plexiform layer underlies the surround-mediated responses of AII amacrine cells in the mammalian retina

    PubMed Central

    Völgyi, Béla; Xin, Daiyan; Bloomfield, Stewart A

    2002-01-01

    Intracellular recordings were made from narrow-field, bistratified AII amacrine cells in the isolated, superfused retina-eyecup of the rabbit. Pharmacological agents were applied to neurons to dissect the synaptic pathways subserving AII cells so as to determine the circuitry generating their off-surround responses. Application of the GABA antagonists, picrotoxin, bicuculline and 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid (TPMPA) all increased the on-centre responses of AII amacrine cells, but attenuated the off-surround activity. At equal concentrations, picrotoxin was approximately twice as effective as bicuculline or TPMPA in modifying the response activity of AII amacrine cells. These results indicate that the mechanism underlying surround inhibition of AII amacrine cells includes activation of both GABAA and GABAC receptors in an approximately equal ratio. Application of the GABA antagonists also increased the size of on-centre receptive fields of AII amacrine cells. Again, picrotoxin was most effective, producing, on average, a 54 % increase in the size of the receptive field, whereas bicuculline and TPMPA produced comparable 34 and 33 % increases, respectfully. Application of the voltage-gated sodium channel blocker TTX produced effects on AII amacrine cells qualitatively similar to those of the GABA blockers. Intracellular application of the chloride channel blocker 4,4′-dinitro-stilbene-2,2′-disulphonic acid (DNDS) abolished the direct effects of GABA on AII amacrine cells. Moreover, DNDS increased the amplitude of both the on-centre and off-surround responses. The failure of DNDS to block the off-surround activity indicates that it is not mediated by direct GABAergic inhibition. Taken together, our results suggest that surround receptive fields of AII amacrine cells are generated indirectly by the GABAergic, reciprocal feedback synapses from S1/S2 amacrine cells to the axon terminals of rod bipolar cells. PMID:11882691

  10. Relative contributions of rod and cone bipolar cell inputs to AII amacrine cell light responses in the mouse retina

    PubMed Central

    Pang, Ji-Jie; Abd-El-Barr, Muhammad M; Gao, Fan; Bramblett, Debra E; Paul, David L; Wu, Samuel M

    2007-01-01

    AII amacrine cells (AIIACs) are crucial relay stations for rod-mediated signals in the mammalian retina and they receive synaptic inputs from depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) as well as from other amacrine cells. Using whole-cell voltage-clamp technique in conjunction with pharmacological tools, we found that the light-evoked current response of AIIACs in the mouse retina is almost completely mediated by two DBC synaptic inputs: a 6,7-dinitro-quinoxaline-2,3-dione (DNQX)-resistant component mediated by cone DBCs (DBCCs) through an electrical synapse, and a DNQX-sensitive component mediated by rod DBCs (DBCRs). This scheme is supported by AIIAC current responses recorded from two knockout mice. The dynamic range of the AIIAC light response in the Bhlhb4−/− mouse (which lacks DBCRs) resembles that of the DNQX-resistant component, and that of the connexin36 (Cx36)−/− mouse resembles the DNQX-sensitive component. By comparing the light responses of the DBCCs with the DNQX-resistant AIIAC component, and light responses of the DBCRs with the DNQX-sensitive AIIAC component, we obtained the input–output relations of the DBCC→AIIAC electrical synapse and the DBCR→AIIAC chemical synapse. Similar to other glutamatergic chemical synapses in the retina, the DBCR→AIIAC synapse is non-linear. Its highest voltage gain (approximately 5) is found near the dark membrane potential, and it saturates for presynaptic signals larger than 5.5 mV. The DBCC→AIIAC electrical synapse is approximately linear (voltage gain of 0.92), consistent with the linear junctional conductance found in retinal electrical synapses. Moreover, relative DBCR and DBCC contributions to the AIIAC response at various light intensity levels are determined. PMID:17255172

  11. Relative contributions of rod and cone bipolar cell inputs to AII amacrine cell light responses in the mouse retina.

    PubMed

    Pang, Ji-Jie; Abd-El-Barr, Muhammad M; Gao, Fan; Bramblett, Debra E; Paul, David L; Wu, Samuel M

    2007-04-15

    AII amacrine cells (AIIACs) are crucial relay stations for rod-mediated signals in the mammalian retina and they receive synaptic inputs from depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) as well as from other amacrine cells. Using whole-cell voltage-clamp technique in conjunction with pharmacological tools, we found that the light-evoked current response of AIIACs in the mouse retina is almost completely mediated by two DBC synaptic inputs: a 6,7-dinitro-quinoxaline-2,3-dione (DNQX)-resistant component mediated by cone DBCs (DBC(C)s) through an electrical synapse, and a DNQX-sensitive component mediated by rod DBCs (DBC(R)s). This scheme is supported by AIIAC current responses recorded from two knockout mice. The dynamic range of the AIIAC light response in the Bhlhb4-/- mouse (which lacks DBC(R)s) resembles that of the DNQX-resistant component, and that of the connexin36 (Cx36)-/- mouse resembles the DNQX-sensitive component. By comparing the light responses of the DBC(C)s with the DNQX-resistant AIIAC component, and light responses of the DBC(R)s with the DNQX-sensitive AIIAC component, we obtained the input-output relations of the DBC(C)-->AIIAC electrical synapse and the DBC(R)-->AIIAC chemical synapse. Similar to other glutamatergic chemical synapses in the retina, the DBC(R)-->AIIAC synapse is non-linear. Its highest voltage gain (approximately 5) is found near the dark membrane potential, and it saturates for presynaptic signals larger than 5.5 mV. The DBC(C)-->AIIAC electrical synapse is approximately linear (voltage gain of 0.92), consistent with the linear junctional conductance found in retinal electrical synapses. Moreover, relative DBC(R) and DBC(C) contributions to the AIIAC response at various light intensity levels are determined.

  12. Connexin30.2: In Vitro Interaction with Connexin36 in HeLa Cells and Expression in AII Amacrine Cells and Intrinsically Photosensitive Ganglion Cells in the Mouse Retina

    PubMed Central

    Meyer, Arndt; Tetenborg, Stephan; Greb, Helena; Segelken, Jasmin; Dorgau, Birthe; Weiler, Reto; Hormuzdi, Sheriar G.; Janssen-Bienhold, Ulrike; Dedek, Karin

    2016-01-01

    Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36—both expressed in AII amacrine cells—are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners. PMID:27303262

  13. Parvalbumin-immunoreactive amacrine cells of macaque retina

    PubMed Central

    Klump, Kathryn E.; Zhang, Ai-Jun; Wu, Samuel M.; Marshak, David W.

    2012-01-01

    A number of authors have observed amacrine cells containing high levels of immunoreactive parvalbumin in primate retinas. The experiments described here were designed to identify these cells morphologically, to determine their neurotransmitter, to record their light responses, and to describe the other cells that they contact. Macaque retinas were fixed in paraformaldehyde and labeled with antibodies to parvalbumin and one or two other markers, and this double- and triple-labeled material was analyzed by confocal microscopy. In their morphology and dendritic stratification patterns, the parvalbumin-positive cells closely resembled the knotty type 2 amacrine cells described using the Golgi method in macaques. They contained immunoreactive glycine transporter, but not immunoreactive γ-aminobutyric acid, and therefore, they use glycine as their neurotransmitter. Their spatial density was relatively high, roughly half that of AII amacrine cells. They contacted lobular dendrites of AII cells, and they are expected to be presynaptic to AII cells based on earlier ultrastructural studies. They also made extensive contacts with axon terminals of OFF midget bipolar cells whose polarity cannot be predicted with certainty. A macaque amacrine cell of the same morphological type depolarized at the onset of increments in light intensity, and it was well coupled to other amacrine cells. Previously, we described amacrine cells like these that contacted OFF parasol ganglion cells and OFF starburst amacrine cells. Taken together, these findings suggest that one function of these amacrine cells is to inhibit the transmission of signals from rods to OFF bipolar cells via AII amacrine cells. Another function may be inhibition of the OFF pathway following increments in light intensity. PMID:19435546

  14. Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina

    PubMed Central

    Lee, Sammy C.S.; Meyer, Arndt; Schubert, Timm; Hüser, Laura; Dedek, Karin; Haverkamp, Silke

    2015-01-01

    Amacrine cells comprise ~30 morphological types in the mammalian retina. The synaptic connectivity and function of a few GABAergic wide-field amacrine cells have recently been studied, however, with the exception of the rod pathway-specific AII amacrine cell the connectivity of glycinergic small-field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small-field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A-type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. Taken together, the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway-specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A-type ganglion cells. PMID:25630271

  15. Intracellular control of axial shape in non-uniform neurites: a serial electron microscopic analysis of organelles and microtubules in AI and AII retinal amacrine neurites

    PubMed Central

    1984-01-01

    AI and AII cat retinal amacrine cells have highly varicose non-uniform, neuritic processes. Processes of both types were reconstructed via a computer system using serial electron micrographs. These reconstructions were analyzed for (a) varicosity volume, surface area, and length, (b) "neck" volume, surface area, and length, (c) number of microtubules within the varicosity, (d) number of microtubules within the "neck," and (e) volume and surface area of mitochondria and smooth endoplasmic reticulum and large smooth vesicular bodies within the processes. Correlation of these parameters revealed a linear relationship between the number of microtubules in the necks and mean neck cross-sectional area (rs = 0.780, P less than 0.001), while microtubule number within the varicosities showed no correlation with varicosity volume (rs = 0.239, P greater than 0.2). Varicosity volume did, however, correlate strongly with the summed volume of mitochondria and smooth vesicular bodies contained within the varicosity for both cell types examined. The ratio between membranous organelle volume and varicosity volume for AI amacrine processes of 1:6.97 (rs = 0.927), differed from the ratio of 1:1.80 for the AII amacrine processes (rs = 0.987). Similar relationships were observed in other nonvaricose neurites such as optic tract axons. Membranous organelles appear to contribute an additional obligatory volume to the cytosol that can be as much as seven times the organelles' direct volume. These observations suggest that both the cytoskeletal components, and the membrane organelles play a direct role in determining neurite shape. PMID:6538879

  16. Expression of brain-derived neurotrophic factor in cholinergic and dopaminergic amacrine cells in the rat retina and the effects of constant light rearing.

    PubMed

    Fujieda, Hiroki; Sasaki, Hiroshi

    2008-02-01

    Brain-derived neurotrophic factor (BDNF) regulates many aspects of neuronal development, including survival, axonal and dendritic growth and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF in retinal development, the retinal cell types expressing BDNF remains poorly defined. The goal of the present study was to determine the localization of BDNF in the mammalian retina, with special focus on the subtypes of amacrine cells, and to characterize, at the cellular level, the effects of constant light exposure during early postnatal period on retinal expression of BDNF. Retinas from 3-week-old rats reared in a normal light cycle or constant light were subjected to double immunofluorescence staining using antibodies to BDNF and retinal cell markers. BDNF immunoreactivity was localized to ganglion cells, cholinergic amacrine cells and dopaminergic amacrine cells, but not to AII amacrine cells regardless of rearing conditions. Approximately 75% of BDNF-positive cells in the inner nuclear layer were cholinergic amacrine cells in animals reared in a normal lighting condition. While BDNF immunoreactivity in ganglion cells and cholinergic amacrine cells was significantly increased by constant light rearing, which in dopaminergic amacrine cells was apparently unaltered. The overall structure of the retina and the density of ganglion cells, cholinergic amacrine cells and AII amacrine cells were unaffected by rearing conditions, whereas the density of dopaminergic amacrine cells was significantly increased by constant light rearing. The present results indicate that cholinergic amacrine cells are the primary source of BDNF in the inner nuclear layer of the rat retina and provide the first evidence that cholinergic amacrine cells may be involved in the visual activity-dependent regulation of retinal development through the production of BDNF. The present data also suggest that the production or survival of dopaminergic amacrine

  17. Development of Retinal Amacrine Cells and Their Dendritic Stratification

    PubMed Central

    Balasubramanian, Revathi

    2014-01-01

    Themammalian retina containsmultiple neurons, each of which contributes differentially to visual processing. Of these retinal neurons, amacrine cells have recently come to prime light since they facilitate majority of visual processing that takes place in the retina. Amacrine cells are also the most diverse group of neurons in the retina, classified majorly based on the neurotransmitter type they express and morphology of their dendritic arbors. Currently, little is known about the molecular basis contributing to this diversity during development. Amacrine cells also contribute to most of the synapses in the inner plexiform layer and mediate visual information input from bipolar cells onto retinal ganglion cells. In this review, we will describe the current understanding of amacrine cell and cell subtype development. Furthermore, we will address the molecular basis of retinal lamination at the inner plexiform layer. Overall, our review will provide a developmental perspective of amacrine cell subtype classification and their dendritic stratification. PMID:25170430

  18. Angiotensin type-1 receptor inhibition is neuroprotective to amacrine cells in a rat model of retinopathy of prematurity.

    PubMed

    Downie, Laura E; Hatzopoulos, Kate M; Pianta, Michael J; Vingrys, Algis J; Wilkinson-Berka, Jennifer L; Kalloniatis, Michael; Fletcher, Erica L

    2010-01-01

    Retinopathy of prematurity (ROP) is characterized by deficits in the scotopic pathway, although the cellular locus for these deficits is not clear. Here we examined neurochemical and cellular changes that develop during oxygen-induced retinopathy, a model of ROP. In addition, we examined whether treatment with the angiotensin II type-1 receptor inhibitor, valsartan, prevented these changes. Newborn Sprague-Dawley rats were exposed from postnatal day (P) 0 to 11 to 80%:20% O(2) (22:2 hr/day) and then room air until P18. Valsartan (40 mg/kg/day) was administered from P12-P18. Pattern recognition analysis of overlapping amino acid profiles was used to provide a statistically robust and spatially complete classification of neural elements for each experimental condition. Classification yielded 12 neuronal theme classes in controls and nine classes following ROP. ROP was associated with a reduction in the number of amacrine and bipolar cell theme classes. The reduction in theme classes was confirmed as true neuronal loss by quantifying anatomical changes and using an apoptotic marker. ROP was associated with shifts in amino acid levels across all neuronal populations except for horizontal cells. A reduction in the density of glycine-immunoreactive amacrine cells, and particularly parvalbumin-immunoreactive AII amacrine cells, was observed following ROP. Valsartan treatment during ROP prevented loss of theme classes and loss of AII amacrine cells. This study suggests that deficits in scotopic vision during ROP may be associated with loss of AII amacrine cells. In addition, this study highlights the potential of AT(1)R blockade in preventing neuronal anomalies in this condition.

  19. The projective field of a retinal amacrine cell

    PubMed Central

    de Vries, Saskia E. J.; Baccus, Stephen A.; Meister, Markus

    2011-01-01

    In sensory systems, neurons are generally characterized by their receptive field, namely the sensitivity to activity patterns at the circuit's input. To assess the neuron's role in the system, one must also know its projective field, namely the spatio-temporal effects the neuron exerts on all the circuit's outputs. We studied both the receptive and projective fields of an amacrine interneuron in the salamander retina. This amacrine type has a sustained OFF response with a small receptive field, but its output projects over a much larger region. Unlike other amacrines, this type is remarkably promiscuous and affects nearly every ganglion cell within reach of its dendrites. Its activity modulates the sensitivity of visual responses in ganglion cells, while leaving their kinetics unchanged. The projective field displays a center-surround structure: Depolarizing a single amacrine suppresses the visual sensitivity of ganglion cells nearby, and enhances it at greater distances. This change in sign is seen even within the receptive field of one ganglion cell; thus the modulation occurs presynaptically on bipolar cell terminals, most likely via GABAB receptors. Such an antagonistic projective field could contribute to the retina's mechanisms for predictive coding. PMID:21653863

  20. Intrinsic oscillatory activity arising within the electrically coupled AII amacrine–ON cone bipolar cell network is driven by voltage-gated Na+ channels

    PubMed Central

    Trenholm, Stuart; Borowska, Joanna; Zhang, Jiawei; Hoggarth, Alex; Johnson, Kyle; Barnes, Steven; Lewis, Timothy J; Awatramani, Gautam B

    2012-01-01

    In the rd1 mouse model for retinal degeneration, the loss of photoreceptors results in oscillatory activity (∼10–20 Hz) within the remnant electrically coupled network of retinal ON cone bipolar and AII amacrine cells. We tested the role of hyperpolarization-activated currents (Ih), voltage-gated Na+ channels and gap junctions in mediating such oscillatory activity. Blocking Ih (1 mm Cs+) hyperpolarized the network and augmented activity, while antagonizing voltage-dependent Na+ channels (1 μm TTX) abolished oscillatory activity in the AII amacrine–ON cone bipolar cell network. Voltage-gated Na+ channels were only observed in AII amacrine cells, implicating these cells as major drivers of activity. Pharmacologically uncoupling the network (200 μm meclofenamic acid (MFA)) blocked oscillations in all cells indicating that Na+ channels exert their influence over multiple cell types within the network. In wt retina, occluding photoreceptor inputs to bipolar cells (10 μm NBQX and 50 μm l-AP4) resulted in a mild (∼10 mV) hyperpolarization and the induction of oscillatory activity within the AII amacrine–ON cone bipolar cell network. These oscillations had similar properties to those observed in rd1 retina, suggesting that no major degeneration-induced network rewiring is required to trigger spontaneous oscillations. Finally, we constructed a simplified computational model that exhibited Na+ channel-dependent network oscillations. In this model, mild heterogeneities in channel densities between individual neurons reproduced our experimental findings. These results indicate that TTX-sensitive Na+ channels in AII amacrine cells trigger degeneration-induced network oscillations, which provide a persistent synaptic drive to downstream remnant neurons, thus appearing to replace photoreceptors as the principal drivers of retinal activity. PMID:22393249

  1. Effects of histamine on light responses of amacrine cells in tiger salamander retina.

    PubMed

    Yu, Yongchun; Satoh, Hiromasa; Vila, Alejandro; Wu, Samuel M; Marshak, David W

    2011-04-01

    Using immunofluorescence, we showed that histamine receptor 1 is expressed by horizontal cell axons and a subset of amacrine cells in the tiger salamander retina. The effects of histamine on light responses of amacrine cells were studied in slice preparations. Histamine modulated the light responses of many salamander amacrine cells, depending upon the morphological type. The most pronounced effects of histamine were decreases in the light responses of broadly stratified amacrine cells, particularly those having medium-sized dendritic field diameters. To determine whether the effects of histamine were direct, Co(++) was substituted for Ca(++) in the extracellular medium to block synaptic transmission. Histamine still affected broadly stratified amacrine cells, but not narrowly stratified amacrine cells under these conditions. Taken together, these findings suggest that inhibitory interactions between strata of the IPL and within the classical receptive fields of the ganglion cells would be particularly sensitive to histamine released from retinopetal axons.

  2. Effects of Histamine on Light Responses of Amacrine Cells in Tiger Salamander Retina

    PubMed Central

    Yu, Yongchun; Satoh, Hiromasa; Vila, Alejandro; Wu, Samuel M.; Marshak, David W.

    2011-01-01

    Using immunofluorescence, we showed that histamine receptor 1 is expressed by horizontal cell axons and a subset of amacrine cells in the tiger salamander retina. The effects of histamine on light responses of amacrine cells were studied in slice preparations. Histamine modulated the light responses of many salamander amacrine cells, depending upon the morphological type. The most pronounced effects of histamine were decreases in the light responses of broadly stratified amacrine cells, particularly those having medium-sized dendritic field diameters. To determine whether the effects of histamine were direct, Co++ was substituted for Ca++ in the extracellular medium to block synaptic transmission. Histamine still affected broadly stratified amacrine cells, but not narrowly stratified amacrine cells under these conditions. Taken together, these findings suggest that inhibitory interactions between strata of the IPL and within the classical receptive fields of the ganglion cells would be particularly sensitive to histamine released from retinopetal axons. PMID:20878231

  3. The Lgr5 transgene is expressed specifically in glycinergic amacrine cells in the mouse retina.

    PubMed

    Sukhdeo, Kumar; Koch, Catherine E; Miller, Tyler E; Zhou, Hannah; Rivera, Maricruz; Yan, Kenneth; Cepko, Constance L; Lathia, Justin D; Rich, Jeremy N

    2014-02-01

    Retinal amacrine cells are a diverse set of interneurons within the inner nuclear layer. The canonical Wnt pathway is highly active within mature amacrine cells, but its role remains unclear. Leucine-rich repeat containing G-protein receptor 5 (Lgr5) is a newly identified component of the Wnt receptor complex that potentiates beta-catenin signaling. In multiple epithelial organs Lgr5 marks adult tissue stem cells. We investigated the expression of this gene using Lgr5-eGFP-IRES-CreER transgenic reporter mice. In the eye, Lgr5 was exclusively expressed in glycinergic amacrine cells in adult mice. Amacrine cells are post-mitotic and represent the first neuronal and non-stem cell lineage to express Lgr5. We further interrogated the spatiotemporal labeling of individual amacrine cells with controlled fluorophore expression. This "fluorofilling" technique provides a tool to study amacrine morphology and dissect neural networks.

  4. The Lgr5 Transgene is Expressed Specifically in Glycinergic Amacrine Cells in the Mouse Retina

    PubMed Central

    Sukhdeo, Kumar; Koch, Catherine E.; Miller, Tyler E.; Zhou, Hannah; Rivera, Maricruz; Yan, Kenneth; Cepko, Constance L.; Lathia, Justin D.; Rich, Jeremy N.

    2014-01-01

    Retinal amacrine cells are a diverse set of interneurons within the inner nuclear layer. The canonical Wnt pathway is highly active within mature amacrine cells, but its role remains unclear. Leucine-rich repeat containing G-protein receptor 5 (Lgr5) is a newly identified component of the Wnt receptor complex that potentiates beta-catenin signaling. In multiple epithelial organs Lgr5 marks adult tissue stem cells. We investigated the expression of this gene using Lgr5-eGFP-IRES-CreER transgenic reporter mice. In the eye, Lgr5 was exclusively expressed in glycinergic amacrine cells in adult mice. Amacrine cells are post-mitotic and represent the first neuronal and non-stem cell lineage to express Lgr5. We further interrogated the spatiotemporal labeling of individual amacrine cells with controlled fluorophore expression. This “fluorofilling” technique provides a tool to study amacrine morphology and dissect neural networks. PMID:24246263

  5. Localization of neurotensin-like immunoreactive amacrine cells in the larval tiger salamander retina.

    PubMed

    Yang, S Z; Watt, C B; Lam, D M; Wu, S M

    1988-01-01

    Light microscopic immunocytochemistry was used to localize the populations of NT-like immunoreactive amacrine cells in the larval tiger salamander retina. Seventy-nine percent of NT-immunostained cells observed in transverse cryo-prepared sections were classified as Type 1 amacrine cells. Another 6% were classified as Type 2 amacrine cells, while 15% of the NT-cells had their cell bodies situated in the ganglion cell layer and were tentatively designated as displaced amacrine cells. Each type of NT-like immunoreactive cell was observed in the central and peripheral retina. NT-immunostained processes were observed to ramify in sublayers 3 and 5 of the inner plexiform layer. An examination of retinal whole mounts revealed that NT-amacrine cells were distributed throughout the center and periphery of the retina at a density of 82 +/- 24 cells/mm2. The dendritic fields of NT-immunostained amacrine and displayed amacrine cells were observed to be either symmetrically or asymmetrically distributed about their somas. Symmetrical dendritic fields were generally oval-shaped and ranged in diameter from 250 to 500 micron (major axis) by 150 to 250 micron (minor axis). Asymmetrical dendritic fields were observed to encompass one-half or less of an imaginary circle surrounding their soma of origin and were orientated in all directions. The processes forming asymmetrical dendritic fields ranged from 75 to 260 micron in length. Furthermore, partial overlap was often observed between the dendritic fields of adjacent NT-amacrine cells.

  6. An amacrine cell circuit for signaling steady illumination in the retina

    PubMed Central

    Jacoby, Jason; Zhu, Yongling; DeVries, Steven H.; Schwartz, Gregory

    2015-01-01

    Summary Decades of research have focused on the circuit connectivity between retinal neurons, yet only a handful of amacrine cells have been described functionally and placed in the context of a specific retinal circuit. Here we identify a circuit where inhibition from a specific amacrine cell plays a vital role in shaping the feature selectivity of a postsynaptic ganglion cell. We record from transgenically labeled CRH-1 amacrine cells and identify a postsynaptic target for CRH-1 amacrine cell inhibition in an atypical retinal ganglion cell (RGC) in mouse retina, the Suppressed-by-Contrast (SbC) RGC. Unlike other RGC types, SbC RGCs spike tonically in steady illumination and are suppressed by both increases and decreases in illumination. Inhibition from GABAergic CRH-1 amacrine cells shapes this unique contrast response profile to positive contrast. We show the existence and impact of this circuit with both paired recordings and cell-type specific ablation. PMID:26711334

  7. Narrow and wide field amacrine cells fire action potentials in response to depolarization and light stimulation.

    PubMed

    Heflin, Stephanie J; Cook, Paul B

    2007-01-01

    Action potentials in amacrine cells are important for lateral propagation of signals across the inner retina, but it is unclear how many subclasses of amacrine cells contain voltage-gated sodium channels or can fire action potentials. This study investigated the ability of amacrine cells with narrow ( <200 microm) and wide (>200 microm) dendritic fields to fire action potentials in response to depolarizing current injections and light stimulation. The pattern of action potentials evoked by current injections revealed two distinct classes of amacrine cells; those that responded with a single action potential (single-spiking cells) and those that responded with repetitive action potentials (repetitive-spiking cells). Repetitive-spiking cells differed from single-spiking cells in several regards: Repetitive-spiking cells were more often wide field cells, while single-spiking cells were more often narrow field cells. Repetitive-spiking cells had larger action potential amplitudes, larger peak voltage-gated NaV currents lower action potential thresholds, and needed less current to induce action potentials. However, there was no difference in the input resistance, holding current or time constant of these two classes of cells. The intrinsic capacity to fire action potentials was mirrored in responses to light stimulation; single-spiking amacrine cells infrequently fired action potentials to light steps, while repetitive-spiking amacrine cells frequently fired numerous action potentials. These results indicate that there are two physiologically distinct classes of amacrine cells based on the intrinsic capacity to fire action potentials.

  8. Dopaminergic modulation of tracer coupling in a ganglion-amacrine cell network

    PubMed Central

    MILLS, STEPHEN L.; XIA, XIAO-BO; HOSHI, HIDEO; FIRTH, SALLY I.; RICE, MARGARET E.; FRISHMAN, LAURA J.; MARSHAK, DAVID W.

    2008-01-01

    Many retinal ganglion cells are coupled via gap junctions with neighboring amacrine cells and ganglion cells. We investigated the extent and dynamics of coupling in one such network, the OFF α ganglion cell of rabbit retina and its associated amacrine cells. We also observed the relative spread of Neurobiotin injected into a ganglion cell in the presence of modulators of gap junctional permeability. We found that gap junctions between amacrine cells were closed via stimulation of a D1 dopamine receptor, while the gap junctions between ganglion cells were closed via stimulation of a D2 dopamine receptor. The pairs of hemichannels making up the heterologous gap junctions between the ganglion and amacrine cells were modulated independently, so that elevations of cAMP in the ganglion cell open the ganglion cell hemichannels, while elevations of cAMP in the amacrine cell close its hemichannels. We also measured endogenous dopamine release from an eyecup preparation and found a basal release from the dark-adapted retina of approximately 2 pmol/min during the day. Maximal stimulation with light increased the rate of dopamine release from rabbit retina by 66%. The results suggest that coupling between members of the OFF α ganglion cell/amacrine cell network is differentially modulated with changing levels of dopamine. PMID:17711603

  9. Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina

    PubMed Central

    Stanke, Jennifer J; Lehman, Bret; Fischer, Andy J

    2008-01-01

    Background Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT). Results We found that as ChAT-immunoreactive cells differentiate they expressed the homeodomain transcription factors Pax6 and Islet1, and the cell-cycle inhibitor p27kip1. As differentiation proceeds, type-II cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein (CRABP) and neurofilament, while type-I cells in the inner nuclear layer did not. Although there is a 1:1 ratio of type-I to type-II cells in vivo, in dissociated cell cultures the type-I cells (ChAT-positive and CRABP-negative) out-numbered the type-II cells (ChAT and CRABP-positive cells) by 2:1. The relative abundance of type-I to type-II cells was not influenced by Sonic Hedgehog (Shh), but was affected by compounds that act at muscarinic acetylcholine receptors. In addition, the abundance and mosaic patterning of type-II cholinergic amacrine cells is disrupted by interfering with muscarinic signaling. Conclusion We conclude that: (1) during development type-I and type-II cholinergic amacrine cells are not homotypic, (2) the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and (3) appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning. PMID:18254959

  10. Kainate-induced endocytosis in retinal amacrine cells.

    PubMed

    Borsello, Tiziana; Bressoud, Raymond; Mottier, Vincent; González, Nicolas; Gomez, Gabriel; Clarke, Peter G H

    2003-10-13

    Endocytosis is enhanced in some cases of neuronal death. We report for the first time that intraocular injections, in chick embryos, of excitotoxic doses of kainate induce strong endocytosis in retinal amacrine cells destined to die and that even subtoxic doses can induce some degree of endocytosis. That the uptake was due to endocytosis rather than passive diffusion through the plasma membrane was shown ultrastructurally. The endocytosis was demonstrated by using three unrelated tracers--horseradish peroxidase, microperoxidase, and 4.4-kDa fluorescein isothiocyanate (FITC)-labeled dextran--suggesting that it does not depend on the binding of the tracers to a particular receptor. However, it appears to be surprisingly sensitive to the size of the ligand, because a heavier (42-kDa) FITC-dextran was not endocytosed. The induction of endocytosis by kainate can occur even when protein synthesis is blocked. These results indicate that toxic or near-toxic doses of kainate induce endocytosis, raising the question of whether this is mechanistically implicated in causing or preventing excitotoxic neuronal death. Copyright 2003 Wiley-Liss, Inc.

  11. Lgr5⁺ amacrine cells possess regenerative potential in the retina of adult mice.

    PubMed

    Chen, Mengfei; Tian, Shenghe; Glasgow, Nathan G; Gibson, Gregory; Yang, Xiaoling; Shiber, Christen E; Funderburgh, James; Watkins, Simon; Johnson, Jon W; Schuman, Joel S; Liu, Hongjun

    2015-08-01

    Current knowledge indicates that the adult mammalian retina lacks regenerative capacity. Here, we show that the adult stem cell marker, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), is expressed in the retina of adult mice. Lgr5(+) cells are generated at late stages of retinal development and exhibit properties of differentiated amacrine interneurons (amacrine cells). Nevertheless, Lgr5(+) amacrine cells contribute to regeneration of new retinal cells in the adult stage. The generation of new retinal cells, including retinal neurons and Müller glia from Lgr5(+) amacrine cells, begins in early adulthood and continues as the animal ages. Together, these findings suggest that the mammalian retina is not devoid of regeneration as previously thought. It is rather dynamic, and Lgr5(+) amacrine cells function as an endogenous regenerative source. The identification of such cells in the mammalian retina may provide new insights into neuronal regeneration and point to therapeutic opportunities for age-related retinal degenerative diseases.

  12. Regenerative amacrine cell depolarization and formation of on-off ganglion cell response.

    PubMed Central

    Werblin, F S

    1977-01-01

    1. Recordings from amacrine and ganglion cells in the mudpuppy retina suggest mechanisms whereby the relatively slow, sustained light responses measured in bipolar cells are converted to rapid, brief, transient activity in the on-off ganglion cells. 2. Double-barrel electrodes were used to control the membrane potential under voltage clamp. The clamp revealed synaptic currents, but eliminated the otherwise obvious spike activity elicited by steps of illumination in both amacrine and ganglion cells, suggesting that the spikes are initiated near the somata. 3. The synaptic current in the on-off ganglion cells was biphasic: a brief inward (depolarizing) membrane current preceded a transient outward (hyperpolarizing) membrane current by about 20 msec. Each component could be isolated by polarizing the membrane to a level near the reversal potential for the other. Each was apparently due to a transient conductance increase of sawtooth shape with a 40 msec time to peak and a decay longer than 400 msec. 4. Synaptic membrane current in amacrine cells was monophasic and inward (depolarizing) of similar sawtooth shape at all potential levels. It was apparently mediated by a conductance increase to ions with a reversal potential more positive than the dark level. 5. When amacrine cells were depolarized in the dark under voltage clamp, a large transient inward membrane current with threshold within 4 mV of the dark level was generated. This regenerative event is capable of boosting a small, 4 mV e.p.s.p. to more than 30 mV in a few milliseconds, thereby generating the leading edge of a rapid sawtooth response. 6. The results suggest that the rapid transient on-off activity in ganglion cells is mediated by opposing sawtooth shaped synaptic currents with different latencies. It is inferred that each of these antagonistic imputs is generated by a regenerative depolarization in amacrine cells which then form synaptic inputs to the ganglion cells. PMID:845823

  13. Pten Regulates Retinal Amacrine Cell Number by Modulating Akt, Tgfβ, and Erk Signaling.

    PubMed

    Tachibana, Nobuhiko; Cantrup, Robert; Dixit, Rajiv; Touahri, Yacine; Kaushik, Gaurav; Zinyk, Dawn; Daftarian, Narsis; Biernaskie, Jeff; McFarlane, Sarah; Schuurmans, Carol

    2016-09-07

    All tissues are genetically programmed to acquire an optimal size that is defined by total cell number and individual cellular dimensions. The retina contains stereotyped proportions of one glial and six neuronal cell types that are generated in overlapping waves. How multipotent retinal progenitors know when to switch from making one cell type to the next so that appropriate numbers of each cell type are generated is poorly understood. Pten is a phosphatase that controls progenitor cell proliferation and differentiation in several lineages. Here, using a conditional loss-of-function strategy, we found that Pten regulates retinal cell division and is required to produce the full complement of rod photoreceptors and amacrine cells in mouse. We focused on amacrine cell number control, identifying three downstream Pten effector pathways. First, phosphoinositide 3-kinase/Akt signaling is hyperactivated in Pten conditional knock-out (cKO) retinas, and misexpression of constitutively active Akt (Akt-CA) in retinal explants phenocopies the reduction in amacrine cell production observed in Pten cKOs. Second, Akt-CA activates Tgfβ signaling in retinal explants, which is a negative feedback pathway for amacrine cell production. Accordingly, Tgfβ signaling is elevated in Pten cKO retinas, and epistatic analyses placed Pten downstream of TgfβRII in amacrine cell number control. Finally, Pten regulates Raf/Mek/Erk signaling levels to promote the differentiation of all amacrine cell subtypes, which are each reduced in number in Pten cKOs. Pten is thus a positive regulator of amacrine cell production, acting via multiple downstream pathways, highlighting its diverse actions as a mediator of cell number control. Despite the importance of size for optimal organ function, how individual cell types are generated in correct proportions is poorly understood. There are several ways to control cell number, including readouts of organ function (e.g., secreted hormones reach functional

  14. Sox2 regulates cholinergic amacrine cell positioning and dendritic stratification in the retina.

    PubMed

    Whitney, Irene E; Keeley, Patrick W; St John, Ace J; Kautzman, Amanda G; Kay, Jeremy N; Reese, Benjamin E

    2014-07-23

    The retina contains two populations of cholinergic amacrine cells, one positioned in the ganglion cell layer (GCL) and the other in the inner nuclear layer (INL), that together comprise ∼1/2 of a percent of all retinal neurons. The present study examined the genetic control of cholinergic amacrine cell number and distribution between these two layers. The total number of cholinergic amacrine cells was quantified in the C57BL/6J and A/J inbred mouse strains, and in 25 recombinant inbred strains derived from them, and variations in their number and ratio (GCL/INL) across these strains were mapped to genomic loci. The total cholinergic amacrine cell number was found to vary across the strains, from 27,000 to 40,000 cells, despite little variation within individual strains. The number of cells was always lower within the GCL relative to the INL, and the sizes of the two populations were strongly correlated, yet there was variation in their ratio between the strains. Approximately 1/3 of that variation in cell ratio was mapped to a locus on chromosome 3, where Sex determining region Y box 2 (Sox2) was identified as a candidate gene due to the presence of a 6-nucleotide insertion in the protein-coding sequence in C57BL/6J and because of robust and selective expression in cholinergic amacrine cells. Conditionally deleting Sox2 from the population of nascent cholinergic amacrine cells perturbed the normal ratio of cells situated in the GCL versus the INL and induced a bistratifying morphology, with dendrites distributed to both ON and OFF strata within the inner plexiform layer.

  15. Sox2 Regulates Cholinergic Amacrine Cell Positioning and Dendritic Stratification in the Retina

    PubMed Central

    Whitney, Irene E.; Keeley, Patrick W.; St. John, Ace J.; Kautzman, Amanda G.; Kay, Jeremy N.

    2014-01-01

    The retina contains two populations of cholinergic amacrine cells, one positioned in the ganglion cell layer (GCL) and the other in the inner nuclear layer (INL), that together comprise ∼1/2 of a percent of all retinal neurons. The present study examined the genetic control of cholinergic amacrine cell number and distribution between these two layers. The total number of cholinergic amacrine cells was quantified in the C57BL/6J and A/J inbred mouse strains, and in 25 recombinant inbred strains derived from them, and variations in their number and ratio (GCL/INL) across these strains were mapped to genomic loci. The total cholinergic amacrine cell number was found to vary across the strains, from 27,000 to 40,000 cells, despite little variation within individual strains. The number of cells was always lower within the GCL relative to the INL, and the sizes of the two populations were strongly correlated, yet there was variation in their ratio between the strains. Approximately 1/3 of that variation in cell ratio was mapped to a locus on chromosome 3, where Sex determining region Y box 2 (Sox2) was identified as a candidate gene due to the presence of a 6-nucleotide insertion in the protein-coding sequence in C57BL/6J and because of robust and selective expression in cholinergic amacrine cells. Conditionally deleting Sox2 from the population of nascent cholinergic amacrine cells perturbed the normal ratio of cells situated in the GCL versus the INL and induced a bistratifying morphology, with dendrites distributed to both ON and OFF strata within the inner plexiform layer. PMID:25057212

  16. Localization of enkephalin-like immunoreactive amacrine cells in the larval tiger salamander retina: a light and electron microscopic study.

    PubMed

    Watt, C B; Li, H B; Fry, K R; Lam, D M

    1985-11-08

    Immunohistochemistry was utilized to examine the light and electron microscopic localization of enkephalin-like (enk) immunoreactive amacrine cells in the larval tiger salamander retina. The vast majority of enk-immunoreactive cells were typical amacrine cells whose round or oval cell bodies (14-16 microns) were situated in the innermost cell row of the inner nuclear layer. A relatively small number of enk-stained oval cell bodies (14-22 microns) were located in the ganglion cell layer and were designated as those of displaced amacrine cells. Enkephalin immunostaining was observed in the inner plexiform layer as a fine plexus in sublamina 1 and as a dense network of fibers in sublamina 5. In both the center and periphery of the retina the density of typical enk-amacrine cells was determined to be 250 +/- 16.36 cells per mm2 surface area of the retina. At the ultrastructural level typical enk-stained amacrine cells possessed a round, indented nuclear membrane. Enk-immunoreactive processes sometimes contained dense-core vesicles (60-115 nm) in addition to a rather homogeneous population of small, round, agranular synaptic vesicles (25-35 nm). In sublamina 1 the processes of enk-amacrine cells were presynaptic to amacrine and bipolar cells. They also contacted processes devoid of synaptic vesicles which possibly arise from ganglion cells. As the postsynaptic element in sublamina 1, they received synaptic input from amacrine cells. In sublamina 5 the processes of enk-amacrine cells were presynaptic to amacrine cells, bipolar cells, and the somas of cells situated in the ganglion cell layer.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Two types of glutamate receptors differentially excite amacrine cells in the tiger salamander retina.

    PubMed Central

    Dixon, D B; Copenhagen, D R

    1992-01-01

    1. Excitatory inputs to amacrine cells in the salamander retinal slice preparation were examined using whole-cell patch pipette voltage-clamp techniques. In strychnine (500 nM) and bicuculline (100 microM), two types of amacrine cell were easily distinguished by their light-evoked excitatory responses: transient and sustained. 2. In transient amacrine cells the current-voltage (I-V) relation for the peak light-evoked current was non-linear with a negative slope region between -50 and -70 mV. Responses reversed near +10 mV and were prolonged at more positive holding potentials. 3. In DL-2-amino-phosphonoheptanoate (AP7, 30 microM), a selective N-methyl-D-aspartate (NMDA) receptor antagonist, both the negatively sloped region of the light I-V relation and the prolongation of the response at positive potentials were eliminated. In 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 2 microM), a selective non-NMDA receptor antagonist, light-evoked currents at the most hyperpolarized holding potentials were eliminated. At potentials positive to -85 mV the light-evoked currents lacked a fast onset. The light I-V relation in CNQX had a negative slope region between -35 and -80 mV. 4. With synaptic transmission blocked, kainate evoked responses in transient cells with a resultant I-V relation that was nearly linear, whereas glutamate and NMDA elicited responses with non-linear I-V relations. 5. Light-evoked currents in sustained amacrine cells had a nearly linear I-V relation and reversed near +10 mV. AP7 at a concentration of 30 microM did not affect the light-evoked currents in sustained cells, but 2 microM-CNQX eliminated all light-evoked currents in these cells. 6. With synaptic transmission blocked, sustained amacrine cells responded only to glutamate and kainate, not NMDA. The resultant I-V relations were linear. 7. We conclude that the light-evoked responses of transient amacrine cells are mediated by concomitant activation of both non-NMDA and NMDA receptors whereas the

  18. All spiking, sustained ON displaced amacrine cells receive gap-junction input from melanopsin ganglion cells

    PubMed Central

    Reifler, Aaron N.; Chervenak, Andrew P.; Dolikian, Michael E.; Benenati, Brian A.; Li, Benjamin Y.; Wachter, Rebecca D.; Lynch, Andrew M.; Demertzis, Zachary D.; Meyers, Benjamin S.; Abufarha, Fady S.; Jaeckel, Elizabeth R.; Flannery, Michael P.; Wong, Kwoon Y.

    2015-01-01

    SUMMARY Retinal neurons exhibit sustained vs. transient light responses, which are thought to encode low- and high-frequency stimuli respectively. This dichotomy has been recognized since the earliest intracellular recordings from the 1960s, but the underlying mechanisms are not yet fully understood. We report that in the ganglion cell layer of rat retinas, all spiking amacrine interneurons with sustained ON photoresponses receive gap-junction input from intrinsically photosensitive retinal ganglion cells (ipRGCs), recently discovered photoreceptors that specialize in prolonged irradiance detection. We have identified three morphological varieties of such ipRGC-driven displaced amacrine cells: 1) monostratified cells with dendrites terminating exclusively in sublamina S5 of the inner plexiform layer; 2) bistratified cells with dendrites in both S1 and S5; and 3) polyaxonal cells with dendrites and axons stratifying in S5. Most of these amacrine cells are wide-field, although some are medium-field. The three classes respond to light differently, suggesting they probably perform diverse functions. These results demonstrate that ipRGCs are a major source of tonic visual information within the retina and exert widespread intraretinal influence. They also add to recent evidence that ganglion cells signal not only to the brain. PMID:26441349

  19. Brain-derived neurotrophic factor regulates expression of vasoactive intestinal polypeptide in retinal amacrine cells.

    PubMed

    Cellerino, Alessandro; Arango-González, Blanca; Pinzón-Duarte, Germán; Kohler, Konrad

    2003-12-01

    Brain-derived neurotrophic-factor (BDNF) is expressed in the retina and controls the development of subtypes of amacrine cells. In the present study we investigated the effects of BDNF on amacrine cells expressing vasoactive intestinal polypeptide (VIP). Rats received three intraocular injections of BDNF on postnatal days (P) 16, 18, and 20. The animals were sacrificed on P22, P40, P60, P80, and P120, and VIP expression in their retinas was detected by immunohistochemistry (P22, P40) and by radioimmunoassay (RIA; P22, P40, P60, P80, P120) to assess the time course of BDNF effects on VIP. A significant increase in the density of VIP-positive amacrine cells was detected in BDNF-treated retinas, and VIP concentration was up-regulated by 150% both at P22 and at P40 with respect to untreated controls. VIP concentration then slowly declined in the treated retinas over a period of 3 months; however, a statistically significant increase of 50% was still detectable on P120. The impact of endogenous BDNF on the regulation of VIP expression in the retina was analyzed in mice homozygous for a targeted deletion of the BDNF gene locus (bdnf-/-). VIP immunohistochemistry revealed a marked reduction of VIP-positive amacrine cells and of VIP-immunopositive processes in the inner plexiform layer of the BDNF knockout mice. Mice lacking BDNF expressed only 5% of the VIP protein in their retinas compared with the retinas of wild-type mice as measured by RIA. Our data show that BDNF is a major regulator of VIP expression in retinal amacrine cells and exerts long-lasting effects on VIP content.

  20. Immunocytochemical analysis of cholinergic amacrine cells in the tiger salamander retina.

    PubMed

    Zhang, J; Wu, S M

    2001-05-25

    Cholinergic amacrine cells in the tiger salamander retina were observed for the first time by using antibodies against choline acetyltransferase (ChAT). ChAT-immunoreactive cells were present in the inner nuclear layer (INL) and in the ganglion cell layer (GCL), and the somas of the former population (average diameter = 15.13 microm) were slightly smaller than those of the latter population (average diameter = 16.42 microm). The processes of these cells form two distinct narrow bands in the inner plexiform layer (IPL), one located near 0.2 inner plexiform units (IU) and the other near 0.65-0.7 IU. Soma size, cell density and spatial distribution of ChAT-positive cells were quantitatively analyzed. Our results suggest that cholinergic amacrine cells in the salamander retina are very similar to their counter parts in other species, and they can be used as a model system for studying cholinergic functions in the visual system.

  1. An unconventional glutamatergic circuit in the retina formed by vGluT3 amacrine cells.

    PubMed

    Lee, Seunghoon; Chen, Lujing; Chen, Minggang; Ye, Meijun; Seal, Rebecca P; Zhou, Z Jimmy

    2014-11-19

    In the vertebrate retina, glutamate is traditionally thought to be released only by photoreceptors and bipolar cells to transmit visual signals radially along parallel ON and OFF channels. Lateral interactions in the inner retina are mediated by amacrine cells, which are thought to be inhibitory neurons. Here, we report calcium-dependent glutamate release from vGluT3-expressing amacrine cells (GACs) in the mouse retina. GACs provide an excitatory glutamatergic input to ON-OFF and ON direction-selective ganglion cells (DSGCs) and a subpopulation of W3 ganglion cells, but not to starburst amacrine cells. GACs receive excitatory inputs from both ON and OFF channels, generate ON-OFF light responses with a medium-center, wide-surround receptive field structure, and directly regulate ganglion cell activity. The results reveal a functional glutamatergic circuit that mediates noncanonical excitatory interactions in the retina and probably plays a role in generating ON-OFF responses, crossover excitation, and lateral excitation. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Sphingosine-1-Phosphate Elicits Receptor-Dependent Calcium Signaling in Retinal Amacrine Cells

    PubMed Central

    Crousillac, Scott; Colonna, Jeremy; McMains, Emily; Dewey, Jill Sayes

    2009-01-01

    Evidence is emerging indicating that sphingosine-1-phosphate (S1P) participates in signaling in the retina. To determine whether S1P might be involved in signaling in the inner retina specifically, we examine the effects of this sphingolipid on cultured retinal amacrine cells. Whole cell voltage-clamp recordings reveal that S1P activates a cation current that is dependent on signaling through Gi and phospholipase C. These observations are consistent with the involvement of members of the S1P receptor family of G-protein-coupled receptors in the production of the current. Immunocytochemistry and PCR amplification provide evidence for the expression of S1P1R and S1P3R in amacrine cells. The receptor-mediated channel activity is shown to be highly sensitive to blockade by lanthanides consistent with the behavior of transient receptor potential canonical (TRPC) channels. PCR products amplified from amacrine cells reveal that TRPCs 1 and 3–7 channel subunits have the potential to be expressed. Because TRPC channels provide a Ca2+ entry pathway, we asked whether S1P caused cytosolic Ca2+ elevations in amacrine cells. We show that S1P-dependent Ca2+ elevations do occur in these cells and that they might be mediated by S1P1R and S1P3R. The Ca2+ elevations are partially due to release from internal stores, but the largest contribution is from influx across the plasma membrane. The effect of inhibition of sphingosine kinase suggests that the production of cytosolic S1P underlies the sustained nature of the Ca2+ elevations. Elucidation of the downstream effects of these signals will provide clues to the role of S1P in regulating inner retinal function. PMID:19776367

  3. Cone signals in monostratified and bistratified amacrine cells of adult zebrafish retina.

    PubMed

    Torvund, M M; Ma, T S; Connaughton, V P; Ono, F; Nelson, R F

    2017-05-01

    Strata within the inner plexiform layer (IPL) of vertebrate retinas are suspected to be distinct signaling regions. Functions performed within adult zebrafish IPL strata were examined through microelectrode recording and staining of stratified amacrine types. The stimulus protocol and analysis discriminated the pattern of input from red, green, blue, and UV cones as well as the light-response waveforms in this tetrachromatic species. A total of 36 cells were analyzed. Transient depolarizing waveforms at ON and OFF originated with bistratified amacrine types, whose dendritic planes branched either in IPL sublaminas a & b, or only within sublamina a. Monophasic-sustained depolarizing waveforms originated with types monostratified in IPL s4 (sublamina b). OFF responses hyperpolarized at onset, depolarized at offset, and in some cases depolarized during mid-stimulus. These signals originated with types monostratified in s1 or s2 (sublamina a). Bistratified amacrines received depolarizing signals only from red cones, at both ON and OFF, while s4 stratified ON cells combined red and green cone signals. The s1/s2 stratified OFF cells utilized hyperpolarizing signals from red, red and green, or red and blue cones at ON, but only depolarizing red cone signals at OFF. ON and OFF depolarizing transients from red cones appear widely distributed within IPL strata. "C-type" physiologies, depolarized by some wavelengths, hyperpolarized by others, in biphasic or triphasic spectral patterns, originated with amacrine cells monostratified in s5. Collectively, cells in this stratum processed signals from all cone types. J. Comp. Neurol. 525:1532-1557, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. The area centralis in the chicken retina contains efferent target amacrine cells

    PubMed Central

    Weller, Cynthia; Lindstrom, Sarah H.; De Grip, Willem J.; Wilson, Martin

    2012-01-01

    The retinas of birds receive a substantial efferent, or centrifugal, input from a midbrain nucleus. The function of this input is presently unclear but previous work in the pigeon has shown that efferent input is excluded from the area centralis, suggesting that the functions of the area centralis and the efferent system are incompatible. Using an antibody specific to rods, we have identified the area centralis in another species, the chicken, and mapped the distribution of the unique amacrine cells that are the postsynaptic partners of efferent fibers. Efferent target amacrine cells are found within the chicken area centralis and their density is continuous across the border of the area centralis. In contrast to the pigeon retina then, we conclude that the chicken area centralis receives efferent input. We suggest that the difference between the 2 species is attributable to the presence of a fovea within the area centralis of the pigeon and its absence from that of the chicken. PMID:19296862

  5. Wide-field Diffuse Amacrine Cells in the Monkey Retina Contain Immunoreactive Cocaine- and Amphetamine-Regulated Transcript (CART)

    PubMed Central

    Liu, Weiley S.; Davis, Elizabeth P.; Lee, Stephen J.; Tseng, Luke; Chuang, Alice Z.; Whitaker, Christopher M.; Massey, Stephen C.; Sherman, Michael B.; Marshak, David W.

    2016-01-01

    The goals of this study were to localize the neuropeptide Cocaine- and Amphetamine-Regulated Transcript (CART) in primate retinas and to describe the morphology, neurotransmitter content and synaptic connections of the neurons that contain it. Using in situ hybridization, light and electron microscopic immunolabeling, CART was localized to GABAergic amacrine cells in baboon retinas. The CART-positive cells had thin, varicose dendrites that gradually descended through the inner plexiform layer and ramified extensively in the innermost stratum. They resembled two types of wide-field diffuse amacrine cells that had been described previously in macaque retinas using the Golgi method and also A17, serotonin-accumulating and waterfall cells of other mammals. The CART-positive cells received synapses from rod bipolar cell axons and made synapses onto the axons in a reciprocal configuration. The CART-positive cells also received synapses from other amacrine cells. Some of these were located on their primary dendrites, and the presynaptic cells there included dopaminergic amacrine cells. Although some CART-positive somas were localized in the ganglion cell layer, they did not contain the ganglion cell marker RNA binding protein with multiple splicing (RBPMS). Based on these results and electrophysiological studies in other mammals, the CART-positive amacrine cells would be expected to play a major role in the primary rod pathway of primates, providing feedback inhibition to rod bipolar cells. PMID:27568514

  6. Wide-field diffuse amacrine cells in the monkey retina contain immunoreactive Cocaine- and Amphetamine-Regulated Transcript (CART).

    PubMed

    Long, Ye; Bordt, Andrea S; Liu, Weiley S; Davis, Elizabeth P; Lee, Stephen J; Tseng, Luke; Chuang, Alice Z; Whitaker, Christopher M; Massey, Stephen C; Sherman, Michael B; Marshak, David W

    2016-10-01

    The goals of this study were to localize the neuropeptide Cocaine- and Amphetamine-Regulated Transcript (CART) in primate retinas and to describe the morphology, neurotransmitter content and synaptic connections of the neurons that contain it. Using in situ hybridization, light and electron microscopic immunolabeling, CART was localized to GABAergic amacrine cells in baboon retinas. The CART-positive cells had thin, varicose dendrites that gradually descended through the inner plexiform layer and ramified extensively in the innermost stratum. They resembled two types of wide-field diffuse amacrine cells that had been described previously in macaque retinas using the Golgi method and also A17, serotonin-accumulating and waterfall cells of other mammals. The CART-positive cells received synapses from rod bipolar cell axons and made synapses onto the axons in a reciprocal configuration. The CART-positive cells also received synapses from other amacrine cells. Some of these were located on their primary dendrites, and the presynaptic cells there included dopaminergic amacrine cells. Although some CART-positive somas were localized in the ganglion cell layer, they did not contain the ganglion cell marker RNA binding protein with multiple splicing (RBPMS). Based on these results and electrophysiological studies in other mammals, the CART-positive amacrine cells would be expected to play a major role in the primary rod pathway of primates, providing feedback inhibition to rod bipolar cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Responses and Receptive Fields of Amacrine Cells and Ganglion Cells in the Salamander Retina

    PubMed Central

    Zhang, Ai-Jun; Wu, Samuel M.

    2013-01-01

    Retinal amacrine cells (ACs) and ganglion cells (GCs) have been shown to display large morphological diversity, and here we show that four types of ACs and three types of GCs exhibit physiologically-distinguishable properties. They are the sustained ON ACs; sustained OFF ACs; transient ON-OFF ACs; transient ON-OFF ACs with wide receptive fields; sustained ON-center/OFF-surround GCs; sustained OFF-center/ON-surround GCs and transient ON-OFF GCs. By comparing response waveforms, receptive fields and relative rod/cone inputs of ACs and GCs with the corresponding parameters of various types of the presynaptic bipolar cells (BCs), we analyze how different types of BCs mediate synaptic inputs to various ACs and GCs. Although more types of third-order retinal neurons may be identified by more refined classification criteria, our observations suggest that many morphologically-distinct ACs and GCs share very similar physiological responses. PMID:20085780

  8. Ionic mechanisms mediating oscillatory membrane potentials in wide-field retinal amacrine cells.

    PubMed

    Vigh, Jozsef; Solessio, Eduardo; Morgans, Catherine W; Lasater, Eric M

    2003-07-01

    Particular types of amacrine cells of the vertebrate retina show oscillatory membrane potentials (OMPs) in response to light stimulation. Historically it has been thought the oscillations arose as a result of circuit properties. In a previous study we found that in some amacrine cells, the ability to oscillate was an intrinsic property of the cell. Here we characterized the ionic mechanisms responsible for the oscillations in wide-field amacrine cells (WFACs) in an effort to better understand the functional properties of the cell. The OMPs were found to be calcium (Ca2+) dependent; blocking voltage-gated Ca2+ channels eliminated the oscillations, whereas elevating extracellular Ca2+ enhanced them. Strong intracellular Ca2+ buffering (10 mM EGTA or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid) eliminated any attenuation in the OMPs as well as a Ca2+-dependent inactivation of the voltage-gated Ca2+ channels. Pharmacological and immunohistochemical characterization revealed that WFACs express L- and N-type voltage-sensitive Ca2+ channels. Block of the L-type channels eliminated the OMPs, but omega-conotoxin GVIA did not, suggesting a different function for the N-type channels. The L-type channels in WFACs are functionally coupled to a set of calcium-dependent potassium (K(Ca)) channels to mediate OMPs. The initiation of OMPs depended on penitrem-A-sensitive (BK) K(Ca) channels, whereas their duration is under apamin-sensitive (SK) K(Ca) channel control. The Ca2+ current is essential to evoke the OMPs and triggering the K(Ca) currents, which here act as resonant currents, enhances the resonance as an amplifying current, influences the filtering characteristics of the cell membrane, and attenuates the OMPs via CDI of the L-type Ca2+ channel.

  9. Double-staining of horizontal and amacrine cells by intracellular injection with lucifer yellow and biocytin in carp retina.

    PubMed

    Teranishi, T; Negishi, K

    1994-03-01

    Horizontal and amacrine cells in the isolated carp retina were impaled with micropipette electrode, identified by their characteristic light responses, and injected iontophoretically with markers for morphological study. Both Lucifer Yellow CH and biocytin were injected simultaneously. Lucifer Yellow was seen by its own fluorescence while biocytin was visualized by binding with Texas Red-linked or horseradish peroxidase-conjugated avidin. For cone-connected horizontal cells, biocytin-coupled cells were found to be approximately five-times more numerous than Lucifer Yellow-coupled cells. Coupling for both tracers was consistently hampered by intravitreally applied dopamine. In untreated retinas, the injected Lucifer Yellow was restricted within one rod-connected horizontal cell, while biocytin revealed several coupled neighbors. Amacrine cells, labeled by the tracers, were morphologically grouped into eight types, based on our earlier classification. Among them, amacrine cells, belonging to three types (Fnd, Pmb or Pma), were confirmed to be Lucifer Yellow-coupled, and the number of biocytin-coupled cells was more numerous (about 2.5 times) than that of Lucifer Yellow-coupled cells. Most amacrine cells (i.e. Pwd, Fnb and Fna) showed biocytin-coupling with no Lucifer Yellow-coupling. A few classified (i.e. Pwb and Fwa) and unclassified cells did not show any coupling. Since the tracer coupling takes place via gap junctions, the majority of amacrine cells, belonging to certain homologous types, appear to be functionally coupled with each other in the inner plexiform layer. However, dopamine did not influence the range of tracer coupling between amacrine cells in the carp retina under the present experimental conditions.

  10. Cholecystokinin-like immunoreactive amacrine cells in the rat retina

    PubMed Central

    Firth, Sally I.; Varela, Carolina; De La Villa, Pedro; Marshak, David W.

    2012-01-01

    High levels of endogenous cholecystokinin (CCK) are present in the rat retina (Eskay & Beinfeld, 1982), but the cellular localization and physiological actions of CCK in the rat retina are uncertain. The goals of this study were to characterize the cells containing CCK, identify cell types that interact with CCK cells, and investigate the effects of CCK on rod bipolar cells. Rat retinas were labeled with antibody to gastrin-CCK (gCCK) using standard immunofluorescence techniques. Patch-clamp methods were used to record from dissociated rod bipolar cells from rats and mice. Gastrin-CCK immunoreactive (-IR) axons were evenly distributed throughout the retina in stratum 5 of the inner plexiform layer of the rat retina. However, the gCCK-IR somata were only detected in the ganglion cell layer in the peripheral retina. The gCCK-IR cells contained glutamate decarboxylase, and some of them also contained immunoreactive substance P. Labeled axons contacted PKC-IR rod bipolar cells, and recoverin-IR ON-cone bipolar cells. CCK-octapeptide inhibits GABAC but not GABAA mediated currents in dissociated rod bipolar cells. PMID:12511085

  11. The role of apoproteins AI and AII in binding of high-density lipoprotein3 to membranes derived from bovine aortic endothelial cells.

    PubMed Central

    Vadiveloo, P K; Fidge, N H

    1992-01-01

    Although binding of high-density lipoproteins (HDL) to a variety of cells in culture has been widely reported, the mechanism of this binding has yet to be fully elucidated. The aim of the current studies was to explore the roles of apoproteins (apo) AI and AII in HDL3 binding to membranes derived from bovine aortic endothelial cells. Binding studies showed that HDL3 (which contains both apo AI and apo AII) and AII-HDL3 (which contain only apo AII) bound to membranes with similar affinity (44 +/- 6 and 41 +/- 9 micrograms/ml respectively) and capacity (673 +/- 97 and 969 +/- 101 ng bound/mg of membrane protein respectively). In contrast with these results, HDL3 [AI w/o AII] (which contain apo AI, but not apo AII) bound to the membranes with a significantly higher capacity (2228 +/- 206 ng bound/mg of membrane protein) and lower affinity (65 +/- 3 micrograms/ml) as compared with HDL3 or AII-HDL3. Therefore, although both apo AI and apo AII appear capable of facilitating HDL3 binding, the mechanisms involved probably differ. A model which fits the data postulates that a common receptor exists which binds both apo AI and apo AII, and that a particle containing AII can occupy up to four receptors (partly owing to each AII molecule containing two binding domains), whereas an HDL3 [AI w/o AII] particle can occupy only one. Images Fig. 3. PMID:1599393

  12. Nitric oxide releases Cl− from acidic organelles in retinal amacrine cells

    PubMed Central

    Krishnan, Vijai; Gleason, Evanna

    2015-01-01

    Determining the factors regulating cytosolic Cl− in neurons is fundamental to our understanding of the function of GABA- and glycinergic synapses. This is because the Cl− distribution across the postsynaptic plasma membrane determines the sign and strength of postsynaptic voltage responses. We have previously demonstrated that nitric oxide (NO) releases Cl− into the cytosol from an internal compartment in both retinal amacrine cells and hippocampal neurons. Furthermore, we have shown that the increase in cytosolic Cl− is dependent upon a decrease in cytosolic pH. Here, our goals were to confirm the compartmental nature of the internal Cl− store and to test the hypothesis that Cl− is being released from acidic organelles (AO) such as the Golgi, endosomes or lysosomes. To achieve this, we made whole cell voltage clamp recordings from cultured chick retinal amacrine cells and used GABA-gated currents to track changes in cytosolic Cl−. Our results demonstrate that intact internal proton gradients are required for the NO-dependent release of internal Cl−. Furthermore, we demonstrate that increasing the pH of AO leads to release of Cl− into the cytosol. Intriguingly, the elevation of organellar pH results in a reversal in the effects of NO. These results demonstrate that cytosolic Cl− is closely linked to the regulation and maintenance of organellar pH and provide evidence that acidic compartments are the target of NO. PMID:26106295

  13. Ganglion Cell and Displaced Amacrine Cell Density Distribution in the Retina of the Howler Monkey (Alouatta caraya)

    PubMed Central

    Muniz, José Augusto Pereira Carneiro; de Athaide, Luana Modesto; Gomes, Bruno Duarte; Finlay, Barbara L.; Silveira, Luiz Carlos de Lima

    2014-01-01

    Unlike all other New World (platyrrine) monkeys, both male and female howler monkeys (Alouatta sp.) are obligatory trichromats. In all other platyrrines, only females can be trichromats, while males are always dichromats, as determined by multiple behavioral, electrophysiological, and genetic studies. In addition to obligatory trichromacy, Alouatta has an unusual fovea, with substantially higher peak cone density in the foveal pit than every other diurnal anthropoid monkey (both platyrrhines and catarrhines) and great ape yet examined, including humans. In addition to documenting the general organization of the retinal ganglion cell layer in Alouatta, the distribution of cones is compared to retinal ganglion cells, to explore possible relationships between their atypical trichromacy and foveal specialization. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from five Alouatta caraya. Ganglion cell density peaked at 0.5 mm between the fovea and optic nerve head, reaching 40,700–45,200 cells/mm2. Displaced amacrine cell density distribution peaked between 0.5–1.75 mm from the fovea, reaching mean values between 2,050–3,100 cells/mm2. The mean number of ganglion cells was 1,133,000±79,000 cells and the mean number of displaced amacrine cells was 537,000±61,800 cells, in retinas of mean area 641±62 mm2. Ganglion cell and displaced amacrine cell density distribution in the Alouatta retina was consistent with that observed among several species of diurnal Anthropoidea, both platyrrhines and catarrhines. The principal alteration in the Alouatta retina appears not to be in the number of any retinal cell class, but rather a marked gradient in cone density within the fovea, which could potentially support high chromatic acuity in a restricted central region. PMID:25546077

  14. Ganglion cell and displaced amacrine cell density distribution in the retina of the howler monkey (Alouatta caraya).

    PubMed

    Muniz, José Augusto Pereira Carneiro; de Athaide, Luana Modesto; Gomes, Bruno Duarte; Finlay, Barbara L; Silveira, Luiz Carlos de Lima

    2014-01-01

    Unlike all other New World (platyrrine) monkeys, both male and female howler monkeys (Alouatta sp.) are obligatory trichromats. In all other platyrrines, only females can be trichromats, while males are always dichromats, as determined by multiple behavioral, electrophysiological, and genetic studies. In addition to obligatory trichromacy, Alouatta has an unusual fovea, with substantially higher peak cone density in the foveal pit than every other diurnal anthropoid monkey (both platyrrhines and catarrhines) and great ape yet examined, including humans. In addition to documenting the general organization of the retinal ganglion cell layer in Alouatta, the distribution of cones is compared to retinal ganglion cells, to explore possible relationships between their atypical trichromacy and foveal specialization. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from five Alouatta caraya. Ganglion cell density peaked at 0.5 mm between the fovea and optic nerve head, reaching 40,700-45,200 cells/mm2. Displaced amacrine cell density distribution peaked between 0.5-1.75 mm from the fovea, reaching mean values between 2,050-3,100 cells/mm2. The mean number of ganglion cells was 1,133,000±79,000 cells and the mean number of displaced amacrine cells was 537,000±61,800 cells, in retinas of mean area 641±62 mm2. Ganglion cell and displaced amacrine cell density distribution in the Alouatta retina was consistent with that observed among several species of diurnal Anthropoidea, both platyrrhines and catarrhines. The principal alteration in the Alouatta retina appears not to be in the number of any retinal cell class, but rather a marked gradient in cone density within the fovea, which could potentially support high chromatic acuity in a restricted central region.

  15. Glycinergic input of widefield, displaced amacrine cells of the mouse retina

    PubMed Central

    Majumdar, Sriparna; Weiss, Jan; Wässle, Heinz

    2009-01-01

    Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyRα1 (Glra1spd-ot, ‘oscillator’ mouse), the GlyRα2 (Glra2−/−) and the GlyRα3 subunit (Glra3−/−). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, τ∼25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1spd-ot and Glra3−/− mice (τ∼25 ms); however, glycinergic currents were absent in group II cells of Glra2−/− mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (τ∼50–70 ms). We propose that GlyRs of group II cells are dominated by the α2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the α4 subunit. PMID:19528249

  16. Light-evoked synaptic activity of retinal ganglion and amacrine cells is regulated in developing mouse retina

    PubMed Central

    He, Quanhua; Wang, Ping; Tian, Ning

    2010-01-01

    Recent studies have shown a continued maturation of visual responsiveness and synaptic activity of retina after eye opening, including the size of receptive fields of retinal ganglion cells (RGCs), light-evoked synaptic output of RGCs, bipolar cell spontaneous synaptic inputs to RGCs, and the synaptic connections between RGCs and ON and OFF bipolar cells. Light deprivation retarded some of these age-dependent changes. However, many other functional and morphological features of RGCs are not sensitive to visual experience. To determine whether light-evoked synaptic responses of RGCs undergo developmental change, we directly examined the light-evoked synaptic inputs from ON and OFF synaptic pathways to RGCs in developing retinas and found that both light-evoked excitatory and inhibitory synaptic currents decreased, but not increased, with age. We also examined the light-evoked synaptic inputs from ON and OFF synaptic pathways to amacrine cells in developing retinas and found that the light-evoked synaptic input of amacrine cells is also down-regulated in developing mouse retina. Different from the developmental changes of RGC spontaneous synaptic activity, dark rearing has little effect on the developmental changes of light-evoked synaptic activity of both RGCs and amacrine cells. Therefore, we concluded that the synaptic mechanisms mediating spontaneous and light-evoked synaptic activity of RGCs and amacrine cells are likely to be different. PMID:21091802

  17. Vesicular γ-Aminobutyric Acid Transporter Expression in Amacrine and Horizontal Cells

    PubMed Central

    Cueva, Juan G.; Haverkamp, Silke; Reimer, Richard J.; Edwards, Robert; Wässle, Heinz; Brecha, Nicholas C.

    2010-01-01

    The vesicular γ-aminobutyric acid (GABA) transporter (VGAT), which transports the inhibitory amino acid transmitters GABA and glycine, is localized to synaptic vesicles in axon terminals. The localization of VGAT immunoreactivity to mouse and rat retina was evaluated with light and electron microscopy by using well-characterized VGAT antibodies. Specific VGAT immunoreactivity was localized to numerous varicose processes in all laminae of the inner plexiform layer (IPL) and to the outer plexiform layer (OPL). Amacrine cell somata characterized by weak VGAT immunoreactivity in the cytoplasm were located in the ganglion cell layer and proximal inner nuclear layer (INL) adjacent to the IPL. In rat retina, VGAT-immunoreactive cell bodies also contained GABA, glycine, or parvalbumin (PV) immunoreactivity, suggesting vesicular uptake of GABA or glycine by these cells. A few varicose VGAT-immunoreactive processes entered the OPL from the IPL. VGAT immunoreactivity in the OPL was predominantly localized to horizontal cell processes. VGAT and calcium binding protein-28K immunoreactivities (CaBP; a marker for horizontal cells) were colocalized in processes and terminals distributed to the OPL. Furthermore, VGAT immunoreactivity overlapped or was immediately adjacent to postsynaptic density-95 (PSD-95) immunoreactivity, which is prominent in photoreceptor terminals. Preem-bedding immunoelectron microscopy of mouse and rat retinae showed that VGAT immunoreactivity was localized to horizontal cell processes and their terminals. Immunoreactivity was distributed throughout the cytoplasm of the horizontal cell processes. Taken together, these findings demonstrate VGAT immunoreactivity in both amacrine and horizontal cell processes, suggesting these cells contain vesicles that accumulate GABA and glycine, possibly for vesicular release. PMID:11920703

  18. Starburst amacrine cells express parvalbumin but not calbindin and calretinin in rabbit retina.

    PubMed

    Lee, Eun-Shil; Jeon, Chang-Jin

    2013-11-13

    Calcium-binding proteins (CBPs) are important components in calcium-mediated cellular signal transduction. Among the many CBPs, at least three EF-hand CBPs, calbindin-D28K (CB), calretinin (CR), and parvalbumin (PV), have been extensively studied in the retina. In the present study, we investigated the expression patterns of these three CBPs in cholinergic starburst amacrine cells (SACs), which are the most important element for direction selectivity in the rabbit retina. Double-label immunocytochemical analysis of vibratome sections and single-cell injection after immunocytochemical analysis on whole mounts were carried out in rabbit retinas. We found that all SACs in the inner nuclear layer and the ganglion cell layer contained PV. However, none of the SACs in the inner nuclear layer or ganglion cell layer contained either CB or CR. These results suggest that PV, but not CR or CB, may act as a calcium-buffering protein in the SACs of the rabbit retina.

  19. Parallel Inhibition of Dopamine Amacrine Cells and Intrinsically Photosensitive Retinal Ganglion Cells in a Non-Image-Forming Visual Circuit of the Mouse Retina.

    PubMed

    Vuong, Helen E; Hardi, Claudia N; Barnes, Steven; Brecha, Nicholas C

    2015-12-02

    An inner retinal microcircuit composed of dopamine (DA)-containing amacrine cells and melanopsin-containing, intrinsically photosensitive retinal ganglion cells (M1 ipRGCs) process information about the duration and intensity of light exposures, mediating light adaptation, circadian entrainment, pupillary reflexes, and other aspects of non-image-forming vision. The neural interaction is reciprocal: M1 ipRGCs excite DA amacrine cells, and these, in turn, feed inhibition back onto M1 ipRGCs. We found that the neuropeptide somatostatin [somatotropin release inhibiting factor (SRIF)] also inhibits the intrinsic light response of M1 ipRGCs and postulated that, to tune the bidirectional interaction of M1 ipRGCs and DA amacrine cells, SRIF amacrine cells would provide inhibitory modulation to both cell types. SRIF amacrine cells, DA amacrine cells, and M1 ipRGCs form numerous contacts. DA amacrine cells and M1 ipRGCs express the SRIF receptor subtypes sst(2A) and sst4 respectively. SRIF modulation of the microcircuit was investigated with targeted patch-clamp recordings of DA amacrine cells in TH-RFP mice and M1 ipRGCs in OPN4-EGFP mice. SRIF increases K(+) currents, decreases Ca(2+) currents, and inhibits spike activity in both cell types, actions reproduced by the selective sst(2A) agonist L-054,264 (N-[(1R)-2-[[[(1S*,3R*)-3-(aminomethyl)cyclohexyl]methyl]amino]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]spiro[1H-indene-1,4'-piperidine]-1'-carboxamide) in DA amacrine cells and the selective sst4 agonist L-803,087 (N(2)-[4-(5,7-difluoro-2-phenyl-1H-indol-3-yl)-1-oxobutyl]-L-arginine methyl ester trifluoroacetate) in M1 ipRGCs. These parallel actions of SRIF may serve to counteract the disinhibition of M1 ipRGCs caused by SRIF inhibition of DA amacrine cells. This allows the actions of SRIF on DA amacrine cells to proceed with adjusting retinal DA levels without destabilizing light responses by M1 ipRGCs, which project to non-image-forming targets in the brain. Copyright © 2015

  20. Amacrine cells coupled to ganglion cells via gap junctions are highly vulnerable in glaucomatous mouse retinas.

    PubMed

    Akopian, Abram; Kumar, Sandeep; Ramakrishnan, Hariharasubramanian; Viswanathan, Suresh; Bloomfield, Stewart A

    2016-07-13

    We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol., 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice

    SciTech Connect

    Fox, Donald A.; Hamilton, W. Ryan; Johnson, Jerry E.; Xiao, Weimin; Chaney, Shawntay; Mukherjee, Shradha; Miller, Diane B.; O'Callaghan, James P.

    2011-11-15

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was {<=} 1, {<=} 10, {approx} 25 and {approx} 40 {mu}g/dL, respectively, on PN10 and by PN30 all were {<=} 1 {mu}g/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. -- Highlights: Black-Right-Pointing-Pointer Peak [BPb] in control, low-, moderate- and high-dose newborn mice with gestational lead exposure: {<=} 1, {<=} 10, 25 and 40 {mu}g/dL Black

  2. Cat retinal ganglion cell receptive-field alterations after 6-hydroxydopamine induced dopaminergic amacrine cell lesions

    SciTech Connect

    Maguire, G.W.; Smith, E.L. III

    1985-06-01

    Optic tract single-unit recordings were used to study ganglion cell response functions of the intact cat eye after 6-hydroxydopamine (6-OHDA) lesioning of the dopaminergic amacrine cell (AC) population of the inner retina. The impairment of the dopaminergic AC was verified by high pressure-liquid chromatography with electrochemical detection of endogenous dopamine content and by (/sup 3/H)dopamine high-affinity uptake; the dopaminergic ACs of the treated eyes demonstrated reduced endogenous dopamine content and reduced (/sup 3/H)dopamine uptake compared with that of their matched controls. Normal appearing (/sup 3/H)GABA and (/sup 3/H)-glycine uptake in the treated retinas suggests the absence of any nonspecific action of the 6-OHDA on the neural retina. The impairment of the dopaminergic AC population was found to alter a number of response properties in off-center ganglion cells, but this impairment had only a modest effect on the on-center cells. An abnormally high proportion of the off-center ganglion cells in the 6-OHDA treated eyes possessed nonlinear, Y-type receptive fields. These cells also possessed shift-responses of greater than normal amplitude, altered intensity-response functions, reduced maintained activities, and more transient center responses. Of the on-center type cells, only the Y-type on-center cells were affected by 6-OHDA, possessing higher than normal maintained activities and altered intensity-response functions. The on-center X-cells were unaffected by 6-OHDA treatment. The dopaminergic AC of the photopically adapted cat retina therefore modulates a number of ganglion cell response properties and within the limits of this study is most prominent in off-center ganglion cell circuitry.

  3. Immuocytochemical analysis of spatial organization of photoreceptors and amacrine and ganglion cells in the tiger salamander retina.

    PubMed

    Zhang, Jian; Yang, Zhuo; Wu, Samuel M

    2004-01-01

    In the present study, using double- or triple-label immunocytochemistry in conjunction with confocal microscopy, we aimed to examine the population and distribution of photoreceptors, GABAergic and glycinergic amacrine cells, and ganglion cells, which are basic but important parameters for studying the structure-function relationship of the salamander retina. We found that the outer nuclear layer (ONL) contained 82,019 +/- 3203 photoreceptors, of which 52% were rods and 48% were cones. The density of photoreceptors peaked at approximately 8000 cells/mm2 in the ventral and dropped to approximately 4000 cells/mm2 in the dorsal retina. In addition, the rod/cone ratio was less than 1 in the central retina but larger than I in the periphery. Moreover, in the proximal region of the inner nuclear layer (INL3), the total number of cells was 50,576 +/- 8400. GABAergic and glycinergic amacrine cells made up approximately 78% of all cells in this layer, including 43% GABAergic, 32% glycinergic, and 3% GABA/glycine colocalized amacrine cells. The density of these amacrine cells was approximately 6500 cells/mm2 in the ventral and approximately 3200 cells/mm2 in the dorsal area. The ratio of GABAergic to glycinergic amacrine cells was larger than 1. Furthermore, in the ganglion cell layer (GCL), among a total of 36,007 +/- 2010 cells, ganglion cells accounted for 65.7 +/- 1.5% of the total cells, whereas displaced GABAergic and glycinergic amacrine cells comprised about 4% of the cells in this layer. The ganglion cell density was approximately 1800 cells/mm2 in the ventral and approximately 600 cells/mm2 in the dorsal retina. Our data demonstrate that all three major cell types are not uniformly distributed across the salamander retina. Instead, they exhibit a higher density in the ventral than in the dorsal retina and their spatial arrangement is associated with the retinal topography. These findings provide a basic anatomical reference for the electrophysiological study of

  4. Visual stimulation switches the polarity of excitatory input to starburst amacrine cells

    PubMed Central

    Vlasits, Anna L.; Bos, Rémi; Morrie, Ryan D.; Fortuny, Cécile; Flannery, John G.; Feller, Marla B.; Rivlin-Etzion, Michal

    2014-01-01

    Summary Direction-selective ganglion cells (DSGCs) are tuned to motion in one direction. Starburst amacrine cells (SACs) are thought to mediate this direction selectivity through precise anatomical wiring to DSGCs. Nevertheless, we previously found that visual adaptation can reverse DSGCs’ directional tuning, overcoming the circuit anatomy. Here we explore the role of SACs in the generation and adaptation of direction selectivity. First, using pharmaco-genetics and two-photon calcium imaging, we validate that SACs are necessary for direction selectivity. Next, we demonstrate that exposure to an adaptive stimulus dramatically alters SACs’ synaptic inputs. Specifically, after visual adaptation, On-SACs lose their excitatory input during light onset but gain an excitatory input during light offset. Our data suggest that visual stimulation alters the interactions between rod and cone-mediated inputs that converge on the terminals of On cone BCs. These results demonstrate how the sensory environment can modify computations performed by anatomically-defined neuronal circuits. PMID:25155960

  5. Zebrafish Tg(7.2mab21l2:EGFP)ucd2 transgenics reveal a unique population of retinal amacrine cells.

    PubMed

    Cederlund, Maria L; Morrissey, Maria E; Baden, Tom; Scholz, Dimitri; Vendrell, Victor; Lagnado, Leon; Connaughton, Victoria P; Kennedy, Breandán N

    2011-03-01

    Amacrine cells constitute a diverse, yet poorly characterized, cell population in the inner retina. Here, the authors sought to characterize the morphology, molecular physiology, and electrophysiology of a subpopulation of EGFP-expressing retinal amacrine cells identified in a novel zebrafish transgenic line. After 7.2 kb of the zebrafish mab21l2 promoter was cloned upstream of EGFP, it was used to create the Tg(7.2mab21l2:EGFP)ucd2 transgenic line. Transgenic EGFP expression was analyzed by fluorescence microscopy in whole mount embryos, followed by detailed analysis of EGFP-expressing amacrine cells using fluorescence microscopy, immunohistochemistry, and electrophysiology. A 7.2-kb fragment of the mab21l2 promoter region is sufficient to drive transgene expression in the developing lens and tectum. Intriguingly, EGFP was also observed in differentiated amacrine cells. EGFP-labeled amacrine cells in Tg(7.2mab21l2:EGFP)ucd2 constitute a novel GABA- and glycine-negative amacrine subpopulation. Morphologically, EGFP-expressing cells stratify in sublamina 1 to 2 (type 1 OFF) or sublamina 3 to 4 (type 1 ON) or branch diffusely (type 2). Electrophysiologically, these cells segregate into amacrine cells with somas in the vitreal part of the INL and linear responses to current injection or, alternatively, amacrine cells with somas proximal to the IPL and active oscillatory voltage signals. CONCLUSIONS; The novel transgenic line Tg(7.2mab21l2:EGFP)ucd2 uncovers a unique subpopulation of retinal amacrine cells.

  6. Activation of nicotinic receptors on GABAergic amacrine cells in the rabbit retina indirectly stimulates dopamine release.

    PubMed

    Neal, M J; Cunningham, J R; Matthews, K L

    2001-01-01

    The retina possesses subpopulations of amacrine cells, which utilize different transmitters, including acetylcholine (ACh), GABA, and dopamine. We have examined interactions between these neurones by studying the effects of nicotinic agonists on GABA and dopamine release. Isolated rabbit retinas were incubated with [3H]dopamine and then superfused. Fractions of the superfusate (2 min) were collected and the [3H]dopamine in each sample was measured. Endogenous GABA release was examined by incubating retinas in a small chamber. At 5-min intervals, the medium was changed and the GABA measured by high-pressure liquid chromatography (HPLC). Exposure of the retina to nicotine, epibatidine, and other nicotinic agonists increased the release of both GABA and dopamine. The effects of nicotine and epibatidine were blocked by mecamylamine, confirming an action on nicotinic receptors. The action of epibatidine on dopamine release was unaffected by glutamate antagonists but was blocked by picrotoxin and gabazine. These results suggested that nicotine might increase dopamine release indirectly by stimulating the release of GABA, which in turn inhibited the release of an inhibitory transmitter acting tonically on the dopaminergic amacrines. Exposure of the retina to GABA caused a small increase in dopamine release. This hypothetical inhibitory transmitter was not GABA, an opioid, adenosine, glycine, nociceptin, a cannabinoid, or nitric oxide because appropriate antagonists did not affect the resting release of dopamine. However, metergoline, a 5HT1/5HT2 receptor antagonist, and ketanserin, a 5HT2A receptor antagonist, but not the 5HT1A antagonist WAY100635, increased the resting release of dopamine and blocked the effects of nicotine. The 5HT1A/5HT7 agonist 8-hydroxy DPAT inhibited both the nicotine and GABA-evoked release of dopamine. We conclude that nicotinic agonists directly stimulate the release of GABA, but the evoked release of dopamine is indirect, and arises from GABA

  7. Survey on Amacrine Cells Coupling to Retrograde-Identified Ganglion Cells in the Mouse Retina

    PubMed Central

    Pang, Ji-Jie; Paul, David L.; Wu, Samuel M.

    2013-01-01

    Purpose. Retinal amacrine cells (ACs) may make inhibitory chemical synapses and potentially excitatory gap junctions on ganglion cells (GCs). The total number and subtypes of ACs coupled to the entire GC population were investigated in wild-type and three lines of transgenic mice. Methods. GCs and GC-coupled ACs were identified by the previously established LY-NB (Lucifer yellow–Neurobiotin) retrograde double-labeling technique, in conjunction with specific antibodies and confocal microscopy. Results. GC-coupled ACs (NB-positive and LY-negative) comprised nearly 11% of displaced ACs and 4% of conventional ACs in wild-type mice, and were 9% and 4% of displaced ACs in Cx45−/− and Cx36/45−/− mice, respectively. Their somas were small in Cx36/45−/− mice, but variable in other strains. They were mostly γ-aminobutyric acid (GABA)-immunoreactive (IR) and located in the GC layer. They comprised only a small portion in the AC subpopulations, including GABA-IR, glycine-IR, calretinin-IR, 5-HT-accumulating, and ON-type choline acetyltransferase (ChAT) ACs in wild-type and ChAT transgenic mice (ChAT- tdTomato). In the distal 80% of the inner plexiform layer (IPL), dense GC dendrites coexisted with rich glycine-IR and GABA-IR. In the inner 20% of the IPL, sparse GC dendrites presented with a major GABA band and sparse glycine-IR. Conclusions. Various subtypes of ACs may couple to GCs. ACs of the same immunoreactivity may either couple or not couple to GCs. Cx36 and Cx45 dominate GC-AC coupling except for small ACs. The overall potency of GC-AC coupling is moderate, especially in the proximal 20% of the IPL, where inhibitory chemical signals are dominated by GABA ACs. PMID:23821205

  8. Asymmetrical developmental pattern of uptake of Lucifer Yellow into amacrine cells in the embryonic chick retina.

    PubMed

    Layer, P G; Kotz, S

    1983-08-01

    Whole chick embryo eyes between embryonic day 10.5 and 12 (E10.5-E12) were incubated in a solution of Lucifer Yellow and examined in frozen sections. Starting around day 10.5, brightly stained cells were observed in the innermost part of the inner nuclear layer. Their size, shape and location suggest that most of them represent a subclass of amacrine cells. A distinct spatio-temporal development of Lucifer Yellow-staining of this subpopulation of cells within the inner nuclear layer between E10.5 and E12 is revealed in detail using 3-dimensional models of Lucifer Yellow-stained eyes. The staining can be first observed at a specific location at the ventro-posterior side not far from the fundus. It then spreads radially in a complex pattern reaching the ora serrata first on the ventro-posterior side, then sequentially reaching into the dorso-posterior, the ventro-anterior and finally the dorso-anterior quadrants of the retina. Our results suggest that a horizontal (posterior-anterior) axis and a vertical (ventro-dorsal) axis function as a coordinate system during differentiation of the tissue. We conclude that there are precise spatio-temporal patterns of Lucifer Yellow-staining which most probably reflect spatio-temporal patterns of cell differentiation within the chicken retina. The correlation of these findings with other data on spatio-temporal patterns during differentiation of the chicken retina is discussed.

  9. Loss of CPEB3 Upregulates MEGF10 to Impair Mosaic Development of ON Starburst Amacrine Cells

    PubMed Central

    Chen, Yin-Peng; Bai, Geng-Shuo; Wu, Meng-Fang; Chiao, Chuan-Chin; Huang, Yi-Shuian

    2016-01-01

    Cytoplasmic polyadenylation element binding protein 3 (CPEB3) regulates target RNA translation in neurons. Here, we examined CPEB3 distribution and function in the mouse retina. CPEB3 is expressed in retinal neurons, including those located in the inner nuclear layer (INL) and ganglion cell layer (GCL) but not in cone and rod photoreceptors in the outer nuclear layer (ONL). A previous study found CPEB3 expressed in cholinergic starburst amacrine cells (SACs). We first examined these cells and observed aberrant SAC mosaicism in CPEB3-knockout (KO) retinas. Retinal neurons showed orderly spatial arrangements. Many individual subtypes are organized non-randomly in patterns called mosaics. Despite CPEB3 being expressed in both populations of SACs, OFF SACs in the INL and ON SACs in the GCL, aberrant mosaic regularity was observed in only ON SACs of CPEB3-KO retinas. Molecular characterization revealed that translation of multiple epidermal growth factor 10 (Megf10) RNA is suppressed by CPEB3 during the first week of postnatal development, when MEGF10 is primarily expressed in SACs and mediates homotypic repulsive interactions to define intercellular spacing of SACs. Thus, elevated MEGF10 expression in the absence of the translational repressor CPEB3 may account for the defective spatial organization of ON SACs. Our findings uncover for the first time that translational control plays a role in shaping retinal mosaic arrangement. PMID:27822178

  10. Retinal input to efferent target amacrine cells in the avian retina

    PubMed Central

    Lindstrom, Sarah H.; Azizi, Nason; Weller, Cynthia; Wilson, Martin

    2012-01-01

    The bird visual system includes a substantial projection, of unknown function, from a midbrain nucleus to the contralateral retina. Every centrifugal, or efferent, neuron originating in the midbrain nucleus makes synaptic contact with the soma of a single, unique amacrine cell, the target cell (TC). By labeling efferent neurons in the midbrain we have been able to identify their terminals in retinal slices and make patch clamp recordings from TCs. TCs generate Na+ based action potentials triggered by spontaneous EPSPs originating from multiple classes of presynaptic neurons. Exogenously applied glutamate elicited inward currents having the mixed pharmacology of NMDA, kainate and inward rectifying AMPA receptors. Exogenously applied GABA elicited currents entirely suppressed by GABAzine, and therefore mediated by GABAA receptors. Immunohistochemistry showed the vesicular glutamate transporter, vGluT2, to be present in the characteristic synaptic boutons of efferent terminals, whereas the GABA synthetic enzyme, GAD, was present in much smaller processes of intrinsic retinal neurons. Extracellular recording showed that exogenously applied GABA was directly excitatory to TCs and, consistent with this, NKCC, the Cl− transporter often associated with excitatory GABAergic synapses, was identified in TCs by antibody staining. The presence of excitatory retinal input to TCs implies that TCs are not merely slaves to their midbrain input; instead, their output reflects local retinal activity and descending input from the midbrain. PMID:20650017

  11. Functional polarity of dendrites and axons of primate A1 amacrine cells

    PubMed Central

    Davenport, Christopher M.; Detwiler, Peter B.; Dacey, Dennis M.

    2011-01-01

    The A1 cell is an axon-bearing amacrine cell of the primate retina with a diffusely stratified, moderately branched dendritic tree (~400 µm diameter). Axons arise from proximal dendrites forming a second concentric, larger arborization (>4 mm diameter) of thin processes with bouton-like swellings along their length. A1 cells are ON-OFF transient cells that fire a brief high frequency burst of action potentials in response to light (Stafford & Dacey, 1997). It has been hypothesized that A1 cells receive local input to their dendrites, with action potentials propagating output via the axons across the retina, serving a global inhibitory function. To explore this hypothesis we recorded intracellularly from A1 cells in an in vitro macaque monkey retina preparation. A1 cells have an antagonistic center-surround receptive field structure for the ON and OFF components of the light response. Blocking the ON pathway with L-AP4 eliminated ON center responses but not OFF center responses or ON or OFF surround responses. Blocking GABAergic inhibition with picrotoxin increased response amplitudes without affecting receptive field structure. TTX abolished action potentials, with little effect on the sub-threshold light response or basic receptive field structure. We also used multi-photon laser scanning microscopy to record light-induced calcium transients in morphologically identified dendrites and axons of A1 cells. TTX completely abolished such calcium transients in the axons but not in the dendrites. Together these results support the current model of A1 function, whereby the dendritic tree receives synaptic input that determines the center-surround receptive field; and action potentials arise in the axons, which propagate away from the dendritic field across the retina. PMID:17550636

  12. Reprogramming amacrine and photoreceptor progenitors into retinal ganglion cells by replacing Neurod1 with Atoh7.

    PubMed

    Mao, Chai-An; Cho, Jang-Hyeon; Wang, Jing; Gao, Zhiguang; Pan, Ping; Tsai, Wen-Wei; Frishman, Laura J; Klein, William H

    2013-02-01

    The specification of the seven retinal cell types from a common pool of retina progenitor cells (RPCs) involves complex interactions between the intrinsic program and the environment. The proneural basic helix-loop-helix (bHLH) transcriptional regulators are key components for the intrinsic programming of RPCs and are essential for the formation of the diverse retinal cell types. However, the extent to which an RPC can re-adjust its inherent program and the mechanisms through which the expression of a particular bHLH factor influences RPC fate is unclear. Previously, we have shown that Neurod1 inserted into the Atoh7 locus activates the retinal ganglion cell (RGC) program in Atoh7-expressing RPCs but not in Neurod1-expressing RPCs, suggesting that Atoh7-expressing RPCs are not able to adopt the cell fate determined by Neurod1, but rather are pre-programmed to produce RGCs. Here, we show that Neurod1-expressing RPCs, which are destined to produce amacrine and photoreceptor cells, can be re-programmed into RGCs when Atoh7 is inserted into the Neurod1 locus. These results suggest that Atoh7 acts dominantly to convert a RPC subpopulation not destined for an RGC fate to adopt that fate. Thus, Atoh7-expressing and Neurod1-expressing RPCs are intrinsically different in their behavior. Additionally, ChIP-Seq analysis identified an Atoh7-dependent enhancer within the intronic region of Nrxn3. The enhancer recognized and used Atoh7 in the developing retina to regulate expression of Nrxn3, but could be forced to use Neurod1 when placed in a different regulatory context. The results indicate that Atoh7 and Neurod1 activate distinct sets of genes in vivo, despite their common DNA-binding element.

  13. Morphological study of a connexin 43-GFP reporter mouse highlights glial heterogeneity, amacrine cells, and olfactory ensheathing cells.

    PubMed

    Theofilas, Panos; Steinhäuser, Christian; Theis, Martin; Derouiche, Amin

    2017-03-30

    Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal Müller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100ß, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP(+) astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. © 2017 Wiley Periodicals, Inc.

  14. Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells

    PubMed Central

    Elgueta, Claudio; Vielma, Alex H.; Palacios, Adrian G.; Schmachtenberg, Oliver

    2015-01-01

    Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca2+ accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca2+ stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing. PMID:25709566

  15. Morphology of primate's dopaminergic amacrine cells as revealed by TH-like immunoreactivity on retinal flat-mounts.

    PubMed

    Nguyen-Legros, J; Botteri, C; Phuc, L H; Vigny, A; Gay, M

    1984-03-12

    Dopaminergic (DA) cells have been revealed by immunohistochemical localization of tyrosine hydroxylase in the retina of cynomolgus monkey, chimpanzee and human. The DA neurons were visualized in cross-sections as well as in flat-mounts of retina. The comparison revealed a striking morphological similarity between the DA neurons in the three species. When observed in flat-mounts, they were of stellate type; when observed in cross-sections, except for a few displaced cells, they were unistratified amacrine cells branching in the outermost sublayer of the inner plexiform layer. Observations in sections suggested the existence of DA-interplexiform cells in ape and human retinas.

  16. Depletion of cholinergic amacrine cells by a novel immunotoxin does not perturb the formation of segregated on and off cone bipolar cell projections.

    PubMed

    Gunhan, Emine; Choudary, Prabhakara V; Landerholm, Thomas E; Chalupa, Leo M

    2002-03-15

    Cone bipolar cells are the first retinal neurons that respond in a differential manner to light onset and offset. In the mature retina, the terminal arbors of On and Off cone bipolar cells terminate in different sublaminas of the inner plexiform layer (IPL) where they form synapses with the dendrites of On and Off retinal ganglion cells and with the stratified processes of cholinergic amacrine cells. Here we first show that cholinergic processes within the On and Off sublaminas of the IPL are present early in development, being evident in the rat on the day of birth, approximately 10 d before the formation of segregated cone bipolar cell axons. This temporal sequence, as well as our previous finding that the segregation of On and Off cone bipolar cell inputs occurs in the absence of retinal ganglion cells, suggested that cholinergic amacrine cells could provide a scaffold for the subsequent in-growth of bipolar cell axons. To test this hypothesis directly, a new cholinergic cell immunotoxin was constructed by conjugating saporin, the ribosome-inactivating protein toxin, to an antibody against the vesicular acetylcholine transporter. A single intraocular injection of the immunotoxin caused a rapid, complete, and selective loss of cholinergic amacrine cells from the developing rat retina. On and Off cone bipolar cells were visualized using an antibody against recoverin, the calcium-binding protein that labels the soma and processes of these interneurons. After complete depletion of cholinergic amacrine cells, cone bipolar cell axon terminals still formed their two characteristic strata within the IPL. These findings demonstrate that the presence of cholinergic amacrine cells is not required for the segregation of recoverin-positive On and Off cone bipolar cell projections.

  17. Type 2 wide-field amacrine cells in TH::GFP mice show a homogenous synapse distribution and contact small ganglion cells.

    PubMed

    Brüggen, Bianca; Meyer, Arndt; Boven, Franziska; Weiler, Reto; Dedek, Karin

    2015-03-01

    In vertebrate retinas, wide-field amacrine cells represent a diverse class of interneurons, important for the extraction of selective features, like motion or objects, from the visual scene. Most types of wide-field amacrine cells lack dedicated output processes, whereas some types spatially segregate outputs from inputs. In the tyrosine hydroxylase (TH)::green fluorescent protein (GFP) mouse line, two types of GFP-expressing wide-field amacrine cells have been described: dopaminergic type 1 and γ-aminobutyric acid-ergic type 2 cells (TH2). TH2 cells possess short and long radial processes stratifying in the middle of the inner plexiform layer, where they collect excitatory and inhibitory inputs from bipolar cells and other amacrine cells, respectively. Although it was shown that these inputs lead to ON-OFF light responses, their spatial distribution along TH2 cell processes is unknown. Also, the postsynaptic targets of TH2 cells have not been identified so far. Here, we analysed the synapse distribution of these cells in TH::GFP mice and show that they form a weakly coupled network. Electrical synapses (made of connexin36) and chemical (excitatory and inhibitory) synapses are uniformly distributed along TH2 dendrites, independent of dendrite length or distance from soma. Moreover, we reveal that TH2 cells contact at least two types of small ganglion cells; one of them is the W3 cell, a ganglion cell sensitive to object motion. Contacts were often associated with markers of inhibitory synapses. Thus, TH2 wide-field amacrine cells likely provide postsynaptic inhibition to W3 ganglion cells and may contribute to object-motion detection in the mouse retina. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  18. The Synaptic and Morphological Basis of Orientation Selectivity in a Polyaxonal Amacrine Cell of the Rabbit Retina.

    PubMed

    Murphy-Baum, Benjamin L; Taylor, W Rowland

    2015-09-30

    Much of the computational power of the retina derives from the activity of amacrine cells, a large and diverse group of GABAergic and glycinergic inhibitory interneurons. Here, we identify an ON-type orientation-selective, wide-field, polyaxonal amacrine cell (PAC) in the rabbit retina and demonstrate how its orientation selectivity arises from the structure of the dendritic arbor and the pattern of excitatory and inhibitory inputs. Excitation from ON bipolar cells and inhibition arising from the OFF pathway converge to generate a quasi-linear integration of visual signals in the receptive field center. This serves to suppress responses to high spatial frequencies, thereby improving sensitivity to larger objects and enhancing orientation selectivity. Inhibition also regulates the magnitude and time course of excitatory inputs to this PAC through serial inhibitory connections onto the presynaptic terminals of ON bipolar cells. This presynaptic inhibition is driven by graded potentials within local microcircuits, similar in extent to the size of single bipolar cell receptive fields. Additional presynaptic inhibition is generated by spiking amacrine cells on a larger spatial scale covering several hundred microns. The orientation selectivity of this PAC may be a substrate for the inhibition that mediates orientation selectivity in some types of ganglion cells. Significance statement: The retina comprises numerous excitatory and inhibitory circuits that encode specific features in the visual scene, such as orientation, contrast, or motion. Here, we identify a wide-field inhibitory neuron that responds to visual stimuli of a particular orientation, a feature selectivity that is primarily due to the elongated shape of the dendritic arbor. Integration of convergent excitatory and inhibitory inputs from the ON and OFF visual pathways suppress responses to small objects and fine textures, thus enhancing selectivity for larger objects. Feedback inhibition regulates the

  19. A role for TREK1 in generating the slow afterhyperpolarization in developing starburst amacrine cells

    PubMed Central

    Ford, Kevin J.; Arroyo, David A.; Kay, Jeremy N.; Lloyd, Eric E.; Bryan, Robert M.; Sanes, Joshua R.

    2013-01-01

    Slow afterhyperpolarizations (sAHPs) play an important role in establishing the firing pattern of neurons that in turn influence network activity. sAHPs are mediated by calcium-activated potassium channels. However, the molecular identity of these channels and the mechanism linking calcium entry to their activation are still unknown. Here we present several lines of evidence suggesting that the sAHPs in developing starburst amacrine cells (SACs) are mediated by two-pore potassium channels. First, we use whole cell and perforated patch voltage clamp recordings to characterize the sAHP conductance under different pharmacological conditions. We find that this conductance was calcium dependent, reversed at EK, blocked by barium, insensitive to apamin and TEA, and activated by arachidonic acid. In addition, pharmacological inhibition of calcium-activated phosphodiesterase reduced the sAHP. Second, we performed gene profiling on isolated SACs and found that they showed strong preferential expression of the two-pore channel gene kcnk2 that encodes TREK1. Third, we demonstrated that TREK1 knockout animals exhibited an altered frequency of retinal waves, a frequency that is set by the sAHPs in SACs. With these results, we propose a model in which depolarization-induced decreases in cAMP lead to disinhibition of the two-pore potassium channels and in which the kinetics of this biochemical pathway dictate the slow activation and deactivation of the sAHP conductance. Our model offers a novel pathway for the activation of a conductance that is physiologically important. PMID:23390312

  20. Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus.

    PubMed

    Bonci, D M O; Lima, S M A de; Grötzner, S R; Ribeiro, C A Oliveira; Hamassaki, D E; Ventura, D F

    2006-03-01

    To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 microg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 microg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 +/- 393 cells/mm2) compared to control (1886 +/- 892 cells/mm2; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 +/- 56 cells/mm2 (2 microg/g) and 845 +/- 82 cells/mm2 (6 microg/g), also lower than control (1312 +/- 31 cells/mm2; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 microg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

  1. Mechanism of Generation of Spontaneous Miniature Outward Currents (SMOCs) in Retinal Amacrine Cells

    PubMed Central

    Mitra, Pratip; Slaughter, Malcolm M.

    2002-01-01

    A subtype of retinal amacrine cells displayed a distinctive array of K+ currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of −60 to −40 mV. Depolarizations above approximately −40 mV were associated with the disappearance of SMOCs and the appearance of transient (Ito) and sustained (Iso) outward K+ currents. Ito appeared at about −40 mV and its apparent magnitude was biphasic with voltage, whereas Iso appeared near −30 mV and increased linearly. SMOCs, Ito, and a component of Iso were Ca2+ dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of −20 mV and above. Their mean amplitude was Nernstian with respect to [K+]ext and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca2+-free solutions, indicative of BK-type Ca2+-activated K+ currents. Dihydropyridine Ca2+ channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca2+ permeation through the kainic acid receptor had no effect. Blockade of organelle Ca2+ channels by ryanodine, or intracellular Ca2+ store depletion with caffeine, eradicated SMOCs. Internal Ca2+ chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca2+ influx through L-type Ca2+ channels and its subsequent amplification by Ca2+-induced Ca2+ release via the ryanodine receptor leads to a localized elevation of internal Ca2+. This amplified Ca2+ signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs. PMID:11929886

  2. Mechanism of generation of spontaneous miniature outward currents (SMOCs) in retinal amacrine cells.

    PubMed

    Mitra, Pratip; Slaughter, Malcolm M

    2002-04-01

    A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.

  3. Photopic ERG negative response from amacrine cell signaling in RCS rat retinal degeneration.

    PubMed

    Machida, Shigeki; Raz-Prag, Dorit; Fariss, Robert N; Sieving, Paul A; Bush, Ronald A

    2008-01-01

    The authors investigated photopic electroretinographic changes during degeneration in the Royal College of Surgeons (RCS) and transgenic P23H rhodopsin rat models, including the cellular origins of a large corneal-negative component that persists in the RCS rat. Photopic and scotopic electroretinograms (ERGs) were recorded from dystrophic RCS (RCS-p(+)/Lav) rats (4-18 weeks old) and transgenic rhodopsin Pro23His line 1 (P23H) rats (4-30 weeks old). Age-matched congenic (RCS-rdy(+)p(+)/Lav) and Sprague-Dawley rats were used as controls. N-methyl-DL-aspartic acid (NMA), dopamine, and gamma-aminobutyric acid (GABA) were injected intravitreally, and optic nerve sectioning (ONS) was performed to suppress or remove inner retinal neuron activity. Retinal morphology for cone cell counts and immunohistochemistry for quantification of Kir4.1 channels were performed at various stages of degeneration. As degeneration progressed, the photopic ERG of RCS dystrophic rats was distinctly different from that of P23H rats, primarily because of the growth of a corneal-negative response (RCS-NPR) after the b-wave in RCS rats. This response had a peak time similar to the photopic negative response (PhNR) in controls but with a more gradual recovery phase, and it was not affected by ONS. The PhNR in P23H rats declined linearly with the b-wave. NMA and GABA eliminated the RCS-NPR and uncovered a larger b-wave in RCS rats at late stages of degeneration, but NMA had little effect on the ERG in P23H rats. The NMA-sensitive negative response in RCS rats declined with age more slowly than did the NMA-isolated b-wave. The density of Kir4.1 channels at the endfeet of Müller cells and in the proximal retina increased significantly between 6 to 10 weeks and 14 weeks of age in the RCS rat retina but not in the P23H rat retina. The photopic ERG of the dystrophic RCS rat retina becomes increasingly electronegative because of an aberrant negative response, originating from amacrine cell activity

  4. Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina

    PubMed Central

    Vila, Alejandro; Huynh, Uyen-Chi N.; Brecha, Nicholas C.

    2008-01-01

    Purpose To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. Methods CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD67), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. Results CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 μm in diameter, and they had a density of 2636±347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD67, GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. Conclusions The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells. PMID:18728756

  5. An excitatory amacrine cell detects object motion and provides feature-selective input to ganglion cells in the mouse retina

    PubMed Central

    Kim, Tahnbee; Soto, Florentina; Kerschensteiner, Daniel

    2015-01-01

    Retinal circuits detect salient features of the visual world and report them to the brain through spike trains of retinal ganglion cells. The most abundant ganglion cell type in mice, the so-called W3 ganglion cell, selectively responds to movements of small objects. Where and how object motion sensitivity arises in the retina is incompletely understood. In this study, we use 2-photon-guided patch-clamp recordings to characterize responses of vesicular glutamate transporter 3 (VGluT3)-expressing amacrine cells (ACs) to a broad set of visual stimuli. We find that these ACs are object motion sensitive and analyze the synaptic mechanisms underlying this computation. Anatomical circuit reconstructions suggest that VGluT3-expressing ACs form glutamatergic synapses with W3 ganglion cells, and targeted recordings show that the tuning of W3 ganglion cells' excitatory input matches that of VGluT3-expressing ACs' responses. Synaptic excitation of W3 ganglion cells is diminished, and responses to object motion are suppressed in mice lacking VGluT3. Object motion, thus, is first detected by VGluT3-expressing ACs, which provide feature-selective excitatory input to W3 ganglion cells. DOI: http://dx.doi.org/10.7554/eLife.08025.001 PMID:25988808

  6. A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina.

    PubMed

    Ozaita, Ander; Petit-Jacques, Jerome; Völgyi, Béla; Ho, Chi Shun; Joho, Rolf H; Bloomfield, Stewart A; Rudy, Bernardo

    2004-08-18

    Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K(+) currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells

  7. Patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

    PubMed Central

    Tu, Hung-Ya; Hsu, Chih-Chun; Chen, Yu-Jiun

    2016-01-01

    SHORT ABSTRACT This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation. LONG ABSTRACT The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs). PMID:27768050

  8. Transcription factor PRDM8 is required for rod bipolar and type 2 OFF-cone bipolar cell survival and amacrine subtype identity.

    PubMed

    Jung, Cynthia C; Atan, Denize; Ng, David; Ploder, Lynda; Ross, Sarah E; Klein, Martin; Birch, David G; Diez, Eduardo; McInnes, Roderick R

    2015-06-09

    Retinal bipolar (BP) cells mediate the earliest steps in image processing in the visual system, but the genetic pathways that regulate their development and function are incompletely known. We identified PRDI-BF1 and RIZ homology domain containing 8 (PRDM8) as a highly conserved transcription factor that is abundantly expressed in mouse retina. During development and in maturity, PRDM8 is expressed strongly in BP cells and a fraction of amacrine and ganglion cells. To determine whether Prdm8 is essential to BP cell development or physiology, we targeted the gene in mice. Prdm8(EGFP/EGFP) mice showed nonprogressive b-wave deficits on electroretinograms, consistent with compromised BP cell function or circuitry resembling the incomplete form of human congenital stationary night blindness (CSNB). BP cell specification was normal in Prdm8(EGFP/EGFP) retina as determined by VSX2(+) cell numbers and retinal morphology at postnatal day 6. BP subtype differentiation was impaired, however, as indicated by absent or diminished expression of BP subtype-specific markers, including the putative PRDM8 regulatory target PKCα (Prkca) and its protein. By adulthood, rod bipolar (RB) and type 2 OFF-cone bipolar (CB) cells were nearly absent from Prdm8-null mice. Although no change was detected in total amacrine cell (AC) numbers, increased PRKCA(+) and cholinergic ACs and decreased GABAergic ACs were seen, suggesting an alteration in amacrine subtype identity. These findings establish that PRDM8 is required for RB and type 2 OFF-CB cell survival and amacrine subtype identity, and they present PRDM8 as a candidate gene for human CSNB.

  9. Distinctive receptive field and physiological properties of a wide-field amacrine cell in the macaque monkey retina

    PubMed Central

    Puller, Christian; Rieke, Fred; Neitz, Jay; Neitz, Maureen

    2015-01-01

    At early stages of visual processing, receptive fields are typically described as subtending local regions of space and thus performing computations on a narrow spatial scale. Nevertheless, stimulation well outside of the classical receptive field can exert clear and significant effects on visual processing. Given the distances over which they occur, the retinal mechanisms responsible for these long-range effects would certainly require signal propagation via active membrane properties. Here the physiology of a wide-field amacrine cell—the wiry cell—in macaque monkey retina is explored, revealing receptive fields that represent a striking departure from the classic structure. A single wiry cell integrates signals over wide regions of retina, 5–10 times larger than the classic receptive fields of most retinal ganglion cells. Wiry cells integrate signals over space much more effectively than predicted from passive signal propagation, and spatial integration is strongly attenuated during blockade of NMDA spikes but integration is insensitive to blockade of NaV channels with TTX. Thus these cells appear well suited for contributing to the long-range interactions of visual signals that characterize many aspects of visual perception. PMID:26133804

  10. Orexin-A differentially modulates AMPA-preferring responses of ganglion cells and amacrine cells in rat retina.

    PubMed

    Zheng, Chao; Deng, Qin-Qin; Liu, Lei-Lei; Wang, Meng-Ya; Zhang, Gong; Sheng, Wen-Long; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2015-06-01

    By activating their receptors (OX1R and OX2R) orexin-A/B regulate wake/sleeping states, feeding behaviors, but the function of these peptides in the retina remains unknown. Using patch-clamp recordings and calcium imaging in rat isolated retinal cells, we demonstrated that orexin-A suppressed α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-preferring receptor-mediated currents (AMPA-preferring currents) in ganglion cells (GCs) through OX1R, but potentiated those in amacrine cells (ACs) through OX2R. Consistently, in rat retinal slices orexin-A suppressed light-evoked AMPA-preferring receptor-mediated excitatory postsynaptic currents in GCs, but potentiated those in ACs. Intracellular dialysis of GDP-β-S or preincubation with the Gi/o inhibitor pertussis toxin (PTX) abolished both the effects. Either cAMP/the protein kinase A (PKA) inhibitor Rp-cAMP or cGMP/the PKG blocker KT5823 failed to alter the orexin-A effects. Whilst both of them involved activation of protein kinase C (PKC), the effects on GCs and ACs were respectively eliminated by the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor and phosphatidylcholine (PC)-PLC inhibitor. Moreover, in GCs orexin-A increased [Ca(2+)]i and the orexin-A effect was blocked by intracellular Ca(2+)-free solution and by inositol 1,4,5-trisphosphate (IP3) receptor antagonists. In contrast, orexin-A did not change [Ca(2+)]i in ACs and the orexin-A effect remained in intracellular or extracellular Ca(2+)-free solution. We conclude that a distinct Gi/o/PI-PLC/IP3/Ca(2+)-dependent PKC signaling pathway, following the activation of OX1R, is likely responsible for the orexin-A effect on GCs, whereas a Gi/o/PC-PLC/Ca(2+)-independent PKC signaling pathway, following the activation of OX2R, mediates the orexin-A effect on ACs. These two actions of orexin-A, while working in concert, provide a characteristic way for modulating information processing in the inner retina.

  11. Calcium-induced transitions between the spontaneous miniature outward and the transient outward currents in retinal amacrine cells.

    PubMed

    Mitra, Pratip; Slaughter, Malcolm M

    2002-04-01

    Spontaneous miniature outward currents (SMOCs) occur in a subset of retinal amacrine cells at membrane potentials between -60 and -40 mV. At more depolarized potentials, a transient outward current (I(to)) appears and SMOCs disappear. Both SMOCs and the I(to) are K(+) currents carried by BK channels. They both arise from Ca(2+) influx through high voltage-activated (HVA) Ca(2+) channels, which stimulates release of internal Ca(2+) from caffeine- and ryanodine-sensitive stores. An increase in Ca(2+) influx resulted in an increase in SMOC frequency, but also led to a decline in SMOC mean amplitude. This reduction showed a temporal dependence: the effect being greater in the latter part of a voltage step. Thus, Ca(2+) influx, although required to generate SMOCs, also produced a negative modulation of their amplitudes. Increasing Ca(2+) influx also led to a decline in the first latency to SMOC occurrence. A combination of these effects resulted in the disappearance of SMOCs, along with the concomitant appearance of the I(to) at high levels of Ca(2+) influx. Therefore, low levels of Ca(2+) influx, arising from low levels of activation of the HVA Ca(2+) channels, produce randomly occurring SMOCs within the range of -60 to -40 mV. Further depolarization leads to greater activation of the HVA Ca(2+) channels, larger Ca(2+) influx, and the disappearance of discontinuous SMOCs, along with the appearance of the I(to). Based on their characteristics, SMOCs in retinal neurons may function as synaptic noise suppressors at quiescent glutamatergic synapses.

  12. Retinal Wave Patterns Are Governed by Mutual Excitation among Starburst Amacrine Cells and Drive the Refinement and Maintenance of Visual Circuits

    PubMed Central

    Xu, Hong-Ping; Burbridge, Timothy J.; Ye, Meijun; Chen, Minggang; Ge, Xinxin; Zhou, Z. Jimmy

    2016-01-01

    Retinal waves are correlated bursts of spontaneous activity whose spatiotemporal patterns are critical for early activity-dependent circuit elaboration and refinement in the mammalian visual system. Three separate developmental wave epochs or stages have been described, but the mechanism(s) of pattern generation of each and their distinct roles in visual circuit development remain incompletely understood. We used neuroanatomical, in vitro and in vivo electrophysiological, and optical imaging techniques in genetically manipulated mice to examine the mechanisms of wave initiation and propagation and the role of wave patterns in visual circuit development. Through deletion of β2 subunits of nicotinic acetylcholine receptors (β2-nAChRs) selectively from starburst amacrine cells (SACs), we show that mutual excitation among SACs is critical for Stage II (cholinergic) retinal wave propagation, supporting models of wave initiation and pattern generation from within a single retinal cell type. We also demonstrate that β2-nAChRs in SACs, and normal wave patterns, are necessary for eye-specific segregation. Finally, we show that Stage III (glutamatergic) retinal waves are not themselves necessary for normal eye-specific segregation, but elimination of both Stage II and Stage III retinal waves dramatically disrupts eye-specific segregation. This suggests that persistent Stage II retinal waves can adequately compensate for Stage III retinal wave loss during the development and refinement of eye-specific segregation. These experiments confirm key features of the “recurrent network” model for retinal wave propagation and clarify the roles of Stage II and Stage III retinal wave patterns in visual circuit development. SIGNIFICANCE STATEMENT Spontaneous activity drives early mammalian circuit development, but the initiation and patterning of activity vary across development and among modalities. Cholinergic “retinal waves” are initiated in starburst amacrine cells and

  13. Retinal Wave Patterns Are Governed by Mutual Excitation among Starburst Amacrine Cells and Drive the Refinement and Maintenance of Visual Circuits.

    PubMed

    Xu, Hong-Ping; Burbridge, Timothy J; Ye, Meijun; Chen, Minggang; Ge, Xinxin; Zhou, Z Jimmy; Crair, Michael C

    2016-03-30

    Retinal waves are correlated bursts of spontaneous activity whose spatiotemporal patterns are critical for early activity-dependent circuit elaboration and refinement in the mammalian visual system. Three separate developmental wave epochs or stages have been described, but the mechanism(s) of pattern generation of each and their distinct roles in visual circuit development remain incompletely understood. We used neuroanatomical,in vitroandin vivoelectrophysiological, and optical imaging techniques in genetically manipulated mice to examine the mechanisms of wave initiation and propagation and the role of wave patterns in visual circuit development. Through deletion of β2 subunits of nicotinic acetylcholine receptors (β2-nAChRs) selectively from starburst amacrine cells (SACs), we show that mutual excitation among SACs is critical for Stage II (cholinergic) retinal wave propagation, supporting models of wave initiation and pattern generation from within a single retinal cell type. We also demonstrate that β2-nAChRs in SACs, and normal wave patterns, are necessary for eye-specific segregation. Finally, we show that Stage III (glutamatergic) retinal waves are not themselves necessary for normal eye-specific segregation, but elimination of both Stage II and Stage III retinal waves dramatically disrupts eye-specific segregation. This suggests that persistent Stage II retinal waves can adequately compensate for Stage III retinal wave loss during the development and refinement of eye-specific segregation. These experiments confirm key features of the "recurrent network" model for retinal wave propagation and clarify the roles of Stage II and Stage III retinal wave patterns in visual circuit development. Spontaneous activity drives early mammalian circuit development, but the initiation and patterning of activity vary across development and among modalities. Cholinergic "retinal waves" are initiated in starburst amacrine cells and propagate to retinal ganglion cells

  14. Double-labeling techniques demonstrate that rod bipolar cells are under GABAergic control in the inner plexiform layer of the rat retina.

    PubMed

    Kim, I B; Lee, M Y; Oh, S; Kim, K Y; Chun, M

    1998-04-01

    The synaptic connectivity between rod bipolar cells and GABAergic neurons in the inner plexiform layer (IPL) of the rat retina was studied using two immunocytochemical markers. Rod bipolar cells were stained with an antibody specific for protein kinase C (PKC, alpha isoenzyme), and GABAergic neurons were stained with an antiserum specific for glutamic-acid decarboxylase (GAD). Some amacrine cells were also labeled with the anti-PKC antiserum. All PKC-labeled amacrine cells examined showed GABA immunoreactivity, indicating that PKC-labeled amacrine cells constitute a subpopulation of GABAergic amacrine cells in the rat retina. A total of 150 ribbon synapses established by rod bipolar cells were observed in the IPL. One member of the postsynaptic dyads was always an unlabeled AII amacrine cell process, and the other belonged to an amacrine-cell process showing GAD immunoreactivity. The majority (n=92) (61.3%) of these processes made reciprocal synapses back to the axon terminals of rod bipolar cells. In addition, 78 conventional synapses onto rod bipolar axons were observed, and among them 52 (66.7%) were GAD-immunoreactive. Thus GABA provides the major inhibitory input to rod bipolar cells.

  15. AdcAII of Streptococcus pneumoniae Affects Pneumococcal Invasiveness

    PubMed Central

    Brown, Lindsey R.; Gunnell, Steven M.; Cassella, Adam N.; Keller, Lance E.; Scherkenbach, Lisa A.; Mann, Beth; Brown, Matthew W.; Hill, Rebecca; Fitzkee, Nicholas C.; Rosch, Jason W.; Tuomanen, Elaine I.; Thornton, Justin A.

    2016-01-01

    Across bacterial species, metal binding proteins can serve functions in pathogenesis in addition to regulating metal homeostasis. We have compared and contrasted the activities of zinc (Zn2+)-binding lipoproteins AdcA and AdcAII in the Streptococcus pneumoniae TIGR4 background. Exposure to Zn2+-limiting conditions resulted in delayed growth in a strain lacking AdcAII (ΔAdcAII) when compared to wild type bacteria or a mutant lacking AdcA (ΔAdcA). AdcAII failed to interact with the extracellular matrix protein laminin despite homology to laminin-binding proteins of related streptococci. Deletion of AdcA or AdcAII led to significantly increased invasion of A549 human lung epithelial cells and a trend toward increased invasion in vivo. Loss of AdcAII, but not AdcA, was shown to negatively impact early colonization of the nasopharynx. Our findings suggest that expression of AdcAII affects invasiveness of S. pneumoniae in response to available Zn2+ concentrations. PMID:26752283

  16. Involvement of brain-derived neurotrophic factor in early retinal neuropathy of streptozotocin-induced diabetes in rats: therapeutic potential of brain-derived neurotrophic factor for dopaminergic amacrine cells.

    PubMed

    Seki, Masaaki; Tanaka, Takayuki; Nawa, Hiroyuki; Usui, Tomoaki; Fukuchi, Takeo; Ikeda, Kazuhito; Abe, Haruki; Takei, Nobuyuki

    2004-09-01

    Although neurotrophins have been assessed as candidate therapeutic agents for neural complications of diabetes, their involvement in diabetic retinopathy has not been fully characterized. We found that the protein and mRNA levels of brain-derived neurotrophic factor (BDNF) in streptozotocin-induced diabetic rat retinas were reduced to 49% (P < 0.005) and 74% (P < 0.05), respectively, of those of normal control animals. In addition, dopaminergic amacrine cells appeared to be degenerating in the diabetic rat retinas, as revealed by tyrosine hydroxylase (TH) immunoreactivity. Overall TH protein levels in the retina were decreased to one-half that of controls (P < 0.01), reflecting reductions in the density of dopaminergic amacrine cells and the intensity of TH immunoreactivity within them. To confirm the neuropathological implications of BDNF reduction, we administered BDNF protein into the vitreous cavities of diabetic rats. Intraocular administration of BDNF rescued dopaminergic amacrine cells from neurodegeneration and counteracted the downregulation of TH expression, demonstrating its therapeutic potential. These findings suggest that the early retinal neuropathy of diabetes involves the reduced expression of BDNF and can be ameliorated by an exogenous supply of this neurotrophin.

  17. Conditional Knock-Out of Vesicular GABA Transporter Gene from Starburst Amacrine Cells Reveals the Contributions of Multiple Synaptic Mechanisms Underlying Direction Selectivity in the Retina

    PubMed Central

    Pei, Zhe; Chen, Qiang; Koren, David; Giammarinaro, Benno; Acaron Ledesma, Hector

    2015-01-01

    Direction selectivity of direction-selective ganglion cells (DSGCs) in the retina results from patterned excitatory and inhibitory inputs onto DSGCs during motion stimuli. The inhibitory inputs onto DSGCs are directionally tuned to the antipreferred (null) direction and therefore potently suppress spiking during motion in the null direction. However, whether direction-selective inhibition is indispensable for direction selectivity is unclear. Here, we selectively eliminated the directional tuning of inhibitory inputs onto DSGCs by disrupting GABA release from the presynaptic interneuron starburst amacrine cell in the mouse retina. We found that, even without directionally tuned inhibition, direction selectivity can still be implemented in a subset of On-Off DSGCs by direction-selective excitation and a temporal offset between excitation and isotropic inhibition. Our results therefore demonstrate the concerted action of multiple synaptic mechanisms for robust direction selectivity in the retina. SIGNIFICANCE STATEMENT The direction-selective circuit in the retina has been a classic model to study neural computations by the brain. An important but unresolved question is how direction selectivity is implemented by directionally tuned excitatory and inhibitory mechanisms. Here we specifically removed the direction tuning of inhibition from the circuit. We found that direction tuning of inhibition is important but not indispensable for direction selectivity of DSGCs' spiking activity, and that the residual direction selectivity is implemented by direction-selective excitation and temporal offset between excitation and inhibition. Our results highlight the concerted actions of synaptic excitation and inhibition required for robust direction selectivity in the retina and provide critical insights into how patterned excitation and inhibition collectively implement sensory processing. PMID:26400950

  18. Non-linear, high-gain and sustained-to-transient signal transmission from rods to amacrine cells in dark-adapted retina of Ambystoma

    PubMed Central

    Yang, Xiong-Li; Gao, Fan; Wu, Samuel M

    2002-01-01

    In darkness, On-Off amacrine cells (ACs) of the tiger salamander retina exhibited large spontaneous transient depolarizing potentials (sTDPs) with average peak amplitude of 5.05 ± 2.5 mV and average frequency of 0.42 ± 0.25 s−1. Under voltage-clamp conditions the cell displayed large spontaneous postsynaptic currents (sPSCs) with average peak amplitude of 98 ± 39 pA and average frequency of 0.45 ± 0.22 s−1. To a light step, ACs gave rise to a transient ‘On’ response at the light onset and a transient ‘Off’ response at light offset, followed by a train of TDPs (‘After’ response). Near the response threshold (0.3 activated rhodopsin molecules per rod per second), light-evoked TDPs (leTDPs) of similar amplitude and kinetics as the large sTDPs observed in darkness were seen, and about half of these leTDPs elicited a regenerative potential (RP). Brighter light steps gave rise to more leTDPs and higher rates of RPs in the On, Off and After responses. Within the linear response range of the rods, the AC response was non-linear, with the highest gain (676 ± 429) near the dark potential. The amplitude of Off responses increased with the duration of the light step, and ACs may use this to encode speeds of moving stimuli: the faster the light object moves, the smaller the AC Off response. Moreover, the number of leTDPs in the AC After response increased with light intensity, and the onset of the After response coincides with bipolar cell tail response recovery. One possible origin of the large sTDPs and leTDPs is the spontaneous and depolarization-induced regenerative calcium potentials (RCaPs) in bipolar cell synaptic terminals. RCaPs in bipolar cell synaptic terminals cause transient glutamate release that results in the sTDPs in darkness, and leTDPs in On, Off and After responses in ACs. PMID:11850516

  19. Apolipoprotein A-II Plus Lipid Emulsion Enhance Cell Growth via SR-B1 and Target Pancreatic Cancer In Vitro and In Vivo

    PubMed Central

    Thanh LE, Thao N.; Gill, Anthony J.; Bulanadi, Jerikho C.; Patel, Mili; Waddington, Lynne J.; Rye, Kerry-Anne; Moghaddam, Minoo J.; Smith, Ross C.

    2016-01-01

    Background Apolipoprotein A-II (ApoA-II) is down regulated in the sera of pancreatic ductal adenocarcinoma (PDAC) patients, which may be due to increase utilization of high density lipoprotein (HDL) lipid by pancreatic cancer tissue. This study examined the influence of exogenous ApoA-II on lipid uptake and cell growth in pancreatic cancer (PC) both in vitro and in vivo. Methods Cryo transmission electron microscopy (TEM) examined ApoA-II’s influence on morphology of SMOFLipid emulsion. The influence of ApoA-II on proliferation of cancer cell lines was determined by incubating them with lipid+/-ApoA-II and anti-SR-B1 antibody. Lipid was labeled with the fluorophore, DiD, to trace lipid uptake by cancer cells in vitro by confocal microscopy and in vivo in PDAC patient derived xenograft tumours (PDXT) by fluorescence imaging. Scavenger receptor class B type-1(SR-B1) expression in PDAC cell lines and in PDAC PDXT was measured by western blotting and immunohistochemistry, respectively. Results ApoA-II spontaneously converted lipid emulsion into very small unilamellar rHDL like vesicles (rHDL/A-II) and enhanced lipid uptake in PANC-1, CFPAC-1 and primary tumour cells as shown by confocal microscopy. SR-B1 expression was 13.2, 10.6, 3.1 and 2.3 fold higher in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cell lines than the normal pancreatic cell line (HPDE6) and 3.7 fold greater in PDAC tissue than in normal pancreas. ApoA-II plus lipid significantly increased the uptake of labeled lipid and promoted cell growth in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cells which was inhibited by anti SR-B1 antibody. Further, ApoA-II increased the uptake of lipid in xenografts by 3.4 fold. Conclusion Our data suggest that ApoA-II enhance targeting potential of lipid in pancreatic cancer which may have imaging and drug delivery potentialities. PMID:27002321

  20. Apolipoprotein A-II Plus Lipid Emulsion Enhance Cell Growth via SR-B1 and Target Pancreatic Cancer In Vitro and In Vivo.

    PubMed

    Julovi, Sohel M; Xue, Aiqun; Thanh LE, Thao N; Gill, Anthony J; Bulanadi, Jerikho C; Patel, Mili; Waddington, Lynne J; Rye, Kerry-Anne; Moghaddam, Minoo J; Smith, Ross C

    2016-01-01

    Apolipoprotein A-II (ApoA-II) is down regulated in the sera of pancreatic ductal adenocarcinoma (PDAC) patients, which may be due to increase utilization of high density lipoprotein (HDL) lipid by pancreatic cancer tissue. This study examined the influence of exogenous ApoA-II on lipid uptake and cell growth in pancreatic cancer (PC) both in vitro and in vivo. Cryo transmission electron microscopy (TEM) examined ApoA-II's influence on morphology of SMOFLipid emulsion. The influence of ApoA-II on proliferation of cancer cell lines was determined by incubating them with lipid+/-ApoA-II and anti-SR-B1 antibody. Lipid was labeled with the fluorophore, DiD, to trace lipid uptake by cancer cells in vitro by confocal microscopy and in vivo in PDAC patient derived xenograft tumours (PDXT) by fluorescence imaging. Scavenger receptor class B type-1(SR-B1) expression in PDAC cell lines and in PDAC PDXT was measured by western blotting and immunohistochemistry, respectively. ApoA-II spontaneously converted lipid emulsion into very small unilamellar rHDL like vesicles (rHDL/A-II) and enhanced lipid uptake in PANC-1, CFPAC-1 and primary tumour cells as shown by confocal microscopy. SR-B1 expression was 13.2, 10.6, 3.1 and 2.3 fold higher in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cell lines than the normal pancreatic cell line (HPDE6) and 3.7 fold greater in PDAC tissue than in normal pancreas. ApoA-II plus lipid significantly increased the uptake of labeled lipid and promoted cell growth in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cells which was inhibited by anti SR-B1 antibody. Further, ApoA-II increased the uptake of lipid in xenografts by 3.4 fold. Our data suggest that ApoA-II enhance targeting potential of lipid in pancreatic cancer which may have imaging and drug delivery potentialities.

  1. Serotonin released from amacrine neurons is scavenged and degraded in bipolar neurons in the retina

    PubMed Central

    Ghai, Kanika; Zelinka, Christopher; Fischer, Andy J.

    2009-01-01

    The neurotransmitter serotonin is synthesized in the retina by one type of amacrine neuron but accumulates in bipolar neurons in many vertebrates. The mechanisms, functions and purpose underlying of serotonin in bipolar cells remain unknown. Here, we demonstrate that exogenous serotonin transiently accumulates in a distinct type of bipolar neuron. KCl-mediated depolarization causes the depletion of serotonin from amacrine neurons and, subsequently, serotonin is taken-up by bipolar neurons. The accumulation of endogenous or exogenous serotonin by bipolar neurons is blocked by selective reuptake inhibitors. Exogenous serotonin is specifically taken-up by bipolar neurons even when serotonin-synthesizing amacrine neurons are destroyed; excluding the possibility that serotonin diffuses through gap junctions from amacrine into bipolar neurons. Further, inhibition of monoamine oxidase (A) prevents the degradation of serotonin in bipolar neurons, suggesting that MAO(A) is present in these neurons. However, the vesicular monoamine transporter (VMAT2) is present only in amacrine cells suggesting that serotonin is not transported into synaptic vesicles and re-used as a transmitter in the bipolar neurons. We conclude that the serotonin-accumulating bipolar neurons perform glial functions in the retina by actively transporting and degrading serotonin that is synthesized in neighboring amacrine cells. PMID:19619137

  2. Dark-adapted response threshold of OFF ganglion cells is not set by OFF bipolar cells in the mouse retina

    PubMed Central

    Arman, A. Cyrus

    2012-01-01

    The nervous system frequently integrates parallel streams of information to encode a broad range of stimulus strengths. In mammalian retina it is generally believed that signals generated by rod and cone photoreceptors converge onto cone bipolar cells prior to reaching the retinal output, the ganglion cells. Near absolute visual threshold a specialized mammalian retinal circuit, the rod bipolar pathway, pools signals from many rods and converges on depolarizing (AII) amacrine cells. However, whether subsequent signal flow to OFF ganglion cells requires OFF cone bipolar cells near visual threshold remains unclear. Glycinergic synapses between AII amacrine cells and OFF cone bipolar cells are believed to relay subsequently rod-driven signals to OFF ganglion cells. However, AII amacrine cells also make glycinergic synapses directly with OFF ganglion cells. To determine the route for signal flow near visual threshold, we measured the effect of the glycine receptor antagonist strychnine on response threshold in fully dark-adapted retinal cells. As shown previously, we found that response threshold for OFF ganglion cells was elevated by strychnine. Surprisingly, strychnine did not elevate response threshold in any subclass of OFF cone bipolar cell. Instead, in every OFF cone bipolar subclass strychnine suppressed tonic glycinergic inhibition without altering response threshold. Consistent with this lack of influence of strychnine, we found that the dominant input to OFF cone bipolar cells in darkness was excitatory and the response threshold of the excitatory input varied by subclass. Thus, in the dark-adapted mouse retina, the high absolute sensitivity of OFF ganglion cells cannot be explained by signal transmission through OFF cone bipolar cells. PMID:22338022

  3. Dark-adapted response threshold of OFF ganglion cells is not set by OFF bipolar cells in the mouse retina.

    PubMed

    Arman, A Cyrus; Sampath, Alapakkam P

    2012-05-01

    The nervous system frequently integrates parallel streams of information to encode a broad range of stimulus strengths. In mammalian retina it is generally believed that signals generated by rod and cone photoreceptors converge onto cone bipolar cells prior to reaching the retinal output, the ganglion cells. Near absolute visual threshold a specialized mammalian retinal circuit, the rod bipolar pathway, pools signals from many rods and converges on depolarizing (AII) amacrine cells. However, whether subsequent signal flow to OFF ganglion cells requires OFF cone bipolar cells near visual threshold remains unclear. Glycinergic synapses between AII amacrine cells and OFF cone bipolar cells are believed to relay subsequently rod-driven signals to OFF ganglion cells. However, AII amacrine cells also make glycinergic synapses directly with OFF ganglion cells. To determine the route for signal flow near visual threshold, we measured the effect of the glycine receptor antagonist strychnine on response threshold in fully dark-adapted retinal cells. As shown previously, we found that response threshold for OFF ganglion cells was elevated by strychnine. Surprisingly, strychnine did not elevate response threshold in any subclass of OFF cone bipolar cell. Instead, in every OFF cone bipolar subclass strychnine suppressed tonic glycinergic inhibition without altering response threshold. Consistent with this lack of influence of strychnine, we found that the dominant input to OFF cone bipolar cells in darkness was excitatory and the response threshold of the excitatory input varied by subclass. Thus, in the dark-adapted mouse retina, the high absolute sensitivity of OFF ganglion cells cannot be explained by signal transmission through OFF cone bipolar cells.

  4. Overexpression of apolipoprotein AII in transgenic mice converts high density lipoproteins to proinflammatory particles.

    PubMed Central

    Castellani, L W; Navab, M; Van Lenten, B J; Hedrick, C C; Hama, S Y; Goto, A M; Fogelman, A M; Lusis, A J

    1997-01-01

    Previous studies showed that transgenic mice overexpressing either apolipoprotein AI (apoAI) or apolipoprotein AII (apoAII), the major proteins of HDL, exhibited elevated levels of HDL cholesterol, but, whereas the apoAI-transgenic mice were protected against atherosclerosis, the apoAII-transgenic mice had increased lesion development. We now examine the basis for this striking functional heterogeneity. HDL from apoAI transgenics exhibited an enhanced ability to promote cholesterol efflux from macrophages, but HDL from apoAII transgenics and nontransgenics were not discernibly different in efflux studies. In contrast with HDL from nontransgenics and apoAI transgenics, HDL from the apoAII transgenics were unable to protect against LDL oxidation in a coculture model of the artery wall. Furthermore, HDL taken from apoAII-transgenic mice, but not HDL taken from either the apoAI transgenics or nontransgenic littermate controls, by itself stimulated lipid hydroperoxide formation in artery wall cells and induced monocyte transmigration, indicating that the apoAII-transgenic HDL were in fact proinflammatory. This loss in the ability of the apoAII-transgenic HDL to function as an antioxidant/antiinflammatory agent was associated with a decreased content of paraoxonase, an enzyme that protects against LDL oxidation. Reconstitution of the apoAII transgenic HDL with purified paraoxonase restored both paraoxonase activity and the ability to protect against LDL oxidation. We conclude that overexpression of apoAII converts HDL from an anti- to a proinflammatory particle and that paraoxonase plays a role in this transformation. PMID:9218525

  5. Interaction of apolipoprotein AII with the putative high-density lipoprotein receptor.

    PubMed

    Vadiveloo, P K; Allan, C M; Murray, B J; Fidge, N H

    1993-09-14

    There is strong evidence to indicate that binding of HDL by cells is due to recognition of apoproteins residing on the surface of the lipoprotein by the putative HDL receptor(s). Although both of the major HDL apoproteins, AI and AII, are recognized by the putative receptor, the nature of the binding interaction and the domains of the apoproteins involved are largely unknown. Previous data from this laboratory led to the proposal of a model to explain how HDL particles containing AII interacted with the HDL receptor in a different manner as compared to HDL particles which contain apoAI but not apoAII [Vadiveloo, P. K., & Fidge, N. H. (1992) Biochem. J. 284, 145-151]. The model predicted that each chain of the apoAII homodimer contained a binding domain capable of interacting with the HDL receptor. This model was tested in the current study by preparing apoAII monomers, complexing them with phospholipid, and determining the ability of these complexes to bind to putative HDL receptors in rat liver plasma membranes (RLPM) and bovine aortic endothelial cell membranes (BAECM) by ligand blotting. The data showed that these complexes were bound by HB1 and HB2 from RLPM, and to the 110-kDa HDL binding protein from BAECM, providing critical evidence to support the model. Further investigation into the binding interaction revealed that apoAII complexed with phospholipid (apoAII-PC) bound more than delipidated apoAII, which bound more than delipidated apoAII monomers. Thus, optimum binding required the presence of lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Zinc uptake by Streptococcus pneumoniae depends on both AdcA and AdcAII and is essential for normal bacterial morphology and virulence.

    PubMed

    Bayle, Lucie; Chimalapati, Suneeta; Schoehn, Guy; Brown, Jeremy; Vernet, Thierry; Durmort, Claire

    2011-11-01

    Zinc is an essential trace metal for living cells. The ABC transporter AdcABC was previously shown to be required for zinc uptake by Streptococcus pneumoniae. As we have recently described AdcAII as another zinc-binding lipoprotein, we have investigated the role of both AdcA and AdcAII in S. pneumoniae zinc metabolism. Deletion of either adcA or adcAII but not phtD reduced S. pneumoniae zinc uptake, with dual mutation of both adcA and adcAII further decreasing zinc import. For the Δ(adcA/adcAII) mutant, growth and intracellular concentrations of zinc were both greatly reduced in low zinc concentration. When grown in zinc-deficient medium, the Δ(adcA/adcAII) mutant displayed morphological defects related to aberrant septation. Growth and morphology of the Δ(adcA/adcAII) mutant recovered after supplementation with zinc. Dual deletion of adcA and adcAII strongly impaired growth of the pneumococcus in bronchoalveolar lavage fluid and human serum, and prevented S. pneumoniae establishing infection in mouse models of nasopharyngeal colonization, pneumonia and sepsis without altering the capsule. Taken together, our results show that AdcA and AdcAII play an essential and redundant role in specifically importing zinc into the pneumococcus, and that both zinc transporters are required for proper cell division and for S. pneumoniae survival during infection. © 2011 Blackwell Publishing Ltd.

  7. Adaptation to background light enables contrast coding at rod bipolar cell synapses

    PubMed Central

    Ke, Jiang-Bin; Wang, Yanbin V.; Borghuis, Bart G.; Cembrowski, Mark S.; Riecke, Hermann; Kath, William L.; Demb, Jonathan B.; Singer, Joshua H.

    2013-01-01

    SUMMARY Rod photoreceptors contribute to vision over a ~6 log-unit range of light intensities. The wide dynamic range of rod vision is thought to depend upon light intensity-dependent switching between two parallel pathways linking rods to ganglion cells: a rod→rod bipolar (RB) cell pathway that operates at dim backgrounds and a rod→cone→cone bipolar cell pathway that operates at brighter backgrounds. We evaluated this conventional model of rod vision by recording rod-mediated light responses from ganglion and AII amacrine cells and by recording RB-mediated synaptic currents from AII amacrine cells in mouse retina. Contrary to the conventional model, we found that the RB pathway functioned at backgrounds sufficient to activate the rod→cone pathway. As background light intensity increased, the RB’s role changed from encoding the absorption of single photons to encoding contrast modulations around mean luminance. This transition is explained by the intrinsic dynamics of transmission from RB synapses. PMID:24373883

  8. Specific binding of Synechococcus sp. strain PCC 7942 proteins to the enhancer element of psbAII required for high-light-induced expression.

    PubMed Central

    Li, R; Dickerson, N S; Mueller, U W; Golden, S S

    1995-01-01

    The psbAII gene of the cyanobacterium Synechococcus sp. strain PCC 7942 is a member of a three-gene family that encodes the D1 protein of the photosystem II reaction center. Transcription of psbAII is rapidly induced when the light intensity reaching the culture increases from 125 microE.m-2.s-1 (low light) to 750 microE.m-2.s-1 (high light). The DNA segment upstream of psbAII that corresponds to the untranslated leader of its major transcript has enhancer activity and confers high-light induction. We show that one or more soluble proteins from PCC 7942 specifically bind to this region of psbAII (designated the enhancer element). In vivo footprinting showed protein binding to the enhancer element in high-light-exposed cell samples but not in those maintained at low light, even though in vitro mobility shifts were detectable with extracts from low- or high-light-grown cells. When 12 bp were deleted from the psbAII enhancer element, protein binding was impaired and high-light induction of both transcriptional and translational psbAII-lacZ reporters was significantly reduced. This finding indicates that protein binding to this region is required for high-light induction of psbAII. The mutant element also showed impaired enhancer activity when combined with a heterologous promoter. PMID:7836280

  9. Bipolar cell gap junctions serve major signaling pathways in the human retina.

    PubMed

    Kántor, Orsolya; Varga, Alexandra; Nitschke, Roland; Naumann, Angela; Énzsöly, Anna; Lukáts, Ákos; Szabó, Arnold; Németh, János; Völgyi, Béla

    2017-08-01

    Connexin36 (Cx36) constituent gap junctions (GJ) throughout the brain connect neurons into functional syncytia. In the retina they underlie the transmission, averaging and correlation of signals prior conveying visual information to the brain. This is the first study that describes retinal bipolar cell (BC) GJs in the human inner retina, whose function is enigmatic even in the examined animal models. Furthermore, a number of unique features (e.g. fovea, trichromacy, midget system) necessitate a reexamination of the animal model results in the human retina. Well-preserved postmortem human samples of this study are allowed to identify Cx36 expressing BCs neurochemically. Results reveal that both rod and cone pathway interneurons display strong Cx36 expression. Rod BC inputs to AII amacrine cells (AC) appear in juxtaposition to AII GJs, thus suggesting a strategic AII cell targeting by rod BCs. Cone BCs serving midget, parasol or koniocellular signaling pathways display a wealth of Cx36 expression to form homologously coupled arrays. In addition, they also establish heterologous GJ contacts to serve an exchange of information between parallel signaling streams. Interestingly, a prominent Cx36 expression was exhibited by midget system BCs that appear to maintain intimate contacts with bistratified BCs serving other pathways. These findings suggest that BC GJs in parallel signaling streams serve both an intra- and inter-pathway exchange of signals in the human retina.

  10. Dopamine D1 receptor expression is bipolar cell type-specific in the mouse retina.

    PubMed

    Farshi, Pershang; Fyk-Kolodziej, Bozena; Krolewski, David M; Walker, Paul D; Ichinose, Tomomi

    2016-07-01

    In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a-tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type-dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5-2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5-1, 9, and rod bipolar cells did not express Drd1a-tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion-coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type-dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059-2079, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  11. Anterior Inferior Iliac Spine (AIIS) and Subspine Hip Impingement.

    PubMed

    Carton, Patrick; Filan, David

    2016-01-01

    Abnormal morphology of the anterior inferior iliac spine (AIIS) and the subspine region of the acetabular rim are increasingly being recognised as a source of symptomatic extra-articular hip impingement. This review article aims to highlight important differences in the pathogenesis, clinical presentation and management of extra-articular hip impingement from both the AIIS and subspine bony regions, and the outcome following surgical intervention. A literature review was undertaken to examine the supporting evidence for AIIS and subspine hip impingement. A narrative account of the Author's professional experience in this area, including operative technique for arthroscopic correction, is also presented. Abnormal morphology of the AIIS and subspine region has been classified using cadaveric, radiological and arthroscopic means; the clinical presentation and operative treatment has been documented in several case series studies. Dual pathology is often present - recognition and treatment of both intra- and extra-articular components are necessary for good postoperative outcome. AIIS and sub-spine hip impingement should be considered as distinct pathological entities, which may also co-exist. Symptom relief can be expected following arthroscopic deformity correction with the treatment of concomitant intra-articular pathology. Failure to recognise and treat the extra-articular component may affect postoperative outcome. V.

  12. Anterior Inferior Iliac Spine (AIIS) and Subspine Hip Impingement

    PubMed Central

    Carton, Patrick; Filan, David

    2016-01-01

    Summary Background Abnormal morphology of the anterior inferior iliac spine (AIIS) and the subspine region of the acetabular rim are increasingly being recognised as a source of symptomatic extra-articular hip impingement. This review article aims to highlight important differences in the pathogenesis, clinical presentation and management of extra-articular hip impingement from both the AIIS and subspine bony regions, and the outcome following surgical intervention. Methods A literature review was undertaken to examine the supporting evidence for AIIS and subspine hip impingement. A narrative account of the Author’s professional experience in this area, including operative technique for arthroscopic correction, is also presented. Results Abnormal morphology of the AIIS and subspine region has been classified using cadaveric, radiological and arthroscopic means; the clinical presentation and operative treatment has been documented in several case series studies. Dual pathology is often present - recognition and treatment of both intra- and extra-articular components are necessary for good postoperative outcome. Conclusions AIIS and sub-spine hip impingement should be considered as distinct pathological entities, which may also co-exist. Symptom relief can be expected following arthroscopic deformity correction with the treatment of concomitant intra-articular pathology. Failure to recognise and treat the extra-articular component may affect postoperative outcome. Level of evidence V. PMID:28066737

  13. Scavenger receptor B1 (SR-B1) profoundly excludes high density lipoprotein (HDL) apolipoprotein AII as it nibbles HDL-cholesteryl ester.

    PubMed

    Gillard, Baiba K; Bassett, G Randall; Gotto, Antonio M; Rosales, Corina; Pownall, Henry J

    2017-05-26

    Reverse cholesterol transport (transfer of macrophage-cholesterol in the subendothelial space of the arterial wall to the liver) is terminated by selective high density lipoprotein (HDL)-cholesteryl ester (CE) uptake, mediated by scavenger receptor class B, type 1 (SR-B1). We tested the validity of two models for this process: "gobbling," i.e. one-step transfer of all HDL-CE to the cell and "nibbling," multiple successive cycles of SR-B1-HDL association during which a few CEs transfer to the cell. Concurrently, we compared cellular uptake of apoAI with that of apoAII, which is more lipophilic than apoAI, using HDL-[(3)H]CE labeled with [(125)I]apoAI or [(125)I]apoAII. The studies were conducted in CHO-K1 and CHO-ldlA7 cells (LDLR(-/-)) with (CHO-SR-B1) and without SR-B1 overexpression and in human Huh7 hepatocytes. Relative to CE, both apoAI and apoAII were excluded from uptake by all cells. However, apoAII was more highly excluded from uptake (2-4×) than apoAI. To distinguish gobbling versus nibbling mechanisms, media from incubations of HDL with CHO-SR-B1 cells were analyzed by non-denaturing PAGE, size-exclusion chromatography, and the distribution of apoAI, apoAII, cholesterol, and phospholipid among HDL species as a function of incubation time. HDL size gradually decreased, i.e. nibbling, with the concurrent release of lipid-free apoAI; apoAII was retained in an HDL remnant. Our data support an SR-B1 nibbling mechanism that is similar to that of streptococcal serum opacity factor, which also selectively removes CE and releases apoAI, leaving an apoAII-rich remnant. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. N-type and L-type calcium channels mediate glycinergic synaptic inputs to retinal ganglion cells of tiger salamanders.

    PubMed

    Bieda, Mark C; Copenhagen, David R

    2004-01-01

    Synaptically localized calcium channels shape the timecourse of synaptic release, are a prominent site for neuromodulation, and have been implicated in genetic disease. In retina, it is well established that L-type calcium channels play a major role in mediating release of glutamate from the photoreceptors and bipolar cells. However, little is known about which calcium channels are coupled to synaptic exocytosis of glycine, which is primarily released by amacrine cells. A recent report indicates that glycine release from spiking AII amacrine cells relies exclusively upon L-type calcium channels. To identify calcium channel types controlling neurotransmitter release from the population of glycinergic neurons that drive retinal ganglion cells, we recorded electrical and potassium evoked inhibitory synaptic currents (IPSCs) from these postsynaptic neurons in retinal slices from tiger salamanders. The L-channel antagonist nifedipine strongly inhibited release and FPL64176, an L-channel agonist, greatly enhanced it, indicating a significant role for L-channels. omega-Conotoxin MVIIC, an N/P/Q-channel antagonist, strongly inhibited release, indicating an important role for non-L channels. While the P/Q-channel blocker omega-Aga IVA produced only small effects, the N-channel blocker omega-conotoxin GVIA strongly inhibited release. Hence, N-type and L-type calcium channels appear to play major roles, overall, in mediating synaptic release of glycine onto retinal ganglion cells.

  15. A splice-junction mutation responsible for familial apolipoprotein A-II deficiency.

    PubMed Central

    Deeb, S S; Takata, K; Peng, R L; Kajiyama, G; Albers, J J

    1990-01-01

    The first case of familial apolipoprotein A-II (apo A-II) deficiency was recently reported from Hiroshima, Japan, and designated apo A-IIHiroshima. The proband had no immunologically detectable apo A-II in her plasma. DNA sequence analysis showed that the proband was homozygous for a G----A transition at position 1 of intron 3 of the apo A-II gene. A sister of the proband, who had an intermediate level of plasma apo AII, was shown to be heterozygous for this base substitution. This splice-junction alteration is most likely responsible for apo A-II deficiency, since it would be expected to completely block splicing of intron 3 from the primary transcript and therefore prevent formation of functional mRNA. This deficiency seems to have little influence either on lipid and lipoprotein profiles or on the occurrence of coronary artery disease. Images Figure 2 PMID:2107739

  16. In vivo expression of Nurr1/Nr4a2a in developing retinal amacrine subtypes in zebrafish Tg(nr4a2a:eGFP) transgenics.

    PubMed

    Goodings, Liana; He, Jie; Wood, Alasdair J; Harris, William A; Currie, Peter D; Jusuf, Patricia R

    2017-06-01

    The Nuclear receptor subfamily 4 group A member 2 (Nr4a2) is crucial for the formation or maintenance of dopaminergic neurons in the central nervous system including the retina, where dopaminergic amacrine cells contribute to visual function. Little is known about which cells express Nr4a2 at which developmental stage. Furthermore, whether Nr4a2 functions in combination with other genes is poorly understood. Thus, we generated a novel transgenic to visualize Nr4a2 expression in vivo during zebrafish retinogenesis. A 4.1 kb fragment of the nr4a2a promoter was used to drive green fluorescent protein expression in this Tg(nr4a2a:eGFP) line. In situ hybridization showed that transgene expression follows endogenous RNA expression at a cellular level. Temporal expression and lineages were quantified using in vivo time-lapse imaging in embryos. Nr4a2 expressing retinal subtypes were characterized immunohistochemically. Nr4a2a:eGFP labeled multiple neuron subtypes including 24.5% of all amacrine interneurons. Nr4a2a:eGFP labels all tyrosine hydroxylase labeled dopaminergic amacrine cells, and other nondopaminergic GABAergic amacrine populations. Nr4a2a:eGFP is confined to a specific progenitor lineage identified by sequential expression of the bhlh transcription factor Atonal7 (Atoh7) and Pancreas transcription factor 1a (Ptf1a), and labels postmitotic postmigratory amacrine cells. Thus, developmental Nr4a2a expression indicates a role during late differentiation of specific amacrine interneurons. Tg(nr4a2a:eGFP) is an early marker of distinct neurons including dopaminergic amacrine cells. It can be utilized to assess consequences of gene manipulations and understand whether Nr4a2 only carries out its role in the presence of specific coexpressed genes. This will allow Nr4a2 use to be refined for regenerative approaches. © 2017 Wiley Periodicals, Inc.

  17. Apolipoprotein A-I, A-II, and H mRNA and protein accumulation sites in the developing lung in late gestation

    PubMed Central

    2011-01-01

    Background Expression of apolipoprotein A-I (apoA-I), A-II, and H was previously observed at 16 to 50-fold higher levels in the fetal than the adult mouse lung. Here, sites of apoA-I, A-II, and H mRNA and protein accumulation were determined in mouse fetal lungs by in situ hybridization and immunohistochemistry in late gestation. Results Expression sites vary for the three genes and change for the distal epithelium before the end of the canalicular stage, thus where and when the surge of surfactant synthesis occurs. Messenger of apoH, but not those of apoA-I and A-II, was also observed in the proximal epithelium and smooth muscles surrounding arteries. In contrast to apoC-II protein, none of the three studied apolipoproteins accumulated within secretory granule-like structures. Immunohistochemistry revealed that apoA-I and apoH accumulated mainly in capillaries. Three different positive signals with the anti-apoA-II antibody were found: one transient signal in the nucleus of a portion of mesenchymal cells, a second at lower levels throughout the mesenchyme, and another in capillaries with a specific increase from gestation day 17.5/18.5. Conclusion Temporal and geographic co-expression of apoAI, AII, and H genes with surfactant production site suggests that the three apolipoproteins are secreted to play roles supporting the lung-specific surfactant lipid-related metabolism. PMID:21756353

  18. Apolipoprotein AII Is a Regulator of Very Low Density Lipoprotein Metabolism and Insulin Resistance*

    PubMed Central

    Castellani, Lawrence W.; Nguyen, Cara N.; Charugundla, Sarada; Weinstein, Michael M.; Doan, Chau X.; Blaner, William S.; Wongsiriroj, Nuttaporn; Lusis, Aldons J.

    2008-01-01

    Apolipoprotein AII (apoAII) transgenic (apoAIItg) mice exhibit several traits associated with the insulin resistance (IR) syndrome, including IR, obesity, and a marked hypertriglyceridemia. Because treatment of the apoAIItg mice with rosiglitazone ameliorated the IR and hypertriglyceridemia, we hypothesized that the hypertriglyceridemia was due largely to overproduction of very low density lipoprotein (VLDL) by the liver, a normal response to chronically elevated insulin and glucose. We now report in vivo and in vitro studies that indicate that hepatic fatty acid oxidation was reduced and lipogenesis increased, resulting in a 25% increase in triglyceride secretion in the apoAIItg mice. In addition, we observed that hydrolysis of triglycerides from both chylomicrons and VLDL was significantly reduced in the apoAIItg mice, further contributing to the hypertriglyceridemia. This is a direct, acute effect, because when mouse apoAII was injected into mice, plasma triglyceride concentrations were significantly increased within 4 h. VLDL from both control and apoAIItg mice contained significant amounts of apoAII, with ∼4 times more apoAII on apoAIItg VLDL. ApoAII was shown to transfer spontaneously from high density lipoprotein (HDL) to VLDL in vitro, resulting in VLDL that was a poorer substrate for hydrolysis by lipoprotein lipase. These results indicate that one function of apoAII is to regulate the metabolism of triglyceride-rich lipoproteins, with HDL serving as a plasma reservoir of apoAII that is transferred to the triglyceride-rich lipoproteins in much the same way as VLDL and chylomicrons acquire most of their apoCs from HDL. PMID:18160395

  19. Synaptic pathways that shape the excitatory drive in an OFF retinal ganglion cell.

    PubMed

    Buldyrev, Ilya; Puthussery, Theresa; Taylor, W Rowland

    2012-04-01

    Different types of retinal ganglion cells represent distinct spatiotemporal filters that respond selectively to specific features in the visual input. Much about the circuitry and synaptic mechanisms that underlie such specificity remains to be determined. This study examines how N-methyl-d-aspartate (NMDA) receptor signaling combines with other excitatory and inhibitory mechanisms to shape the output of small-field OFF brisk-sustained ganglion cells (OFF-BSGCs) in the rabbit retina. We used voltage clamp to separately resolve NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and inhibitory inputs elicited by stimulation of the receptive field center. Three converging circuits were identified. First is a direct glutamatergic input, arising from OFF cone bipolar cells (CBCs), which is mediated by synaptic NMDA and AMPA receptors. The NMDA input was saturated at 10% contrast, whereas the AMPA input increased monotonically up to 60% contrast. We propose that NMDA inputs selectively enhance sensitivity to low contrasts. The OFF bipolar cells, mediating this direct excitatory input, express dendritic kainate (KA) receptors, which are resistant to the nonselective AMPA/KA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX), but are suppressed by a GluK1- and GluK3-selective antagonist, (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxy-thiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione (UBP-310). The second circuit entails glycinergic crossover inhibition, arising from ON-CBCs and mediated by AII amacrine cells, which modulate glutamate release from the OFF-CBC terminals. The third circuit also comprises glycinergic crossover inhibition, which is driven by the ON pathway; however, this inhibition impinges directly on the OFF-BSGCs and is mediated by an unknown glycinergic amacrine cell that expresses AMPA but not KA receptors.

  20. Angiotensin II receptors labelled with 125I-[Sar1, Ile8]-AII in albino rabbit ocular tissues.

    PubMed

    Mallorga, P; Babilon, R W; Sugrue, M F

    1989-08-01

    High affinity binding sites for the angiotensin II antagonist 125I-[Sar1,Ile8]-AII have been identified and characterized in membrane suspensions of ocular tissues of albino rabbits. Scatchard analysis of the binding indicated a single class of sites with Kd values of 186, 92, 152, 50, 102 pM for the iris + ciliary body, choroid, ciliary process, retina and cornea, respectively. The corresponding concentrations of binding sites were 22, 68, 35, 22 and 4 fmole/mg of protein. The order of potency for several AII analogs to compete with 125I-[Sar1,Ile8]-AII at its binding sites in iris + ciliary body membranes ([Sar1,Leu8]-AII = [Sar1,Ile8]-AII greater than AII = [Sar1, Ala8]-AII greater than AIII greater than AI) resembled the order of potency found for AII receptors in other tissues. The competition curves for this tissue using AII and AIII were best explained by the existence of two populations of binding sites. The addition of the guanine nucleotide, GppNHp, to the assay resulted in a 6.7-fold and 2.3-fold decrease in the respective affinities of AII and AIII for 125I-[Sar1,Ile8]-AII binding sites without a change in the slope of the competition curves. The GppNHp-induced effect was also observed in ciliary process membranes but not in retinal or choroidal membranes. These results indicate the presence of AII receptors regulated by a GTP-binding protein in both the ciliary process and the iris + ciliary body of the rabbit. They also suggest a difference in the guanine nucleotide regulation of AII receptors in different ocular tissues.

  1. Components and properties of the G3 ganglion cell circuit in the rabbit retina.

    PubMed

    Hoshi, Hideo; Mills, Stephen L

    2009-03-01

    Each point on the retina is sampled by about 15 types of ganglion cell, each of which is an element in a circuit also containing specific types of bipolar cell and amacrine cell. Only a few of these circuits are well characterized. We found that intracellular injection of Neurobiotin into a specific ganglion cell type targeted by fluorescent markers also stained an asymmetrically branching ganglion cell. It was also tracer-coupled to an unusual type of amacrine cell whose dendrites were strongly asymmetric, coursing in a narrow bundle from the soma in the dorsal direction only. The dendritic field of the ganglion cell stratifies initially in sublamina b (the ON layers), but with few specializations and branches, and then more extensively in sublamina a (the OFF layers) at the level of the processes of the coupled amacrine cell. Intersections of the ganglion and amacrine cell processes contain puncta immunopositive for Cx36. Additionally, we found that the dopaminergic amacrine cell makes contact with both the ganglion cell and the amacrine cell, and that a bipolar cell immunopositive for calbindin synapses onto the sublamina b processes of the ganglion cell. Dopamine D(1) receptor activation reduced tracer flow to the amacrine cells. We have thus targeted and characterized two poorly understood retinal cell types and placed them with two other cell types in a substantial portion of a new retinal circuit. This unique circuit comprised of pronounced asymmetries in the ganglion cell and amacrine cell dendritic fields may result in a substantial orientation bias.

  2. Caloric restriction reduces the systemic progression of mouse AApoAII amyloidosis

    PubMed Central

    Ding, Xin; Yang, Mu; Xu, Zhe; Miyahara, Hiroki; Mori, Masayuki; Higuchi, Keiichi

    2017-01-01

    In mouse senile amyloidosis, apolipoprotein (Apo) A-II is deposited extracellularly in many organs in the form of amyloid fibrils (AApoAII). Reduction of caloric intake, known as caloric restriction (CR), slows the progress of senescence and age-related disorders in mice. In this study, we intravenously injected 1 μg of isolated AApoAII fibrils into R1.P1-Apoa2c mice to induce experimental amyloidosis and investigated the effects of CR for the next 16 weeks. In the CR group, AApoAII amyloid deposits in the liver, tongue, small intestine and skin were significantly reduced compared to those of the ad libitum feeding group. CR treatment led to obvious reduction in body weight, improvement in glucose metabolism and reduction in the plasma concentration of ApoA-II. Our molecular biological analyses of the liver suggested that CR treatment might improve the symptoms of inflammation, the unfolded protein response induced by amyloid deposits and oxidative stress. Furthermore, we suggest that CR treatment might improve mitochondrial functions via the sirtuin 1-peroxisome proliferator-activated receptor γ coactivator 1α (SIRT1-PGC-1α) pathway. We suggest that CR is a promising approach for treating the onset and/or progression of amyloidosis, especially for systemic amyloidosis such as senile AApoAII amyloidosis. Our analysis of CR treatment for amyloidosis should provide useful information for determining the cause of amyloidosis and developing effective preventive treatments. PMID:28225824

  3. Molecular identification of aiiA homologous gene from endophytic Enterobacter species and in silico analysis of putative tertiary structure of AHL-lactonase.

    PubMed

    Rajesh, P S; Rai, V Ravishankar

    2014-01-03

    The aiiA homologous gene known to encode AHL- lactonase enzyme which hydrolyze the N-acylhomoserine lactone (AHL) quorum sensing signaling molecules produced by Gram negative bacteria. In this study, the degradation of AHL molecules was determined by cell-free lysate of endophytic Enterobacter species. The percentage of quorum quenching was confirmed and quantified by HPLC method (p<0.0001). Amplification and sequence BLAST analysis showed the presence of aiiA homologous gene in endophytic Enterobacter asburiae VT65, Enterobacter aerogenes VT66 and Enterobacter ludwigii VT70 strains. Sequence alignment analysis revealed the presence of two zinc binding sites, "HXHXDH" motif as well as tyrosine residue at the position 194. Based on known template available at Swiss-Model, putative tertiary structure of AHL-lactonase was constructed. The result showed that novel endophytic strains of Enterobacter genera encode the novel aiiA homologous gene and its structural importance for future study. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. 78 FR 64396 - Mixed Straddles; Straddle-by-Straddle Identification Under Section 1092(b)(2)(A)(i)(I); Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-29

    ... Under Section 1092(b)(2)(A)(i)(I); Correction AGENCY: Internal Revenue Service (IRS), Treasury. ACTION... straddles; straddle-by-straddle identification under section 1092(b)(2)(A)(i)(I) (Temporary). * * * * * (b... regulations relating to guidance for taxpayers electing to establish a mixed straddle using straddle-by...

  5. Apolipoprotein A-II polymorphism: relationships to behavioural and hormonal mediators of obesity

    USDA-ARS?s Scientific Manuscript database

    Background: The interaction between apolipoprotein A-II (APOA2) m265 genotype and saturated fat for obesity traits has been more extensively demonstrated than for any other locus, but behavioural and hormonal mechanisms underlying this relationship are unexplored. In this study, we evaluated relatio...

  6. An apolipoprotein A-II polymorphism (-265T/C, rs5082), regulates postprandial response to a saturated fat overload in healthy men

    USDA-ARS?s Scientific Manuscript database

    Apolipoprotein (Apo) A-II is an apolipoprotein with an unknown role in lipid metabolism. It has been suggested that the presence of the less frequent allele of a single nucleotide polymorphism (Apo A-II -265T/C, rs5082) reduces the transcription rate of Apo A-II and enhances VLDL postprandial cleara...

  7. Francisella tularensis Subtype A.II Genomic Plasticity in Comparison with Subtype A.I

    PubMed Central

    Larson, Marilynn A.; Nalbantoglu, Ufuk; Sayood, Khalid; Zentz, Emily B.; Bartling, Amanda M.; Francesconi, Stephen C.; Fey, Paul D.; Dempsey, Michael P.; Hinrichs, Steven H.

    2015-01-01

    Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed F. tularensis type A genomes. In contrast with the F. tularensis A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced F. tularensis A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96-3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that F. tularensis A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving F. tularensis genetic diversity and plasticity is needed. PMID:25918839

  8. AiiA-mediated quorum quenching does not affect virulence or toxoflavin expression in Burkholderia glumae SL2376.

    PubMed

    Park, J Y; Lee, Y H; Yang, K Y; Kim, Y C

    2010-12-01

    The primary objective of this study was to determine the effects of quorum quenching in the pathogenicity and toxoflavin production of Burkholderia glumae causing bacterial rice grain rot. An acyl-homoserine lactonase (aiiA) gene from Bacillus sp. was expressed in B. glumae under the control of a constitutive promoter. Acyl-homoserine lactone production in the aiiA-transformants was reduced significantly, and the aiiA-expressing B. glumae strain reduced the severity of soft rot when the strain was co-inoculated with a soft-rot pathogen, Pectobacterium carotovorum ssp. carotovorum SCCI. However, the aiiA-transformant still caused rice seedling rot and rice grain rot. The aiiA-expressing strains had wild-type levels of transcription from the genes in the toxoflavin biosynthetic operon, and as well as wild-type levels of toxin production. Our results show that aiiA-mediated quorum quenching does not affect virulence or toxoflavin production in B. glumae. Indirect quorum quenching may prove an ineffective approach to the control of rice grain rot, because it reduces, but does not eliminate entirely homoserine lactones in B. glumae. Virulence of rice grain rot was retained despite reduction in homoserine lactones by the expression of aiiA in B. glumae. © 2010 The Authors. Letters in Applied Microbiology 51, 619-624 © 2010 The Society for Applied Microbiology.

  9. Covalent structure of apolipoprotein A-II from Macaca mulatta serum high-density lipoproteins.

    PubMed

    Edelstein, C; Noyes, C; Keim, P; Heinrikson, R L; Fellows, R E; Scanu, A M

    1976-03-23

    The covalent structure of apolipoprotein A-II, isolated from the serum high-density lipoprotein of a single male Rhesus monkey (Macaca mulatta), was determined. The amino acid sequence of this 77-residue polypeptide is: less than Glu-Ala-Glu-Glu-Pro5-Ser-Val-Glu-Ser-Leu10-Val-Ser-Gln-Tyr-Phe15-Gln-Thr-Val-Thr-Asp20-Tyr-Gly-Lys-Asp-Leu25-Met-Glu-Lys-Val-Lys30-Ser-Pro-Glu-Leu-Gln35-Ala-Gln-Ala-Lys-Ala40-Tyr-Phe-Glu-Lys-Ser45-Lys-Glu-Gln-Leu-Thr50-Pro-Leu-Val-Lys-Lys55-Ala-Gly-Thr-Asp-Leu60-Val-Asn-Phe-Leu-Ser65-Tyr-Phe-Val-Glu-Leu70-Arg-Thr-Gln-Pro-Ala75-Thr-Gln-COOH. A comparison of this structure to that of the monomeric form of human apolipoprotein A-II reveals a high degree of homology except for six conservative amino acid replacements (positions 3, 6, 40, 53, 59, and 71). Of particular structural significance is the replacement of cysteine by serine in position 6. This explaines why Rhesus A-II exists in monomeric form, contrary to the established dimeric nature of the human protein.

  10. Goldfish Leptin-AI and Leptin-AII: Function and Central Mechanism in Feeding Control.

    PubMed

    Yan, Ai-Fen; Chen, Ting; Chen, Shuang; Ren, Chun-Hua; Hu, Chao-Qun; Cai, Yi-Ming; Liu, Fang; Tang, Dong-Sheng

    2016-05-30

    In mammals, leptin is a peripheral satiety factor that inhibits feeding by regulating a variety of appetite-related hormones in the brain. However, most of the previous studies examining leptin in fish feeding were performed with mammalian leptins, which share very low sequence homologies with fish leptins. To elucidate the function and mechanism of endogenous fish leptins in feeding regulation, recombinant goldfish leptin-AI and leptin-AII were expressed in methylotrophic yeast and purified by immobilized metal ion affinity chromatography (IMAC). By intraperitoneal (IP) injection, both leptin-AI and leptin-AII were shown to inhibit the feeding behavior and to reduce the food consumption of goldfish in 2 h. In addition, co-treatment of leptin-AI or leptin-AII could block the feeding behavior and reduce the food consumption induced by neuropeptide Y (NPY) injection. High levels of leptin receptor (lepR) mRNA were detected in the hypothalamus, telencephalon, optic tectum and cerebellum of the goldfish brain. The appetite inhibitory effects of leptins were mediated by downregulating the mRNA levels of orexigenic NPY, agouti-related peptide (AgRP) and orexin and upregulating the mRNA levels of anorexigenic cocaine-amphetamine-regulated transcript (CART), cholecystokinin (CCK), melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC) in different areas of the goldfish brain. Our study, as a whole, provides new insights into the functions and mechanisms of leptins in appetite control in a fish model.

  11. Goldfish Leptin-AI and Leptin-AII: Function and Central Mechanism in Feeding Control

    PubMed Central

    Yan, Ai-Fen; Chen, Ting; Chen, Shuang; Ren, Chun-Hua; Hu, Chao-Qun; Cai, Yi-Ming; Liu, Fang; Tang, Dong-Sheng

    2016-01-01

    In mammals, leptin is a peripheral satiety factor that inhibits feeding by regulating a variety of appetite-related hormones in the brain. However, most of the previous studies examining leptin in fish feeding were performed with mammalian leptins, which share very low sequence homologies with fish leptins. To elucidate the function and mechanism of endogenous fish leptins in feeding regulation, recombinant goldfish leptin-AI and leptin-AII were expressed in methylotrophic yeast and purified by immobilized metal ion affinity chromatography (IMAC). By intraperitoneal (IP) injection, both leptin-AI and leptin-AII were shown to inhibit the feeding behavior and to reduce the food consumption of goldfish in 2 h. In addition, co-treatment of leptin-AI or leptin-AII could block the feeding behavior and reduce the food consumption induced by neuropeptide Y (NPY) injection. High levels of leptin receptor (lepR) mRNA were detected in the hypothalamus, telencephalon, optic tectum and cerebellum of the goldfish brain. The appetite inhibitory effects of leptins were mediated by downregulating the mRNA levels of orexigenic NPY, agouti-related peptide (AgRP) and orexin and upregulating the mRNA levels of anorexigenic cocaine-amphetamine-regulated transcript (CART), cholecystokinin (CCK), melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC) in different areas of the goldfish brain. Our study, as a whole, provides new insights into the functions and mechanisms of leptins in appetite control in a fish model. PMID:27249000

  12. Apolipoproteins A-I, A-II and E in cholestatic liver disease.

    PubMed

    Florén, C H; Gustafson, A

    1985-04-01

    Apolipoproteins A-I, A-II and E were determined in the plasma of nine patients (five females, four males) with cholestatic liver disease (eight patients with primary biliary cirrhosis and one patient with sclerosing cholangitis). Plasma concentrations were measured by electroimmunoassay in the fasting state, postprandially after ingestion of either 100 g fat as whipping cream or a light mixed meal with or without addition of wheat fibre. Concentrations of apolipoproteins A-I and A-II were low in patients with cholestatic liver disease and A-I levels correlated inversely with the severity of liver disease as measured by bilirubin levels (r = -0.66). No changes in plasma apolipoprotein A-I, A-II or E concentrations occurred postprandially. There was an inverse correlation between plasma concentrations of apolipoproteins A-I and E (p less than 0.05, r = -0.68). A close relation existed between the ratio of apolipoprotein E to apolipoprotein A-I and plasma bile salt concentration (r = 0.80, p less than 0.01) and serum bilirubin (r = 0.76, p less than 0.01). This implies that in cholestatic liver disease apolipoprotein E and A-I levels reflect the degree of cholestasis.

  13. Transgenic Amorphophallus konjac expressing synthesized acyl-homoserine lactonase (aiiA) gene exhibit enhanced resistance to soft rot disease.

    PubMed

    Ban, Huifang; Chai, Xinli; Lin, Yongjun; Zhou, Ying; Peng, Donghai; Zhou, Yi; Zou, Yulan; Yu, Ziniu; Sun, Ming

    2009-12-01

    Amorphophallus konjac is an important economic crop widely used in health products and biomaterials. However, this monocotyledonous plant's production is seriously restricted by soft rot disease. Some Bacillus thuringiensis strains generate an endocellular acyl homoserine lactonase (AiiA), which has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain transgenic A. konjac expressing AiiA protein and exhibiting resistance to soft rot. But till now, there is not any report about exogenous gene transformation in A. konjac. In this research, an Agrobacterium-mediated genetic transformation system was constructed. An aiiA gene was synthesized according to the codon usage in A. konjac. Embryogenic callus was infected with the A. tumefaciens strain EHA105 harboring the plant transformation plasmid pU1301 plus synthesized aiiA gene. After antibiotics screening, 34 plants were obtained. PCR analysis showed that positive amplified fragments were present in 21 out of these 34 lines. Southern blot analysis indicated that aiiA gene had integrated into the genome of A. konjac. Western blotting demonstrated that the target protein of interest was reactive with the antibody against AiiA. Further disease resistance detection revealed that all of the tested transgenic A. konjac lines exhibited high resistance to soft rot bacteria Erwinia carotovora subsp. Carotovora (Ecc) SCG1. The protocol is useful for the quality improvement of A. konjac through genetic transformation.

  14. Cytokinetic studies reveal etiology of cytogenetic genotoxicity produced by a series of angiotensin II (AII) receptor antagonists may be perturbed DNA synthesis

    SciTech Connect

    Selden, J.R.; Miller, J.E.; Dolbeare, F.; Galloway, S.M.; Nichols, W.W. Lawrence Livermore National Lab., CA )

    1993-01-01

    Six of 13 AII receptor antagonists produced chromosomal aberrations in CHO cells. In addition, these six compounds perturbed cellular kinetics (i.e., reduced mitotic indices and cell yields). It was hypothesized that the mechanism of clastogenesis was not due to a direct genotoxic effect, but may result from disruption of DNA replication. Flow cytokinetic studies, using the BrdUrd-FITC/propidium iodide technique, were performed on all six clastogenic compounds, and a seventh candidate from this group. All seven altered CHO cell kinetics as follows: (1) The amount of BrdUrd per S phase cell was reduced; (2) Cell movement within S phase was inhibited; and (3) Lowest doses perturbing CHO cell kinetics were below minimum concentrations producing aberrations. These data provide evidence that this cytogenetic damage is mediated by a mechanism which disrupts cellular DNA synthesis.

  15. Mechanical strain and collagen potentiate mitogenic activity of angiotensin II in rat vascular smooth muscle cells.

    PubMed Central

    Sudhir, K; Wilson, E; Chatterjee, K; Ives, H E

    1993-01-01

    The effects of extracellular matrix proteins and mechanical strain on the mitogenic activity of angiotensins I and II (AI and AII) were examined in cultured rat vascular smooth muscle (VSM) cells. VSM cells on various extracellular matrices were exposed to AII (1 microM) for 48 h. On plastic, AII induced only a 1.6-fold increase in [3H]thymidine incorporation, but on fibronectin- or type I collagen-coated plastic, the response to AII was enhanced from two- to fourfold. On a type I collagen-coated silicone elastomer, to which mechanical strain was applied, [3H]thymidine incorporation dramatically increased to a maximum of 53-fold. Dup 753 (10(-5) M) blocked the AII-induced increase in DNA synthesis. AI also increased DNA synthesis in VSM cells, and this response was also enhanced by mechanical strain. Mitogenic activity of AI was blocked by ramiprilat (10(-5) M), indicating that its mitogenic activity was via conversion to AII. The synergy between AII and strain was completely eliminated by neutralizing antibodies to PDGF AB (3 micrograms/ml). Furthermore, the mitogenic effect of AII in unstrained cells was also synergistic with submaximal concentrations of PDGF AB (1 ng/ml). Thus, the synergy between AII and mechanical strain probably results from synergism between AII and PDGF secreted in response to strain. PMID:8254054

  16. Effects of an inducible aiiA gene on disease resistance in Eucalyptus urophylla × Eucalyptus grandis.

    PubMed

    Ouyang, L J; Li, L M

    2016-08-01

    N-acyl-homoserine lactones (AHLs) are metabolites of mostly gram-negative bacteria and are critical signaling molecules in bacterial quorum-sensing systems. At threshold concentrations, AHLs can activate the expression of pathogenic genes and induce diseases. Therefore, reducing AHL concentrations is a key point of disease control in plants. AHL-lactonase, which is expressed by aiiA, is widespread in Bacillus sp and can hydrolyze AHLs. In the present study, we cloned aiiA from Bacillus subtilis by PCR. A plant expression vector of aiiA was constructed and name Pcam-PPP3-aiiA, in which expression of aiiA was controlled by the pathogen-inducible plant promoter PPP3. The recombinant plasmid was transferred into Eucalyptus × urophylla × E. grandis by an Agrobacterium-mediated transformation. PCR and Southern blotting showed that aiiA was successfully integrated into the E. urophylla × E. grandis genome and its expression was induced by Ralstonia solanacearum 12 h after inoculation, as shown by reverse transcription-PCR. The transcription efficacy of aiiA increased 43.88-, 30.65-, and 18.95-fold after inoculation with R. solanacearum, Erwinia carotovora ssp. zeae (Sabet) and Cylindrocladium quinqueseptatum, respectively as shown by RT-real-time PCR. Transgenic E.urophylla × E.grandis expressing the AIIA protein exhibited significantly enhanced disease resistance compared to non-transgenic plants by delaying the onset of wilting and reducing the disease index.

  17. 49 CFR Appendix A-Ii to Part 541 - Lines With Antitheft Devices Which Are Exempted in-Part From the Parts-Marking Requirements of...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 6 2010-10-01 2010-10-01 false Lines With Antitheft Devices Which Are Exempted in-Part From the Parts-Marking Requirements of This Standard Pursuant to 49 CFR Part 543 A Appendix A-II... STANDARD Pt. 541, App. A-II Appendix A-II to Part 541—Lines With Antitheft Devices Which Are Exempted...

  18. 49 CFR Appendix A-Ii to Part 541 - Lines With Antitheft Devices Which Are Exempted in-Part From the Parts-Marking Requirements of...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 6 2011-10-01 2011-10-01 false Lines With Antitheft Devices Which Are Exempted in-Part From the Parts-Marking Requirements of This Standard Pursuant to 49 CFR Part 543 A Appendix A-II... STANDARD Pt. 541, App. A-II Appendix A-II to Part 541—Lines With Antitheft Devices Which Are Exempted...

  19. Plasma biomarker for detection of early stage pancreatic cancer and risk factors for pancreatic malignancy using antibodies for apolipoprotein-AII isoforms.

    PubMed

    Honda, Kazufumi; Kobayashi, Michimoto; Okusaka, Takuji; Rinaudo, Jo Ann; Huang, Ying; Marsh, Tracey; Sanada, Mitsuaki; Sasajima, Yoshiyuki; Nakamori, Shoji; Shimahara, Masashi; Ueno, Takaaki; Tsuchida, Akihiko; Sata, Naohiro; Ioka, Tatsuya; Yasunami, Yohichi; Kosuge, Tomoo; Miura, Nami; Kamita, Masahiro; Sakamoto, Takako; Shoji, Hirokazu; Jung, Giman; Srivastava, Sudhir; Yamada, Tesshi

    2015-11-09

    We recently reported that circulating apolipoprotein AII (apoAII) isoforms apoAII-ATQ/AT (C-terminal truncations of the apoAII homo-dimer) decline significantly in pancreatic cancer and thus might serve as plasma biomarkers for the early detection of this disease. We report here the development of novel enzyme-linked immunosorbent assays (ELISAs) for measurement of apoAII-ATQ/AT and their clinical applicability for early detection of pancreatic cancer. Plasma and serum concentrations of apoAII-ATQ/AT were measured in three independent cohorts, which comprised healthy control subjects and patients with pancreatic cancer and gastroenterologic diseases (n = 1156). These cohorts included 151 cases of stage I/II pancreatic cancer. ApoAII-ATQ/AT not only distinguished the early stages of pancreatic cancer from healthy controls but also identified patients at high risk for pancreatic malignancy. AUC values of apoAII-ATQ/AT to detect early stage pancreatic cancer were higher than those of CA19-9 in all independent cohorts. ApoAII-ATQ/AT is a potential biomarker for screening patients for the early stage of pancreatic cancer and identifying patients at risk for pancreatic malignancy (161 words).

  20. Plasma biomarker for detection of early stage pancreatic cancer and risk factors for pancreatic malignancy using antibodies for apolipoprotein-AII isoforms

    PubMed Central

    Honda, Kazufumi; Kobayashi, Michimoto; Okusaka, Takuji; Rinaudo, Jo Ann; Huang, Ying; Marsh, Tracey; Sanada, Mitsuaki; Sasajima, Yoshiyuki; Nakamori, Shoji; Shimahara, Masashi; Ueno, Takaaki; Tsuchida, Akihiko; Sata, Naohiro; Ioka, Tatsuya; Yasunami, Yohichi; Kosuge, Tomoo; Miura, Nami; Kamita, Masahiro; Sakamoto, Takako; Shoji, Hirokazu; Jung, Giman; Srivastava, Sudhir; Yamada, Tesshi

    2015-01-01

    We recently reported that circulating apolipoprotein AII (apoAII) isoforms apoAII-ATQ/AT (C-terminal truncations of the apoAII homo-dimer) decline significantly in pancreatic cancer and thus might serve as plasma biomarkers for the early detection of this disease. We report here the development of novel enzyme-linked immunosorbent assays (ELISAs) for measurement of apoAII-ATQ/AT and their clinical applicability for early detection of pancreatic cancer. Plasma and serum concentrations of apoAII-ATQ/AT were measured in three independent cohorts, which comprised healthy control subjects and patients with pancreatic cancer and gastroenterologic diseases (n = 1156). These cohorts included 151 cases of stage I/II pancreatic cancer. ApoAII-ATQ/AT not only distinguished the early stages of pancreatic cancer from healthy controls but also identified patients at high risk for pancreatic malignancy. AUC values of apoAII-ATQ/AT to detect early stage pancreatic cancer were higher than those of CA19–9 in all independent cohorts. ApoAII-ATQ/AT is a potential biomarker for screening patients for the early stage of pancreatic cancer and identifying patients at risk for pancreatic malignancy (161 words). PMID:26549697

  1. Accumulation of pro-apolipoprotein A-II in mouse senile amyloid fibrils.

    PubMed Central

    Higuchi, K; Kogishi, K; Wang, J; Xia, C; Chiba, T; Matsushita, T; Hosokawa, M

    1997-01-01

    Apolipoprotein A-II (apoA-II), the major apoprotein of serum high-density lipoprotein, is deposited as amyloid fibrils (AApoAII) in murine senile amyloidosis. We have identified and purified a more basic amyloid protein from old-mouse liver. N-terminal sequencing of the protein revealed that the pro-segment of five amino acid residues (Ala-Leu-Val-Lys-Arg) extended from the N-terminal glutamine residue of mature apoA-II protein. MS analysis revealed the deposit of intact pro-apoA-II protein (molecular mass 9319 Da). Antiserum was prepared for staining of the AApoAII amyloid deposition. The relative abundance of pro-apoA-II to mature apoA-II in the amyloid-fibril fraction isolated from livers of mice with severe amyloidosis was 14.1%. The similar abundance of pro-apoA-II in the amyloid fibril fraction from the spleen (16.3%) suggested that deposited pro-apoA-II originated from the blood. The concentration of pro-apoA-II was much lower in the serum (1.5% of mature apoA-II) than in the amyloid-fibril fraction. There was no difference in the content of pro-apoA-II between the amyloidogenetic R1.P1-Apoa2c and amyloid-resistant SAMR1 strains at the age of 3 months. The abundance of pro-apoA-II in the amyloid-fibril fraction compared with the serum suggested that it plays a key role in the initialization of mouse senile amyloidosis. PMID:9271085

  2. Atypical Porcine Pestivirus: A Possible Cause of Congenital Tremor Type A-II in Newborn Piglets

    PubMed Central

    de Groof, Ad; Deijs, Martin; Guelen, Lars; van Grinsven, Lotte; van Os-Galdos, Laura; Vogels, Wannes; Derks, Carmen; Cruijsen, Toine; Geurts, Victor; Vrijenhoek, Mieke; Suijskens, Janneke; van Doorn, Peter; van Leengoed, Leo; Schrier, Carla; van der Hoek, Lia

    2016-01-01

    Congenital tremor type A-II in piglets has been regarded as a transmissible disease since the 1970s, possibly caused by a very recently-described virus: atypical porcine pestivirus (APPV). Here, we describe several strains of APPV in piglets with clinical signs of congenital tremor (10 of 10 farms tested). Piglets on a farm with no history of congenital tremor were PCR-negative for the virus. To demonstrate a causal relationship between APPV and disease, three gilts were inoculated via intramuscular injection at day 32 of pregnancy. In two of the three litters, vertical transmission of the virus occurred. Clinical signs of congenital tremor were observed in APPV-infected newborns, yet also two asymptomatic carriers were among the offspring. Piglets of one litter were PCR-negative for the virus, and these piglets were all without congenital tremors. Long-term follow up of farm piglets born with congenital tremors showed that the initially high viremia in serum declines at five months of age, but shedding of the virus in feces continues, which explains why the virus remains present at affected farms and causes new outbreaks. We conclude that trans-placental transmission of APPV and subsequent infection of the fetuses is a very likely cause of congenital tremor type A-II in piglets. PMID:27782037

  3. Atypical Porcine Pestivirus: A Possible Cause of Congenital Tremor Type A-II in Newborn Piglets.

    PubMed

    de Groof, Ad; Deijs, Martin; Guelen, Lars; van Grinsven, Lotte; van Os-Galdos, Laura; Vogels, Wannes; Derks, Carmen; Cruijsen, Toine; Geurts, Victor; Vrijenhoek, Mieke; Suijskens, Janneke; van Doorn, Peter; van Leengoed, Leo; Schrier, Carla; van der Hoek, Lia

    2016-10-04

    Congenital tremor type A-II in piglets has been regarded as a transmissible disease since the 1970s, possibly caused by a very recently-described virus: atypical porcine pestivirus (APPV). Here, we describe several strains of APPV in piglets with clinical signs of congenital tremor (10 of 10 farms tested). Piglets on a farm with no history of congenital tremor were PCR-negative for the virus. To demonstrate a causal relationship between APPV and disease, three gilts were inoculated via intramuscular injection at day 32 of pregnancy. In two of the three litters, vertical transmission of the virus occurred. Clinical signs of congenital tremor were observed in APPV-infected newborns, yet also two asymptomatic carriers were among the offspring. Piglets of one litter were PCR-negative for the virus, and these piglets were all without congenital tremors. Long-term follow up of farm piglets born with congenital tremors showed that the initially high viremia in serum declines at five months of age, but shedding of the virus in feces continues, which explains why the virus remains present at affected farms and causes new outbreaks. We conclude that trans-placental transmission of APPV and subsequent infection of the fetuses is a very likely cause of congenital tremor type A-II in piglets.

  4. Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens

    SciTech Connect

    Liu, D.; Thomas, P.W.; Momb, J.; Hoang, Q.Q.; Petsko, G.A.; Ringe, D.; Fast, W.

    2009-06-03

    N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.

  5. Some OFF bipolar cell types make contact with both rods and cones in macaque and mouse retinas

    PubMed Central

    Tsukamoto, Yoshihiko; Omi, Naoko

    2014-01-01

    This study compared the types of OFF bipolar cells found in the macaque retina with those found in the mouse retina and determined whether these OFF bipolar cells make direct contacts with both rods and cones by serial section transmission electron microscopy. We performed scatter plots and cluster analysis of the morphological variables of their axon terminals such as the stratification level, the arbor thickness, the arbor area, and the number of ribbons. Five OFF bipolar cell types, including the recently discovered DB3b type, were identified in the macaque retina. The macaque OFF bipolar cell types FMB, DB1, DB2, DB3a, and DB3b corresponded to the mouse OFF bipolar cell types 2, 1, 4, 3a, and 3b, respectively. In addition to contacting rod bipolar cells, ~7% of rods in the macaque retina made basal contacts exclusively with one cell type, DB3b, whereas 18% of rods in the mouse retina made basal contacts with one or two of types, 3a, 3b, and 4. Approximately 3% of mouse rods were divergently connected to two OFF bipolar cells of different types, but macaque rods were solely connected to one OFF bipolar cell. Rod-rod gap junctions were localized at rod cell bodies and axons in the outer nuclear layer in both macaque and mouse retinas. The direct rod-OFF bipolar connection system is slightly more developed in the mouse retina than in the macaque retina, possibly as a fine-tuned adaptation to nocturnal conditions. This one-step direct synaptic pathway from rods to OFF bipolar cells may enhance the response speed to OFF light stimuli compared with more indirect pathways via rod-cone gap junctions (a two-step pathway) and via rod bipolar and AII amacrine cells (a three-step pathway). PMID:25309346

  6. 26 CFR 1.1092(b)-3T - Mixed straddles; straddle-by-straddle identification under section 1092(b)(2)(A)(i)(I) (temporary).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... identification under section 1092(b)(2)(A)(i)(I) (temporary). 1.1092(b)-3T Section 1.1092(b)-3T Internal Revenue... identification under section 1092(b)(2)(A)(i)(I) (temporary). (a) In general. Except as otherwise provided, a... constitute independent verification: (i) Separate account. Placement of one or more positions of a section...

  7. 26 CFR 1.1092(b)-3T - Mixed straddles; straddle-by-straddle identification under section 1092(b)(2)(A)(i)(I) (temporary).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... identification under section 1092(b)(2)(A)(i)(I) (temporary). 1.1092(b)-3T Section 1.1092(b)-3T Internal Revenue... identification under section 1092(b)(2)(A)(i)(I) (temporary). (a) In general. Except as otherwise provided, a... constitute independent verification: (i) Separate account. Placement of one or more positions of a section...

  8. 26 CFR 1.1092(b)-3T - Mixed straddles; straddle-by-straddle identification under section 1092(b)(2)(A)(i)(I) (temporary).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... identification under section 1092(b)(2)(A)(i)(I) (temporary). 1.1092(b)-3T Section 1.1092(b)-3T Internal Revenue... identification under section 1092(b)(2)(A)(i)(I) (temporary). (a) In general. Except as otherwise provided, a... constitute independent verification: (i) Separate account. Placement of one or more positions of a section...

  9. 26 CFR 1.1092(b)-3T - Mixed straddles; straddle-by-straddle identification under section 1092(b)(2)(A)(i)(I) (temporary).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... identification under section 1092(b)(2)(A)(i)(I) (temporary). 1.1092(b)-3T Section 1.1092(b)-3T Internal Revenue... section 1092(b)(2)(A)(i)(I) (temporary). (a) In general. Except as otherwise provided, a taxpayer shall... constitute independent verification: (i) Separate account. Placement of one or more positions of a section...

  10. Diversity and Distribution of N-Acylhomoserine Lactone (AHL)-Degrading Activity and AHL-Lactonase (AiiM) in Genus Microbacterium

    PubMed Central

    Wang, Wen-Zhao; Morohoshi, Tomohiro; Someya, Nobutaka; Ikeda, Tsukasa

    2012-01-01

    N-Acylhomoserine lactone (AHL)-degrading enzyme, AiiM, was identified from the potato leaf-associated Microbacterium testaceum StLB037. In this study, we cloned eight aiiM gene homologues from other AHL-degrading Microbacterium strains. The similarity of the chromosomal locus of the aiiM gene is associated with the phylogenetic classification based on 16S rRNA. Degenerate PCR revealed that the aiiM gene was only conserved in AHL-degrading Microbacterium strains, but not in fifteen Microbacterium type strains or two Microbacterium isolates from other plants. These results suggested that the high level of AHL-degrading activity in Microbacterium strains was caused by the aiiM gene encoded on their chromosome. PMID:22446311

  11. Disruption in dopaminergic innervation during photoreceptor degeneration.

    PubMed

    Ivanova, Elena; Yee, Christopher W; Sagdullaev, Botir T

    2016-04-15

    Dopaminergic amacrine cells (DACs) release dopamine in response to light-driven synaptic inputs, and are critical to retinal light adaptation. Retinal degeneration (RD) compromises the light responsiveness of the retina and, subsequently, dopamine metabolism is impaired. As RD progresses, retinal neurons exhibit aberrant activity, driven by AII amacrine cells, a primary target of the retinal dopaminergic network. Surprisingly, DACs are an exception to this physiological change; DACs exhibit rhythmic activity in healthy retina, but do not burst in RD. The underlying mechanism of this divergent behavior is not known. It is also unclear whether RD leads to structural changes in DACs, impairing functional regulation of AII amacrine cells. Here we examine the anatomical details of DACs in three mouse models of human RD to determine how changes to the dopaminergic network may underlie physiological changes in RD. By using rd10, rd1, and rd1/C57 mice we were able to dissect the impacts of genetic background and the degenerative process on DAC structure in RD retina. We found that DACs density, soma size, and primary dendrite length are all significantly reduced. Using a novel adeno-associated virus-mediated technique to label AII amacrine cells in mouse retina, we observed diminished dopaminergic contacts to AII amacrine cells in RD mice. This was accompanied by changes to the components responsible for dopamine synthesis and release. Together, these data suggest that structural alterations of the retinal dopaminergic network underlie physiological changes during RD.

  12. Localization of Neuropeptide Y1 Receptor Immunoreactivity in the Rat Retina and the Synaptic Connectivity of Y1 Immunoreactive Cells

    PubMed Central

    D'Angelo, Iona; Oh, Su-Ja; Chun, Myung-Hoon; Brecha, Nicholas C.

    2010-01-01

    Neuropeptide Y (NPY), an inhibitory neuropeptide expressed by a moderately dense population of wide-field amacrine cells in the rat retina, acts through multiple (Y1–y6) G-protein–coupled receptors. This study determined the cellular localization of Y1 receptors and the synaptic connectivity of Y1 processes in the inner plexiform layer (IPL) of the rat retina. Specific Y1 immunoreactivity was localized to horizontal cell bodies in the distal inner nuclear layer and their processes in the outer plexiform layer. Immunoreactivity was also prominent in cell processes located in strata 2 and 4, and puncta in strata 4 and 5 of the IPL. Double-label immunohistochemical experiments with calbindin, a horizontal cell marker, confirmed Y1 immunostaining in all horizontal cells. Double-label immunohistochemical experiments, using antibodies to choline acetyltransferase and vesicular acetylcholine transporter to label cholinergic amacrine cell processes, demonstrated that Y1 immunoreactivity in strata 2 and 4 of the IPL was localized to cholinergic amacrine cell processes. Electron microscopic studies of the inner retina showed that Y1-immunostained amacrine cell processes and puncta received synaptic inputs from unlabeled amacrine cell processes (65.2%) and bipolar cell axon terminals (34.8%). Y1-immunoreactive amacrine cell processes most frequently formed synaptic outputs onto unlabeled amacrine cell processes (34.0%) and ganglion cell dendrites (54.1%). NPY immunoreactivity in the rat retina is distributed primarily to strata 1 and 5 of the IPL, and the present findings, thus, suggest that NPY acts in a paracrine manner on Y1 receptors to influence both horizontal and amacrine cells. PMID:12455004

  13. Mouse Ganglion-Cell Photoreceptors Are Driven by the Most Sensitive Rod Pathway and by Both Types of Cones

    PubMed Central

    Weng, Shijun; Estevez, Maureen E.; Berson, David M.

    2013-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) are depolarized by light by two mechanisms: directly, through activation of their photopigment melanopsin; and indirectly through synaptic circuits driven by rods and cones. To learn more about the rod and cone circuits driving ipRGCs, we made multielectrode array (MEA) and patch-clamp recordings in wildtype and genetically modified mice. Rod-driven ON inputs to ipRGCs proved to be as sensitive as any reaching the conventional ganglion cells. These signals presumably pass in part through the primary rod pathway, involving rod bipolar cells and AII amacrine cells coupled to ON cone bipolar cells through gap junctions. Consistent with this interpretation, the sensitive rod ON input to ipRGCs was eliminated by pharmacological or genetic disruption of gap junctions, as previously reported for conventional ganglion cells. A presumptive cone input was also detectable as a brisk, synaptically mediated ON response that persisted after disruption of rod ON pathways. This was roughly three log units less sensitive than the rod input. Spectral analysis revealed that both types of cones, the M- and S-cones, contribute to this response and that both cone types drive ON responses. This contrasts with the blue-OFF, yellow-ON chromatic opponency reported in primate ipRGCs. The cone-mediated response was surprisingly persistent during steady illumination, echoing the tonic nature of both the rod input to ipRGCs and their intrinsic, melanopsin-based phototransduction. These synaptic inputs greatly expand the dynamic range and spectral bandpass of the non-image-forming visual functions for which ipRGCs provide the principal retinal input. PMID:23762490

  14. Transient release kinetics of rod bipolar cells revealed by capacitance measurement of exocytosis from axon terminals in rat retinal slices

    PubMed Central

    Oltedal, Leif; Hartveit, Espen

    2010-01-01

    Presynaptic transmitter release has mostly been studied through measurements of postsynaptic responses, but a few synapses offer direct access to the presynaptic terminal, thereby allowing capacitance measurements of exocytosis. For mammalian rod bipolar cells, synaptic transmission has been investigated in great detail by recording postsynaptic currents in AII amacrine cells. Presynaptic measurements of the dynamics of vesicular cycling have so far been limited to isolated rod bipolar cells in dissociated preparations. Here, we first used computer simulations of compartmental models of morphologically reconstructed rod bipolar cells to adapt the ‘Sine + DC’ technique for capacitance measurements of exocytosis at axon terminals of intact rod bipolar cells in retinal slices. In subsequent physiological recordings, voltage pulses that triggered presynaptic Ca2+ influx evoked capacitance increases that were proportional to the pulse duration. With pulse durations ≤100 ms, the increase saturated at ∼10 fF, corresponding to the size of a readily releasable pool of vesicles. Pulse durations ≥400 ms evoked additional capacitance increases, probably reflecting recruitment from additional pools of vesicles. By using Ca2+ tail current stimuli, we separated Ca2+ influx from Ca2+ channel activation kinetics, allowing us to estimate the intrinsic release kinetics of the readily releasable pool, yielding a time constant of ∼1.1 ms and a maximum release rate of 2–3 vesicles (release site)−1 ms−1. Following exocytosis, we observed endocytosis with time constants ranging from 0.7 to 17 s. Under physiological conditions, it is likely that release will be transient, with the kinetics limited by the activation kinetics of the voltage-gated Ca2+ channels. PMID:20211976

  15. Elevated high density lipoprotein cholesterol levels correlate with decreased apolipoprotein A-I and A-II fractional catabolic rate in women.

    PubMed Central

    Brinton, E A; Eisenberg, S; Breslow, J L

    1989-01-01

    High levels of HDL-cholesterol (HDL-C) protect against coronary heart disease susceptibility, but the metabolic mechanisms underlying elevated HDL-C levels are poorly understood. We now report the turnover of isologous radioiodinated HDL apolipoproteins, apo A-I and apo A-II, in 15 female subjects on a metabolic diet with HDL-C levels ranging from 51 to 122 mg/dl. The metabolic parameters, fractional catabolic rate (FCR) and absolute synthetic rate (SR), were determined for apo A-I and apo A-II in all subjects. There was an inverse correlation between plasma HDL-C and the FCR of apo A-I and apo A-II (r = -0.75, P less than 0.001, and r = -0.54, P = 0.036, respectively), but no correlation with the SR of either apo A-I or apo A-II (r = 0.09, and r = -0.16, respectively, both P = NS). Apo A-I levels correlated inversely with apo A-I FCR (r = -0.64, P = 0.01) but not with apo A-I SR (r = 0.30, P = NS). In contrast, plasma levels of apo A-II did not correlate with apo A-II FCR (r = -0.38, P = 0.16), but did correlate with apo A-II SR (r = 0.65, P = 0.009). Further analysis showed that apo A-I and apo A-II FCR were inversely correlated with the HDL-C/apo A-I + A-II ratio (r = -0.69 and -0.61, P = 0.005 and 0.015, respectively). These data suggest that: (a) low HDL apolipoprotein FCR is the predominant metabolic mechanism of elevated HDL-C levels; (b) apo A-I FCR is the primary factor in controlling plasma apo A-I levels, but apo A-II SR is the primary factor controlling plasma apo A-II levels; (c) low HDL apolipoprotein FCR is associated with a lipid-rich HDL fraction. These findings elucidate aspects of HDL metabolism which contribute to high HDL-C levels and which may constitute mechanisms for protection against coronary heart disease. PMID:2500457

  16. Apolipoprotein A-II polymorphism and visceral adiposity in African-American and white women.

    PubMed

    Lara-Castro, Cristina; Hunter, Gary R; Lovejoy, Jennifer C; Gower, Barbara A; Fernández, José R

    2005-03-01

    To determine the association between the -265 T to C substitution in the apolipoprotein A-II (APOA-II) gene and levels of visceral adipose tissue (VAT) in a group of premenopausal African-American and white women, we genotyped 237 women (115 African-American and 122 white) for this polymorphism. Body composition was assessed by DXA, and VAT was determined from a single computed tomography scan. In addition to VAT, we examined the association between the polymorphism and other phenotypes (total body fat, total abdominal adipose tissue, and subcutaneous abdominal adipose tissue). The mutant C allele in the APOA-II gene was less frequent in African-American compared with white women, 23% vs. 36%, respectively (p < 0.01). VAT was significantly higher in carriers of the C allele compared with noncarriers after adjustment for total body fat (p < 0.05). When separate analyses by ethnic group were conducted, the association between the polymorphism and VAT was observed in white (p < 0.05) but not African-American (p = 0.57) women. There was no association between the polymorphism and the other phenotypes. These results indicate a significant association between the T265C APOA-II polymorphism and levels of VAT in premenopausal women. This association is present in white but not African-American women.

  17. Structure of 23S rRNA hairpin 35 and its interaction with the tylosin-resistance methyltransferase RlmAII

    PubMed Central

    Lebars, Isabelle; Yoshizawa, Satoko; Stenholm, Anne R.; Guittet, Eric; Douthwaite, Stephen; Fourmy, Dominique

    2003-01-01

    The bacterial rRNA methyltransferase RlmAII (formerly TlrB) contributes to resistance against tylosin-like 16-membered ring macrolide antibiotics. RlmAII was originally discovered in the tylosin-producer Streptomyces fradiae, and members of this subclass of methyltransferases have subsequently been found in other Gram-positive bacteria, including Streptococcus pneumoniae. In all cases, RlmAII methylates 23S rRNA at nucleotide G748, which is situated in a stem–loop (hairpin 35) at the macrolide binding site of the ribosome. The conformation of hairpin 35 recognized by RlmAII is shown here by NMR spectroscopy to resemble the anticodon loop of tRNA. The loop folds independently of the rest of the 23S rRNA, and is stabilized by a non-canonical G–A pair and a U-turn motif, rendering G748 accessible. Binding of S.pneumoniae RlmAII induces changes in NMR signals at specific nucleotides that are involved in the methyltransferase–RNA interaction. The conformation of hairpin 35 that interacts with RlmAII is radically different from the structure this hairpin adopts within the 50S subunit. This indicates that the hairpin undergoes major structural rearrangement upon interaction with ribosomal proteins during 50S assembly. PMID:12514124

  18. Internalization and lysosomal association of (/sup 125/I)angiotensin II in norepinephrine-containing cells of the rat adrenal medulla

    SciTech Connect

    Bianchi, C.; Gutkowska, J.; Charbonneau, C.; Ballak, M.; Anand-Srivastava, M.B.; De Lean, A.; Genest, J.; Cantin, M.

    1986-10-01

    The morphological localization of (/sup 125/I)angiotensin II (AII) in the rat adrenal medulla (AM) was studied by light- and electron-microscopic radioautography in vivo. With light microscopy the presence of binding sites for AII in both norepinephrine-containing (NE) and epinephrine-containing (E) cells was confirmed. With electron microscopy, it was found that AII binds to the cell surface of NE cells, is progressively internalized, and is associated with lysosomes and Golgi complex within 20 min, whereas in E cells AII seems to be internalized earlier and recycled back to the cell surface within 5 min without any appreciable association with intracellular organelles. These results suggest different intracellular pathways for AII in NE and E cells of the rat AM.

  19. The light microscopic localization of substance P- and somatostatin-like immunoreactive cells in the larval tiger salamander retina.

    PubMed

    Li, H B; Chen, N X; Watt, C B; Lam, D M

    1986-01-01

    Light microscopic immunocytochemistry was utilized to localize the populations of substance P (SP)- and somatostatin (SOM)-like immunoreactive cells in the larval tiger salamander retina. Of 104 SP-immunostained cells observed, 82% were Type 1 amacrine cells. Another 8% of the SP-cells were classified as Type 2 amacrine cells, while 10% of the SP-cells had their cell bodies located in the ganglion cell layer and were designated as displaced amacrine cells. Each type of SP-like immunoreactive cell was observed in the central and peripheral retina. SP-immunopositive processes were observed in the inner plexiform layer as a sparse plexus in sublamina 1 and as a denser network of fibers in sublamina 5. Seventy-eight percent of the 110 somatostatin-immunopositive cells observed were designated as Type 1 amacrine cells. Another 12% of SOM-cells were classified as displaced amacrine cells, while only two SOM-immunopositive Type 2 amacrine cells were observed. Nine percent of the SOM-cells were designated as interplexiform cells, based on their giving rise to processes distributing in the outer plexiform layer as well as processes ramifying in the inner plexiform layer. Each type of SOM-immunoreactive cell was observed in the central and peripheral retina, with the exception of the Type 2 amacrine cells, whose somas were only found in the central retina. Lastly, SOM-immunopositive processes in the inner plexiform layer appeared as a fine plexus in sublamina 1 and as a somewhat denser network of fibers in sublamina 5.

  20. Methylation of 23S rRNA Nucleotide G748 by RlmAII Methyltransferase Renders Streptococcus pneumoniae Telithromycin Susceptible

    PubMed Central

    Sato, Yoshiharu; Shoji, Tatsuma; Yamamoto, Tomoko

    2013-01-01

    Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmAII, which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmAII to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmAII renders S. pneumoniae TEL susceptible. PMID:23716046

  1. Use of aiiA gene amplification for AHL-lactonase production from endophytic bacterium Enterobacter species.

    PubMed

    Rajesh, P S; Rai, V Ravishankar

    2015-01-01

    AHL-lactonase has gained renewed interest due to biotechnological applications such as antiquorum sensing, antibiofilm strategies, biofouling, etc. In our study, the production of AHL-lactonase from endophytic bacteria Enterobacter aerogenes VT66 was optimized by response surface methodology (RSM) using central composite design (CCD) for four different cultural conditions. The relative activity of AHL-lactonase was correlated with amplification of aiiA homologous gene amplification with respect to cultural conditions. Statistical analysis by ANOVA of the quadratic regression model showed that the RSM model constructed is highly significant, as indicated by F-test with a low probability value (p(model) < 0.0001) and high regression coefficient (0.9997) as well as lower coefficient of variation (1.86%) indicate that suitability of variable parameters. The quadratic regression model of AHL-lactonase production in terms of relative activity was built and the optimal cultural conditions for maximum enzyme production were determined as 32.5 °C temperature, pH 7.0, 350 μM of substrate concentration and 33 h of incubation time. The enhanced AHL-lactonase yielded 1.33 fold increases in relative activity and it positively correlated with the amplification of aiiA gene.

  2. Morphology and distribution of neurons in the retinal ganglion cell layer of the adult tammar wallaby--Macropus eugenii.

    PubMed

    Wong, R O; Wye-Dvorak, J; Henry, G H

    1986-11-01

    The morphology of the ganglion cell layer of the adult tammar wallaby has been examined from Nissl-stained retinal flatmounts. From this material, neurons have been classed as ganglion cells or displaced amacrine cells according to the disposition of Nissl substance. A further subdivision of ganglion cells into a separate group of alphalike cells was assisted by determining the range of soma sizes in neurofibrillar-stained flatmounts, a method which, in the cat, has revealed the presence of alpha cells. Isodensity contour maps prepared from the Nissl-stained flatmounts show a well-developed visual streak and an area centralis in the total neuronal population. A similar pattern was also found in the ganglion cells, thus confirming Tancred's (J. Comp. Neurol. 196:585-603, '81) finding, and, as well, in the alphalike ganglion cells and the displaced amacrine cells. The relative proportions of ganglion cells to displaced amacrines (GC:DA) were evaluated from isodensity profiles drawn along and vertical to the visual streak for the two cell types and also from maps showing the variation in the GC:DA ratio throughout the retina. A comparison with results published for other species shows that the visual streak development in the tammar wallaby is consistent with the expectations of the "terrain" theory and that, in its relative proportion of displaced amacrines, the tammar closely resembles the rabbit but contrasts sharply with the cat, which has half as many ganglion cells and three times as many displaced amacrines as the other two species.

  3. Apolipoprotein A-II is a key regulatory factor of HDL metabolism as appears from studies with transgenic animals and clinical outcomes.

    PubMed

    Maïga, Sira Fatoumata; Kalopissis, Athina-Despina; Chabert, Michèle

    2014-01-01

    The structure and metabolism of HDL are linked to their major apolipoproteins (apo) A-I and A-II. HDL metabolism is very dynamic and depends on the constant remodeling by lipases, lipid transfer proteins and receptors. HDL exert several cardioprotective effects, through their antioxidant and antiinflammatory capacities and through the stimulation of reverse cholesterol transport from extrahepatic tissues to the liver for excretion into bile. HDL also serve as plasma reservoir for C and E apolipoproteins, as transport vehicles for a great variety of proteins, and may have more physiological functions than previously recognized. In this review we will develop several aspects of HDL metabolism with emphasis on the structure/function of apo A-I and apo A-II. An important contribution to our understanding of the respective roles of apo A-I and apo A-II comes from studies using transgenic animal models that highlighted the stabilizatory role of apo A-II on HDL through inhibition of their remodeling by lipases. Clinical studies coupled with proteomic analyses revealed the presence of dysfunctional HDL in patients with cardiovascular disease. Beyond HDL cholesterol, a new notion is the functionality of HDL particles. In spite of abundant literature on HDL metabolic properties, a major question remains unanswered: which HDL particle(s) confer(s) protection against cardiovascular risk?

  4. A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis†

    PubMed Central

    Liu, Ce Feng; Liu, Dali; Momb, Jessica; Thomas, Pei W.; Lajoie, Ashley; Petsko, Gregory A.; Fast, Walter; Ringe, Dagmar

    2013-01-01

    AiiA is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity, but shows preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center, but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the presence and absence of N-decanoyl-l-homoserine. F107 is located in a hydrophobic cavity adjacent to the previously identified ligand binding pocket, and F107W results in the formation of an unexpected interaction with the ring-opened product. Notably, the structure reveals a previously unidentified hydrophobic binding pocket for the substrate’s N-acyl chain. Two aromatic residues, F64 and F68 form a hydrophobic clamp, centered around the seventh carbon in the product-bound structure’s decanoyl chain, making an interaction that would also be available for longer substrates, but not for shorter substrates. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the hydrophobic clamp, including insertion of a redox sensitive cysteine pair, confirms the importance of this hydrophobic feature for substrate preference. Identifying the specificity determinants of AiiA will aid the development of more selective quorum-quenching enzymes as tools and as potential therapeutics. PMID:23387521

  5. Apo A-II participates in HDL-liposome interaction by the formation of new pre-β mobility particles and the modification of liposomes.

    PubMed

    Wróblewska, Małgorzata; Czyżewska, Marta; Wolska, Anna; Kortas-Stempak, Barbara; Szutowicz, Andrzej

    2010-12-01

    Interaction between high density lipoproteins (HDL) and liposomes results in both a structural modification of HDL and the generation of new pre-β HDL-like particles. Here, phosphatidylcholine liposomes and human HDL were incubated at liposomal phospholipid/HDL phospholipid (L-PL/HDL-PL) ratios of 1:1, 3:1 and 5:1 with a subsequent assessment of the distribution of apolipoprotein (apo) A-I, apo A-II, free cholesterol (FC) and PL between newly generated pre-β mobility lipoproteins and non-disrupted liposomes. Both at L-PL/HDL-PL ratios of 3:1 and 5:1 the fraction of liposomal-derived PL associated with pre-β fraction was significantly higher than those accepted by α-HDL. We found that 78% of apo A-I released from HDL was incorporated into pre-β mobility fraction. The relative contents of PL and apo A-I in pre-β fraction were constant irrespective of the initial L-PL/HDL-PL ratio in the incubation mixture and accounted for approximately 83 and 11%, respectively. Apo A-II was detached from HDL to a similar extent as apo A-I and distributed evenly between pre-β fraction and non-disrupted liposomes. Apo A-II constituted approximately 1%, by weight, in these fractions at all L-PL/HDL-PL ratios investigated. It corresponded approximately to 10% of pre-β fraction protein mass. Both liposomes and pre-β fraction accepted comparable amounts of FC released from HDL. This data indicated that during the interaction between human HDL and phosphatidylcholine liposome apo A-II participates both in structural modification of liposomes and in the generation of pre-β mobility fraction of constant content of PL, apo A-I and apo A-II. Involvement of apo A-II in HDL-liposome interaction may influence the anti-atherogenic properties of liposomes.

  6. Retinal Physiology: Non-Bipolar-Cell Excitatory Drive in the Inner Retina.

    PubMed

    Baden, Tom; Euler, Thomas

    2016-08-08

    The long-held view that bipolar cells provide the exclusive excitatory drive to the mammalian inner retina has been challenged: new studies indicate that, instead, at least two cells that lack the dendrites characteristic for bipolar cells, and therefore resemble amacrine cells, excite inner retinal circuits using glutamate.

  7. ON inputs to the OFF layer: bipolar cells that break the stratification rules of the retina.

    PubMed

    Hoshi, Hideo; Liu, Wei-Li; Massey, Stephen C; Mills, Stephen L

    2009-07-15

    The vertebrate retina is a distinctly laminar structure. Functionally, the inner plexiform layer, in which bipolar cells synapse onto amacrine and ganglion cells, is subdivided into two sublaminae. Cells that depolarize at light offset ramify in sublamina a; those that depolarize at light onset ramify in sublamina b. The separation of ON and OFF pathways appears to be a fundamental principle of retinal organization that is reflected throughout the entire visual system. We show three clear exceptions to this rule, in which the axons of calbindin-positive ON cone bipolar cells make ribbon synapses as they pass through the OFF layers with three separate cell types: (1) dopaminergic amacrine cells, (2) intrinsically photosensitive ganglion cells, and (3) bistratified diving ganglion cells. The postsynaptic location of the AMPA receptor GluR4 at these sites suggests that ON bipolar cells can make functional synapses as their axons pass through the OFF layers of the inner plexiform layer. These findings resolve a long-standing question regarding the anomalous ON inputs to dopaminergic amacrine cells and suggest that certain ON bipolar cell axons can break the stratification rules of the inner plexiform layer by providing significant synaptic output before their terminal specializations. These outputs are not only to dopaminergic amacrine cells but also to at least two ON ganglion cell types that have dendrites that arborize in sublamina a.

  8. The application of high density microarray for analysis of mitogenic signaling and cell-cycle in the adrenal.

    PubMed

    Wang, C; Francis, R; Harirchian, S; Batlle, D; Mayhew, B; Bassett, M; Rainey, W E; Pestell, R G

    2000-11-01

    Angiotensin II (AII) binds to specific G-protein coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. The H295R human adrenocortical cell line, which expresses AII receptors predominantly of the AT1 subclass, proliferates in response to treatment with AII. The induction and maintenance of cellular proliferation involves a precisely coordinated induction of a variety of genes. As the human genome sequencing projects near completion a variety of high throughput technologies have been developed in order to create dynamic displays of genomic responses. One high throughput method, the gridded cDNA microarray has been developed in which immobilised DNA samples are hybridized on glass slides for the identification of global genomic responses. For this purpose high precision robotic microarrayers have been developed at AECOM. The cyclin D1 gene, which encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), was induced by AII in H295R cells. Abundance of the cyclin D1 gene is rate-limiting in G1 phase progression of the cell-cycle in a variety of cell types. AII induced cyclin D1 promoter activity through a c-Fos and c-Jun binding sequence at -954 bp. Theabundance of c-Fos within this complex was increased by AII treatment. Analysis of AII signaling in adrenal cells by cDNA microarray demonstrated an induction of the human homologue of Xenopus XPMC2 (HXPMC2). The cDNA for XPMC2 was previously shown to rescue mitotic catastrophe in mutant S. Pombe defective in cdc2 kinase function. Further studies are required to determine the requirement for cyclin D1 and XPMC2H in AII-induced cell-cycle progression and cellular proliferation in the adrenal.

  9. In vivo and in vitro phosphorylation of annexin II in T cells: potential regulation by annexin V.

    PubMed Central

    Dubois, T; Oudinet, J P; Russo-Marie, F; Rothhut, B

    1995-01-01

    In order to understand how signal transduction occurs during T cell activation, it is necessary to identify the key regulatory molecules whose function is influenced by phosphorylation. Annexins II (A-II) and V (A-V) belong to a large family of Ca(2+)-dependent phospholipid-binding proteins. Among many putative functions, annexins may be involved in signal transduction during cellular proliferation and differentiation. In the present study we show that A-II is phosphorylated in vivo in the Jurkat human T cell line. Indeed, A-II is phosphorylated after stimulation by phorbol myristate acetate and on serine residues after T cell antigen receptor (TcR) stimulation. In cytosol from Jurkat cells, A-II is phosphorylated only by Ca2+/phospholipid-stimulated kinases such as Ca(2+)-dependent protein kinases C (cPKCs). A-V inhibits the phosphorylation of A-II and other substrates of cPKCs and has no effect on kinases activated only by phospholipids. In conclusion, A-II is phosphorylated both in vitro and in vivo in Jurkat cells, and may play a role as a substrate during signal transduction in lymphocytes via the TcR through the PKC pathway. On the other hand, A-V could act as a potent modulator of cPKCs in Jurkat cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7646452

  10. Differential synaptic organization of GABAergic bipolar cells and non-GABAergic (glutamatergic) bipolar cells in the tiger salamander retina.

    PubMed

    Yang, Chen-Yu; Zhang, Jun; Yazulla, Stephen

    2003-01-06

    The synaptic organizations of gamma-aminobutyric acid-immunoreactive (GABA-IR, GABAergic) and non-GABA-IR (non-IR, glutamatergic) bipolar cells in salamander retina were compared by postembedding immunoelectron microscopy. A total of 238 presynaptic bipolar cell synapses were studied; 61 were GABA-IR and 177 were non-IR. Both groups were similar in that (1). they made asymmetrical ribbon synapses as well as asymmetrical non-ribbon synapses; (2). they made ribbon synapses at dyads, triads, and monads; and (3). the vast majority of ribbon synapses ( approximately 90%) were with dyads. The differences were that synapses of GABA-IR bipolar cells had a higher proportion of (1). direct contact with ganglion cells, (2). non-ribbon synapses, (3). output to GABA-IR amacrine cells, and (4). output in sublamina a. Overall, the output of GABA-IR ribbons was equally split between amacrine and ganglion cell processes, whereas for non-IR ribbons, it was approximately 2:1 in favor of amacrine cells. The ribbon:non-ribbon synapse ratio was approximately 1.2:1 (33:28) for GABA-IR but approximately 2:1 (118:59) for non-IR terminals. Thus, GABA-IR bipolar cells made more direct contacts with ganglion cells and used a higher proportion of non-ribbon synapses. GABA-IR dyads were more likely to contact GABA-IR amacrine profiles (52% vs. 38%). Finally, GABA-IR ribbon synapses were more common in sublamina a than sublamina b (2:1), whereas non-IR synapses were equally present in sublaminas a and b. This differential targeting of ganglion cells and amacrine cells in the OFF vs. ON layers indicates a difference in the role of bipolar cells in the generation of receptive field properties, depending on whether or not they use GABA as well as glutamate for their transmitter.

  11. Role of apolipoprotein A-II in the structure and remodeling of human high-density lipoprotein (HDL): protein conformational ensemble on HDL.

    PubMed

    Gao, Xuan; Yuan, Shujun; Jayaraman, Shobini; Gursky, Olga

    2012-06-12

    High-density lipoproteins (HDL, or "good cholesterol") are heterogeneous nanoparticles that remove excess cell cholesterol and protect against atherosclerosis. The cardioprotective action of HDL and its major protein, apolipoprotein A-I (apoA-I), is well-established, yet the function of the second major protein, apolipoprotein A-II (apoA-II), is less clear. In this review, we postulate an ensemble of apolipoprotein conformations on various HDL. This ensemble is based on the crystal structure of Δ(185-243)apoA-I determined by Mei and Atkinson combined with the "double-hairpin" conformation of apoA-II(dimer) proposed in the cross-linking studies by Silva's team, and is supported by the wide array of low-resolution structural, biophysical, and biochemical data obtained by many teams over decades. The proposed conformational ensemble helps integrate and improve several existing HDL models, including the "buckle-belt" conformation of apoA-I on the midsize disks and the "trefoil/tetrafoil" arrangement on spherical HDL. This ensemble prompts us to hypothesize that endogenous apoA-II (i) helps confer lipid surface curvature during conversion of nascent discoidal HDL(A-I) and HDL(A-II) containing either apoA-I or apoA-II to mature spherical HDL(A-I/A-II) containing both proteins, and (ii) hinders remodeling of HDL(A-I/A-II) by hindering the expansion of the apoA-I conformation. Also, we report that, although endogenous apoA-II circulates mainly on the midsize spherical HDL(A-I/A-II), exogenous apoA-II can bind to HDL of any size, thereby slightly increasing this size and stabilizing the HDL assembly. This suggests distinctly different effects of the endogenous and exogenous apoA-II on HDL. Taken together, the existing results and models prompt us to postulate a new structural and functional role of apoA-II on human HDL.

  12. Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines

    PubMed Central

    Vuong, Helen E.; de Sevilla Müller, Luis Pérez; Hardi, Claudia N.; McMahon, Douglas G.; Brecha, Nicholas C.

    2015-01-01

    Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) line with three catecholamine-related Cre recombinase lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ~6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium somal diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines were generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. PMID:26335381

  13. Elevated glucose and angiotensin II increase 12-lipoxygenase activity and expression in porcine aortic smooth muscle cells.

    PubMed Central

    Natarajan, R; Gu, J L; Rossi, J; Gonzales, N; Lanting, L; Xu, L; Nadler, J

    1993-01-01

    The lipoxygenase (LO) pathway of arachidonate metabolism has been suggested to play a key role in atherosclerosis and in mediating several actions of angiotensin II (AII). However, the relationship between LO activation and factors linked to accelerated diabetic vascular disease such as hyperglycemia and AII is not known. We have investigated the effect of high glucose (HG; 25 mM) and AII on LO activity as well as LO protein and mRNA expression in porcine aortic vascular smooth muscle cells (PVSMCs). We observed that cells cultured in HG had significantly higher levels of the cell-associated LO products 12- and 15-hydroxyeicosatetraenoic acids (HETEs). AII added to cells grown in HG specifically further increased only cell-associated 12-HETE levels. Using immunoblot analysis and reverse transcriptase PCRs, we demonstrated the presence in PVSMCs of porcine leukocyte-type 12-LO protein and mRNA, respectively. Furthermore, the levels of both were markedly upregulated by AII as well as by HG. These studies suggest that enhanced 12-LO activity and expression are mechanisms for accelerated vascular disease produced by HG and AII in diabetes mellitus. Images Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8506339

  14. Bioinformatic Analysis of Plasma Apolipoproteins A-I and A-II Revealed Unique Features of A-I/A-II HDL Particles in Human Plasma

    PubMed Central

    Kido, Toshimi; Kurata, Hideaki; Kondo, Kazuo; Itakura, Hiroshige; Okazaki, Mitsuyo; Urata, Takeyoshi; Yokoyama, Shinji

    2016-01-01

    Plasma concentration of apoA-I, apoA-II and apoA-II-unassociated apoA-I was analyzed in 314 Japanese subjects (177 males and 137 females), including one (male) homozygote and 37 (20 males and 17 females) heterozygotes of genetic CETP deficiency. ApoA-I unassociated with apoA-II markedly and linearly increased with HDL-cholesterol, while apoA-II increased only very slightly and the ratio of apoA-II-associated apoA-I to apoA-II stayed constant at 2 in molar ratio throughout the increase of HDL-cholesterol, among the wild type and heterozygous CETP deficiency. Thus, overall HDL concentration almost exclusively depends on HDL with apoA-I without apoA-II (LpAI) while concentration of HDL containing apoA-I and apoA-II (LpAI:AII) is constant having a fixed molar ratio of 2 : 1 regardless of total HDL and apoA-I concentration. Distribution of apoA-I between LpAI and LpAI:AII is consistent with a model of statistical partitioning regardless of sex and CETP genotype. The analysis also indicated that LpA-I accommodates on average 4 apoA-I molecules and has a clearance rate indistinguishable from LpAI:AII. Independent evidence indicated LpAI:A-II has a diameter 20% smaller than LpAI, consistent with a model having two apoA-I and one apoA-II. The functional contribution of these particles is to be investigated. PMID:27526664

  15. RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility

    PubMed Central

    Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

    2015-01-01

    Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmAII enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmAII, rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmAII in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmAII activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmAII, thereby facilitating TEL binding to the ribosome. PMID:26365244

  16. Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells

    PubMed Central

    Wagner, Carsten A.; Giebisch, Gerhard; Lang, Florian; Geibel, John P.

    1998-01-01

    Two mechanisms of H+ ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H+ ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na+/H+ exchanger and the basolateral Na+/HCO3− cotransporter. In the present study, we examined the effects of AII on H+-ATPase activity in isolated proximal tubule fragments. H+-ATPase activity was assessed by monitoring intracellular pH after Na+ removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A1, removal of Cl−, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na+ and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A1, by removal of Cl−, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl− channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H+-ATPase by a Cl−-dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO3− reabsorption along the proximal tubule when tubules are exposed to AII. PMID:9689138

  17. Regulation of ERK5 by insulin and angiotensin-II in vascular smooth muscle cells

    SciTech Connect

    Sharma, Girish; Goalstone, Marc Lee; E-mail: Marc.Goalstone@uchsc.edu

    2007-03-23

    ERK5 is involved in proliferation of vascular smooth muscle cells (VSMC). The proliferative actions of insulin and angiotensin-II (A-II) in VSMC are mediated in part by ERK1/2. We hypothesized that insulin and A-II also regulate ERK5 activity in VSMC. Acute treatment (<60 min) with insulin or A-II increased phosphorylation of ERK1/2 at 15 min and ERK5 at 5 min. Chronic treatment ({<=}8 h) with insulin increased ERK1/2 phosphorylation by 4 h and ERK5 by 8 h. A-II-stimulated phosphorylation of ERK1/2 by 8 h and ERK5 by 4 h. The EC{sub 50} for insulin treatment effecting ERK1/2 and ERK5 phosphorylation was 1.5 and 0.1 nM, whereas the EC{sub 50} for A-II was 2 nM, each. Insulin plus A-II induced an additive effect only on ERK5 phosphorylation. Inhibition of insulin- and A-II-stimulated phosphorylation of ERK5 and ERK1/2 by PD98059 and Wortmannin exhibited differential and time-dependent effects. Taken together, these data indicate that insulin and A-II regulate the activity of ERK5, but different from that seen for ERK1/2.

  18. MATH5 controls the acquisition of multiple retinal cell fates

    PubMed Central

    2010-01-01

    Math5-null mutation results in the loss of retinal ganglion cells (RGCs) and in a concurrent increase of amacrine and cone cells. However, it remains unclear whether there is a cell fate switch of Math5-lineage cells in the absence of Math5 and whether MATH5 cell-autonomously regulates the differentiation of the above retinal neurons. Here, we performed a lineage analysis of Math5-expressing cells in developing mouse retinas using a conditional GFP reporter (Z/EG) activated by a Math5-Cre knock-in allele. We show that during normal retinogenesis, Math5-lineage cells mostly develop into RGCs, horizontal cells, cone photoreceptors, rod photoreceptors, and amacrine cells. Interestingly, amacrine cells of Math5-lineage cells are predominately of GABAergic, cholinergic, and A2 subtypes, indicating that Math5 plays a role in amacrine subtype specification. In the absence of Math5, more Math5-lineage cells undergo cell fate conversion from RGCs to the above retinal cell subtypes, and occasionally to cone-bipolar cells and Müller cells. This change in cell fate choices is accompanied by an up-regulation of NEUROD1, RXRγ and BHLHB5, the transcription factors essential for the differentiation of retinal cells other than RGCs. Additionally, loss of Math5 causes the failure of early progenitors to exit cell cycle and leads to a significant increase of Math5-lineage cells remaining in cell cycle. Collectively, these data suggest that Math5 regulates the generation of multiple retinal cell types via different mechanisms during retinogenesis. PMID:21087508

  19. Fat3 and Ena/VASP proteins influence the emergence of asymmetric cell morphology in the developing retina.

    PubMed

    Krol, Alexandra; Henle, Steven J; Goodrich, Lisa V

    2016-06-15

    Neurons exhibit asymmetric morphologies throughout development - from migration to the elaboration of axons and dendrites - that are correctly oriented for the flow of information. For instance, retinal amacrine cells migrate towards the inner plexiform layer (IPL) and then retract their trailing processes, thereby acquiring a unipolar morphology with a single dendritic arbor restricted to the IPL. Here, we provide evidence that the Fat-like cadherin Fat3 acts during multiple stages of amacrine cell development in mice to orient overall changes in cell shape towards the IPL. Using a time-lapse imaging assay, we found that developing amacrine cells are less directed towards the IPL in the absence of Fat3, during both migration and retraction. Consistent with its predicted role as a cell-surface receptor, Fat3 functions cell-autonomously and is able to influence the cytoskeleton directly through its intracellular domain, which can bind and localize Ena/VASP family actin regulators. Indeed, a change in Ena/VASP protein distribution is sufficient to recapitulate the Fat3 mutant amacrine cell phenotype. Thus, Fat-like proteins might control the polarized development of tissues by sculpting the cytoskeleton of individual cells. © 2016. Published by The Company of Biologists Ltd.

  20. Fat3 and Ena/VASP proteins influence the emergence of asymmetric cell morphology in the developing retina

    PubMed Central

    Krol, Alexandra; Henle, Steven J.

    2016-01-01

    Neurons exhibit asymmetric morphologies throughout development – from migration to the elaboration of axons and dendrites – that are correctly oriented for the flow of information. For instance, retinal amacrine cells migrate towards the inner plexiform layer (IPL) and then retract their trailing processes, thereby acquiring a unipolar morphology with a single dendritic arbor restricted to the IPL. Here, we provide evidence that the Fat-like cadherin Fat3 acts during multiple stages of amacrine cell development in mice to orient overall changes in cell shape towards the IPL. Using a time-lapse imaging assay, we found that developing amacrine cells are less directed towards the IPL in the absence of Fat3, during both migration and retraction. Consistent with its predicted role as a cell-surface receptor, Fat3 functions cell-autonomously and is able to influence the cytoskeleton directly through its intracellular domain, which can bind and localize Ena/VASP family actin regulators. Indeed, a change in Ena/VASP protein distribution is sufficient to recapitulate the Fat3 mutant amacrine cell phenotype. Thus, Fat-like proteins might control the polarized development of tissues by sculpting the cytoskeleton of individual cells. PMID:27122175

  1. Thyroid Hormone Signaling in the Mouse Retina

    PubMed Central

    Arbogast, Patrick; Flamant, Frédéric; Godement, Pierre; Glösmann, Martin

    2016-01-01

    Thyroid hormone is a crucial regulator of gene expression in the developing and adult retina. Here we sought to map sites of thyroid hormone signaling at the cellular level using the transgenic FINDT3 reporter mouse model in which neurons express β-galactosidase (β-gal) under the control of a hybrid Gal4-TRα receptor when triiodothyronine (T3) and cofactors of thyroid receptor signaling are present. In the adult retina, nearly all neurons of the ganglion cell layer (GCL, ganglion cells and displaced amacrine cells) showed strong β-gal labeling. In the inner nuclear layer (INL), a minority of glycineric and GABAergic amacrine cells showed β-gal labeling, whereas the majority of amacrine cells were unlabeled. At the level of amacrine types, β-gal labeling was found in a large proportion of the glycinergic AII amacrines, but only in a small proportion of the cholinergic/GABAergic ‘starburst’ amacrines. At postnatal day 10, there also was a high density of strongly β-gal-labeled neurons in the GCL, but only few amacrine cells were labeled in the INL. There was no labeling of bipolar cells, horizontal cells and Müller glia cells at both stages. Most surprisingly, the photoreceptor somata in the outer nuclear layer also showed no β-gal label, although thyroid hormone is known to control cone opsin expression. This is the first record of thyroid hormone signaling in the inner retina of an adult mammal. We hypothesize that T3 levels in photoreceptors are below the detection threshold of the reporter system. The topographical distribution of β-gal-positive cells in the GCL follows the overall neuron distribution in that layer, with more T3-signaling cells in the ventral than the dorsal half-retina. PMID:27942035

  2. Early Retinal Neuronal Dysfunction in Diabetic Mice: Reduced Light-Evoked Inhibition Increases Rod Pathway Signaling

    PubMed Central

    Moore-Dotson, Johnnie M.; Beckman, Jamie J.; Mazade, Reece E.; Hoon, Mrinalini; Bernstein, Adam S.; Romero-Aleshire, Melissa J.; Brooks, Heddwen L.; Eggers, Erika D.

    2016-01-01

    Purpose Recent studies suggest that the neural retinal response to light is compromised in diabetes. Electroretinogram studies suggest that the dim light retinal rod pathway is especially susceptible to diabetic damage. The purpose of this study was to determine whether diabetes alters rod pathway signaling. Methods Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of streptozotocin (STZ; 75 mg/kg), and confirmed by blood glucose levels > 200 mg/dL. Six weeks after the first injection, whole-cell voltage clamp recordings of spontaneous and light-evoked inhibitory postsynaptic currents from rod bipolar cells were made in dark-adapted retinal slices. Light-evoked excitatory currents from rod bipolar and AII amacrine cells, and spontaneous excitatory currents from AII amacrine cells were also measured. Receptor inputs were pharmacologically isolated. Immunohistochemistry was performed on whole mounted retinas. Results Rod bipolar cells had reduced light-evoked inhibitory input from amacrine cells but no change in excitatory input from rod photoreceptors. Reduced light-evoked inhibition, mediated by both GABAA and GABAC receptors, increased rod bipolar cell output onto AII amacrine cells. Spontaneous release of GABA onto rod bipolar cells was increased, which may limit GABA availability for light-evoked release. These physiological changes occurred in the absence of retinal cell loss or changes in GABAA receptor expression levels. Conclusions Our results indicate that early diabetes causes deficits in the rod pathway leading to decreased light-evoked rod bipolar cell inhibition and increased rod pathway output that provide a basis for the development of early diabetic visual deficits. PMID:27028063

  3. ApoER2 Function in the Establishment and Maintenance of Retinal Synaptic Connectivity

    PubMed Central

    Trotter, Justin H.; Klein, Martin; Jinwal, Umesh K.; Abisambra, Jose F.; Dickey, Chad A.; Tharkur, Jeremy; Masiulis, Irene; Ding, Jindong; Locke, Kirstin G.; Rickman, Catherine Bowes; Birch, David G.; Weeber, Edwin J.; Herz, Joachim

    2011-01-01

    The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult ApoER2 was expressed by A-II amacrine cells. ApoER2 knockout (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding disabled-1 (Dab1) and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway while the alternatively-spliced exon19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development. VLDLR KO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in both ApoER2 and VLDLR KOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity. PMID:21976526

  4. The -256T>C Polymorphism in the Apolipoprotein A-II Gene Promoter Is Associated with Body Mass Index and Food Intake in the Genetics of Lipid Lowering Drugs and Diet Network Study

    USDA-ARS?s Scientific Manuscript database

    BACKGROUND: Apolipoprotein A-II (APOA2) plays an ambiguous role in lipid metabolism, obesity, and atherosclerosis. METHODS: We studied the association between a functional APOA2 promoter polymorphism (-265T>C) and plasma lipids (fasting and postprandial), anthropometric variables, and food intake in...

  5. ON Cone Bipolar Cell Axonal Synapses in the OFF Inner Plexiform Layer of the Rabbit Retina

    PubMed Central

    Lauritzen, J. Scott; Anderson, James R.; Jones, Bryan W.; Watt, Carl B.; Mohammed, Shoeb; Hoang, John V.; Marc, Robert E.

    2012-01-01

    Analysis of the rabbit retinal connectome RC1 reveals that the division between the ON and OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make non-canonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscope (ATEM) imaging, molecular tagging, tracing, and rendering of ≈ 400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include GABA-positive amacrine cells (γACs), glycine-positive amacrine cells (GACs) and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON-OFF amacrine cells. Many of these ON-OFF GACs target ON cone bipolar cell axons, ON γACs and/or ON-OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC-mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON-OFF ganglion cells in the OFF layer and provide ON drive to polarity-appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells. PMID:23042441

  6. Morphology and Tracer Coupling Pattern of Alpha Ganglion Cells in the Mouse Retina

    PubMed Central

    VÖLGYI, BÉLA; ABRAMS, JOSEPH; PAUL, DAVID L.; BLOOMFIELD, STEWART A.

    2010-01-01

    Alpha cells are a type of ganglion cell whose morphology appears to be conserved across a number of mammalian retinas. In particular, alpha cells display the largest somata and dendritic arbors at a given eccentricity and tile the retina as independent on- (ON) and off-center (OFF) subtypes. Mammalian alpha cells also express a variable tracer coupling pattern, which often includes homologous (same cell type) coupling to a few neighboring alpha cells and extensive heterologous (different cell type) coupling to two to three amacrine cell types. Here, we use the gap junction-permeant tracer Neurobiotin to determine the architecture and coupling pattern of alpha cells in the mouse retina. We find that alpha cells show the same somatic and dendritic architecture described previously in the mammal. However, alpha cells show varied tracer coupling patterns related to their ON and OFF physiologies. ON alpha cells show no evidence of homologous tracer coupling but are coupled heterologously to at least two types of amacrine cell whose somata lie within the ganglion cell layer. In contrast, OFF alpha cells are coupled to one another in circumscribed arrays as well as to two to three types of amacrine cell with somata occupying the inner nuclear layer. We find that homologous coupling between OFF alpha cells is unaltered in the connexin36 (Cx36) knockout (KO) mouse retina, indicating that it is not dependent on Cx36. However, a subset of the heterologous coupling of ON alpha cells and all the heterologous coupling of OFF alpha cells are eliminated in the KO retina, suggesting that Cx36 comprises most of the junctions made with amacrine cells. PMID:16175559

  7. The mechanisms of repetitive spike generation in an axonless retinal interneuron

    PubMed Central

    Cembrowski, Mark S.; Logan, Stephen M.; Tian, Miao; Jia, Li; Li, Wei; Kath, William L.; Riecke, Hermann; Singer, Joshua H.

    2012-01-01

    SUMMARY Several types of retinal interneurons exhibit spikes but lack axons. One such neuron is the AII amacrine cell, in which spikes recorded at the soma exhibit small amplitudes (<10 mV) and broad time courses (>5 ms). Here, we used electrophysiological recordings and computational analysis to examine the mechanisms underlying this atypical spiking. We found that somatic spikes likely represent large, brief action potential-like events initiated in a single, electrotonically-distal dendritic compartment. In this same compartment, spiking undergoes slow modulation, likely by an M-type K conductance. The structural correlate of this compartment is a thin neurite that extends from the primary dendritic tree: local application of TTX to this neurite, or excision of it, eliminates spiking. Thus, the physiology of the axonless AII is much more complex than would be anticipated from morphological descriptions and somatic recordings; in particular, the AII possesses a single dendritic structure that controls its firing pattern. PMID:22832164

  8. Synaptic inputs to the ganglion cells in the tiger salamander retina.

    PubMed

    Wunk, D F; Werblin, F S

    1979-03-01

    The postsynaptic potentials (PSPs) that form the ganglion cell light response were isolated by polarizing the cell membrane with extrinsic currents while stimulating at either the center or surround of the cell's receptive field. The time-course and receptive field properties of the PSPs were correlated with those of the bipolar and amacrine cells. The tiger salamander retina contains four main types of ganglion cell: "on" center, "off" center, "on-off", and a "hybrid" cell that responds transiently to center, but sustainedly, to surround illumination. The results lead to these inferences. The on-ganglion cell receives excitatory synpatic input from the on bipolars and that synapse is "silent" in the dark. The off-ganglion cell receives excitatory synaptic input from the off bipolars with this synapse tonically active in the dark. The on-off and hybrid ganglion cells receive a transient excitatory input with narrow receptive field, not simply correlated with the activity of any presynaptic cell. All cell types receive a broad field transient inhibitory input, which apparently originates in the transient amacrine cells. Thus, most, but not all, ganglion cell responses can be explained in terms of synaptic inputs from bipolar and amacrine cells, integrated at the ganglion cell membrane.

  9. Differential encoding of spatial information among retinal on cone bipolar cells

    PubMed Central

    Purgert, Robert J.

    2015-01-01

    The retina is the first stage of visual processing. It encodes elemental features of visual scenes. Distinct cone bipolar cells provide the substrate for this to occur. They encode visual information, such as color and luminance, a principle known as parallel processing. Few studies have directly examined whether different forms of spatial information are processed in parallel among cone bipolar cells. To address this issue, we examined the spatial information encoded by mouse ON cone bipolar cells, the subpopulation excited by increments in illumination. Two types of spatial processing were identified. We found that ON cone bipolar cells with axons ramifying in the central inner plexiform layer were tuned to preferentially encode small stimuli. By contrast, ON cone bipolar cells with axons ramifying in the proximal inner plexiform layer, nearest the ganglion cell layer, were tuned to encode both small and large stimuli. This dichotomy in spatial tuning is attributable to amacrine cells providing stronger inhibition to central ON cone bipolar cells compared with proximal ON cone bipolar cells. Furthermore, background illumination altered this difference in spatial tuning. It became less pronounced in bright light, as amacrine cell-driven inhibition became pervasive among all ON cone bipolar cells. These results suggest that differential amacrine cell input determined the distinct spatial encoding properties among ON cone bipolar cells. These findings enhance the known parallel processing capacity of the retina. PMID:26203104

  10. Differential encoding of spatial information among retinal on cone bipolar cells.

    PubMed

    Purgert, Robert J; Lukasiewicz, Peter D

    2015-09-01

    The retina is the first stage of visual processing. It encodes elemental features of visual scenes. Distinct cone bipolar cells provide the substrate for this to occur. They encode visual information, such as color and luminance, a principle known as parallel processing. Few studies have directly examined whether different forms of spatial information are processed in parallel among cone bipolar cells. To address this issue, we examined the spatial information encoded by mouse ON cone bipolar cells, the subpopulation excited by increments in illumination. Two types of spatial processing were identified. We found that ON cone bipolar cells with axons ramifying in the central inner plexiform layer were tuned to preferentially encode small stimuli. By contrast, ON cone bipolar cells with axons ramifying in the proximal inner plexiform layer, nearest the ganglion cell layer, were tuned to encode both small and large stimuli. This dichotomy in spatial tuning is attributable to amacrine cells providing stronger inhibition to central ON cone bipolar cells compared with proximal ON cone bipolar cells. Furthermore, background illumination altered this difference in spatial tuning. It became less pronounced in bright light, as amacrine cell-driven inhibition became pervasive among all ON cone bipolar cells. These results suggest that differential amacrine cell input determined the distinct spatial encoding properties among ON cone bipolar cells. These findings enhance the known parallel processing capacity of the retina. Copyright © 2015 the American Physiological Society.

  11. Exploring the retinal connectome

    PubMed Central

    Anderson, James R.; Jones, Bryan W.; Watt, Carl B.; Shaw, Margaret V.; Yang, Jia-Hui; DeMill, David; Lauritzen, James S.; Lin, Yanhua; Rapp, Kevin D.; Mastronarde, David; Koshevoy, Pavel; Grimm, Bradley; Tasdizen, Tolga; Whitaker, Ross

    2011-01-01

    Purpose A connectome is a comprehensive description of synaptic connectivity for a neural domain. Our goal was to produce a connectome data set for the inner plexiform layer of the mammalian retina. This paper describes our first retinal connectome, validates the method, and provides key initial findings. Methods We acquired and assembled a 16.5 terabyte connectome data set RC1 for the rabbit retina at ≈2 nm resolution using automated transmission electron microscope imaging, automated mosaicking, and automated volume registration. RC1 represents a column of tissue 0.25 mm in diameter, spanning the inner nuclear, inner plexiform, and ganglion cell layers. To enhance ultrastructural tracing, we included molecular markers for 4-aminobutyrate (GABA), glutamate, glycine, taurine, glutamine, and the in vivo activity marker, 1-amino-4-guanidobutane. This enabled us to distinguish GABAergic and glycinergic amacrine cells; to identify ON bipolar cells coupled to glycinergic cells; and to discriminate different kinds of bipolar, amacrine, and ganglion cells based on their molecular signatures and activity. The data set was explored and annotated with Viking, our multiuser navigation tool. Annotations were exported to additional applications to render cells, visualize network graphs, and query the database. Results Exploration of RC1 showed that the 2 nm resolution readily recapitulated well known connections and revealed several new features of retinal organization: (1) The well known AII amacrine cell pathway displayed more complexity than previously reported, with no less than 17 distinct signaling modes, including ribbon synapse inputs from OFF bipolar cells, wide-field ON cone bipolar cells and rod bipolar cells, and extensive input from cone-pathway amacrine cells. (2) The axons of most cone bipolar cells formed a distinct signal integration compartment, with ON cone bipolar cell axonal synapses targeting diverse cell types. Both ON and OFF bipolar cells receive

  12. Distribution of muscarinic acetylcholine receptors on processes of isolated retinal cells.

    PubMed

    Townes-Anderson, E; Vogt, B A

    1989-12-15

    Binding of propylbenzilylcholine mustard, a muscarinic acetylcholine receptor antagonist, to isolated retinal cells was examined with light microscopic autoradiography. Dissociation of the adult tiger salamander retina yielded identifiable rod, cone, horizontal, bipolar, amacrine/ganglion, and Müller cells. Preservation of fine structure was assessed with conventional electron microscopy. For all cell types, the plasmalemma was intact and free of adhering debris; in addition, presynaptic ribbon complexes were present in photoreceptor and bipolar axon terminals indicating that synaptic structures were retained. Specific binding to cell bodies and processes was analyzed separately by using morphometric and statistical techniques. The highest grain densities occurred on processes of amacrine/ganglion cells and axons and 2 degrees and 3 degrees dendrites of bipolar neurons. Bipolar cells, however, seemed to be a heterogeneous population because there was great variation in the density of binding sites on both their axons and distal dendrites. Intermediate levels of binding were found on bipolar 1 degree dendrites and horizontal cells. No specific binding was detected on Müller cells and most parts of photoreceptors. Comparisons between cells showed that grain densities were similar for bipolar axons and amacrine/ganglion cell processes but bipolar dendrites were richer in binding sites than horizontal cell dendrites. Thus, muscarinic receptors in the salamander retina are located on amacrine/ganglion, bipolar, and horizontal cells and primarily confined to the processes which compose the two synaptic layers. In the inner plexiform layer, muscarinic receptors reside on processes from all three inner retinal neurons: in the outer synaptic layer, receptors are only on second-order cells and are more numerous along bipolar than horizontal cell dendrites.

  13. Retinal ganglion cell distribution and spatial resolving power in deep-sea lanternfishes (Myctophidae).

    PubMed

    de Busserolles, Fanny; Marshall, N Justin; Collin, Shaun P

    2014-01-01

    Topographic analyses of retinal ganglion cell density are very useful in providing information about the visual ecology of a species by identifying areas of acute vision within the visual field (i.e. areas of high cell density). In this study, we investigated the neural cell distribution in the ganglion cell layer of a range of lanternfish species belonging to 10 genera. Analyses were performed on wholemounted retinas using stereology. Topographic maps were constructed of the distribution of all neurons and both ganglion and amacrine cell populations in 5 different species from Nissl-stained retinas using cytological criteria. Amacrine cell distribution was also examined immunohistochemically in 2 of the 5 species using anti-parvalbumin antibody. The distributions of both the total neuron and the amacrine cell populations were aligned in all of the species examined, showing a general increase in cell density toward the retinal periphery. However, when the ganglion cell population was topographically isolated from the amacrine cell population, which comprised up to 80% of the total neurons within the ganglion cell layer, a different distribution was revealed. Topographic maps of the true ganglion cell distribution in 18 species of lanternfishes revealed well-defined specializations in different regions of the retina. Different species possessed distinct areas of high ganglion cell density with respect to both peak density and the location and/or shape of the specialized acute zone (i.e. elongated areae ventro-temporales, areae temporales and large areae centrales). The spatial resolving power was calculated to be relatively low (varying from 1.6 to 4.4 cycles per degree), indicating that myctophids may constitute one of the less visually acute groups of deep-sea teleosts. The diversity in retinal specializations and spatial resolving power within the family is assessed in terms of possible ecological functions and evolutionary history. © 2014 S. Karger AG, Basel.

  14. Two distinct types of ON directionally selective ganglion cells in the rabbit retina.

    PubMed

    Hoshi, Hideo; Tian, Lian-Ming; Massey, Stephen C; Mills, Stephen L

    2011-09-01

    Mammalian retinas contain about 20 types of ganglion cells that respond to different aspects of the visual scene, including the direction of motion of objects in the visual field. The rabbit retina has long been thought to contain two distinct types of directionally selective (DS) ganglion cell: a bistratified ON-OFF DS ganglion cell that responds to onset and termination of light, and an ON DS ganglion cell, which stratifies only in the ON layer and responds only to light onset. This division is challenged by targeted recordings from rabbit retina, which indicate that ON DS ganglion cells occur in two discriminably different types. One of these is strongly tracer-coupled to amacrine cells; the other is never tracer-coupled. These two types also differ in branching pattern, stratification depth, relative latency, and transience of spiking. The sustained, uncoupled ON DS cell ramifies completely within the lower cholinergic band and responds to nicotine with continuous firing. In contrast, the transient, coupled ON DS ganglion cell stratifies above the cholinergic band and is not positioned to receive major input from cholinergic amacrine cells, consistent with its modest response to the cholinergic agonist nicotine. Much data have accrued that directional responses in the mammalian retina originate via gamma-aminobutyric acid (GABA) release from the dendrites of starburst amacrine cells (Euler et al., 2002). If there is an ON DS ganglion cell that does not stratify in the starburst band, this suggests that its GABA-dependent directional signals may be generated by a mechanism independent of starburst amacrine cells. Copyright © 2011 Wiley-Liss, Inc.

  15. Restraining Erwinia virulence by expression of N-acyl homoserine lactonase gene pro3A-aiiA in Bacillus thuringiensis subsp leesis.

    PubMed

    Zhu, Chenguang; Yu, Ziniu; Sun, Ming

    2006-10-20

    To widen the biological control function of a genetically modified Bacillus thuringiensis subsp leesis strain BMB-005, an acyl homoserine lactonase (AHL lactonase) gene aiiA transcribed by the promoter of insecticidal crystal protein coding gene cry3A, was transformed into strain BMB-005. The amount of AHL lactonase protein produced by transformant BMB821A was 2.4-fold more than that produced by BMB-005. AHL-degradation assay showed that transformant BMB821A could degrade more AHLs molecules than the original strain BMB-005. The result of Erwinia carotovora pathogenicity test showed that the parental strain BMB-005 had no restraint of Erwinia infection, but the transformants exhibited strong restraint of E. carotovora infection on potato slices and cactus stems. Insecticidal bioassay against lepidopteran Spodoptera exigua showed that both strain BMB-005 and transformant BMB821A were toxic to S. exigua. The toxicity of transformant BMB821A (LC(50) was 3.8) was a little attenuated comparing with the toxicity of the original strain BMB-005 (LC(50) was 2.9). The B. thuringiensis strain BMB-005 has high toxicity against Helicoverpa armigera, Plutella xylostella, and S. exigua. This work provided new strategy for developing genetically engineered multi-functional B. thuringiensis strain that possesses insecticidal activity together with restraint of bacterial pathogenicity.

  16. Crosstalk between insulin-like growth factor-1 and angiotensin-II in dopaminergic neurons and glial cells: role in neuroinflammation and aging

    PubMed Central

    Rodriguez-Perez, Ana I.; Borrajo, Ana; Diaz-Ruiz, Carmen; Garrido-Gil, Pablo; Labandeira-Garcia, Jose L.

    2016-01-01

    The local renin-angiotensin system (RAS) and insulin-like growth factor 1 (IGF-1) have been involved in longevity, neurodegeneration and aging-related dopaminergic degeneration. However, it is not known whether IGF-1 and angiotensin-II (AII) activate each other. In the present study, AII, via type 1 (AT1) receptors, exacerbated neuroinflammation and dopaminergic cell death. AII, via AT1 receptors, also increased the levels of IGF-1 and IGF-1 receptors in microglial cells. IGF-1 inhibited RAS activity in dopaminergic neurons and glial cells, and also inhibited the AII-induced increase in markers of the M1 microglial phenotype. Consistent with this, IGF-1 decreased dopaminergic neuron death induced by the neurotoxin MPP+ both in the presence and in the absence of glia. Intraventricular administration of AII to young rats induced a significant increase in IGF-1 expression in the nigral region. However, aged rats showed decreased levels of IGF-1 relative to young controls, even though RAS activity is known to be enhanced in aged animals. The study findings show that IGF-1 and the local RAS interact to inhibit or activate neuroinflammation (i.e. transition from the M1 to the M2 phenotype), oxidative stress and dopaminergic degeneration. The findings also show that this mechanism is impaired in aged animals. PMID:27167199

  17. The nuclear hormone receptor gene Nr2c1 (Tr2) is a critical regulator of early retina cell patterning.

    PubMed

    Olivares, Ana Maria; Han, Yinan; Soto, David; Flattery, Kyle; Marini, Joseph; Molemma, Nissa; Haider, Ali; Escher, Pascal; DeAngelis, Margaret M; Haider, Neena B

    2017-09-01

    Nuclear hormone receptors play a major role in the development of many tissues. This study uncovers a novel role for testicular receptor 2 (Tr2, Nr2c1) in defining the early phase of retinal development and regulating normal retinal cell patterning and topography. The mammalian retina undergoes an overlapping yet biphasic period of development to generate all seven retinal cell types. We discovered that Nr2c1 expression coincides with development of the early retinal cells. Loss of Nr2c1 causes a severe vision deficit and impacts early, but not late retina cell types. Retinal cone cell topography is disrupted with an increase in displaced amacrine cells. Additionally, genetic background significantly impacts phenotypic outcome of cone photoreceptor cells but not amacrine cells. Chromatin-IP experiments reveal NR2C1 regulates early cell transcription factors that regulate retinal progenitor cells during development, including amacrine (Satb2) and cone photoreceptor regulators thyroid and retinoic acid receptors. This study supports a role for Nr2c1 in defining the biphasic period of retinal development and specifically influencing the early phase of retinal cell fate. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Light adaptation alters the source of inhibition to the mouse retinal OFF pathway

    PubMed Central

    Mazade, Reece E.

    2013-01-01

    Sensory systems must avoid saturation to encode a wide range of stimulus intensities. One way the retina accomplishes this is by using both dim-light-sensing rod and bright-light-sensing cone photoreceptor circuits. OFF cone bipolar cells are a key point in this process, as they receive both excitatory input from cones and inhibitory input from AII amacrine cells via the rod pathway. However, in addition to AII amacrine cell input, other inhibitory inputs from cone pathways also modulate OFF cone bipolar cell light signals. It is unknown how these inhibitory inputs to OFF cone bipolar cells change when switching between rod and cone pathways or whether all OFF cone bipolar cells receive rod pathway input. We found that one group of OFF cone bipolar cells (types 1, 2, and 4) receive rod-mediated inhibitory inputs that likely come from the rod-AII amacrine cell pathway, while another group of OFF cone bipolar cells (type 3) do not. In both cases, dark-adapted rod-dominant light responses showed a significant contribution of glycinergic inhibition, which decreased with light adaptation and was, surprisingly, compensated by an increase in GABAergic inhibition. As GABAergic input has distinct timing and spatial spread from glycinergic input, a shift from glycinergic to GABAergic inhibition could significantly alter OFF cone bipolar cell signaling to downstream OFF ganglion cells. Larger GABAergic input could reflect an adjustment of OFF bipolar cell spatial inhibition, which may be one mechanism that contributes to retinal spatial sensitivity in the light. PMID:23926034

  19. Method development and analysis of retail foods and beverages for carotenoid food colouring materials E160a(ii) and E160e.

    PubMed

    Scotter, M J; Castle, L; Croucher, J M; Olivier, L

    2003-02-01

    An analytical method using high-performance liquid chromatography with photodiode array detection was developed and applied to the determination of the permitted food colour additives beta-carotene(E160a(ii)) and beta-apo-8'-carotenal (E160e) in foods and beverages. The scope of previously reported methods has been broadened to cover a wide range of retail foods and enzymatic hydrolysis has been used in place of saponification for high-fat samples. Quantitative results (greater then 0.1 mg kg(-1)) are given for the major colour principals trans-beta-apo-8'-carotenal and trans-beta-carotene. Semiquantitative results are given for the various cis-isomers of each colorant for which authentic reference standards were not available. The method has been used successfully for the analysis of a wide range of foodstuffs with differing fat content without the need for saponification, except for moderate- to high-fat foodstuffs containing significant levels of emulsifiers, for which it was limited. The results suggest that beta-apo-8'-carotenal (E160e) does not have widespread use in the UK. None of the samples exhibited a total beta-carotene content greater than 20 mg kg(-1) and none of the high-fat samples and only one of the 17 low-fat/beverage samples contained total beta-carotene at levels less than 0.1 mg kg(-1). The total beta-carotene contents of the low-fat/beverage samples ranged from 0.4 +/- 0.03 to 8.4 i 0.71 mg kg(-1), and the total beta-carotene contents of the high-fat samples ranged from 0.1 +/- 0.01 (jelly confectionery) to 18.5 +/- 0.98 mg kg(-1) (processed cheese).

  20. A Novel Retinal Oscillation Mechanism in an Autosomal Dominant Photoreceptor Degeneration Mouse Model.

    PubMed

    Tu, Hung-Ya; Chen, Yu-Jiun; McQuiston, Adam R; Chiao, Chuan-Chin; Chen, Ching-Kang

    2015-01-01

    It has been shown in rd1 and rd10 models of photoreceptor degeneration (PD) that inner retinal neurons display spontaneous and rhythmic activities. Furthermore, the rhythmic activity has been shown to require the gap junction protein connexin 36, which is likely located in AII amacrine cells (AII-ACs). In the present study, an autosomal dominant PD model called rhoΔCTA, whose rods overexpress a C-terminally truncated mutant rhodopsin and degenerate with a rate similar to that of rd1, was used to investigate the generality and mechanisms of heightened inner retinal activity following PD. To fluorescently identify cholinergic starburst amacrine cells (SACs), the rhoΔCTA mouse was introduced into a combined ChAT-IRES-Cre and Ai9 background. In this mouse, we observed excitatory postsynaptic current (EPSC) oscillation and non-rhythmic inhibitory postsynaptic current (IPSC) in both ON- and OFF-SACs. The IPSCs were more noticeable in OFF- than in ON-SACs. Similar to reported retinal ganglion cell (RGC) oscillation in rd1 mice, EPSC oscillation was synaptically driven by glutamate and sensitive to blockade of NaV channels and gap junctions. These data suggest that akin to rd1 mice, AII-AC is a prominent oscillator in rhoΔCTA mice. Surprisingly, OFF-SAC but not ON-SAC EPSC oscillation could readily be enhanced by GABAergic blockade. More importantly, weakening the AII-AC gap junction network by activating retinal dopamine receptors abolished oscillations in ON-SACs but not in OFF-SACs. Furthermore, the latter persisted in the presence of flupirtine, an M-type potassium channel activator recently reported to dampen intrinsic AII-AC bursting. These data suggest the existence of a novel oscillation mechanism in mice with PD.

  1. Brain-derived neurotrophic factor modulates the dopaminergic network in the rat retina after axotomy.

    PubMed

    Lee, Eun-Jin; Song, Myoung-Chul; Kim, Hyun-Ju; Lim, Eun-Jin; Kim, In-Beom; Oh, Su-Ja; Moon, Jung-I L; Chun, Myung-Hoon

    2005-11-01

    Dopaminergic cells in the retina express the receptor for brain-derived neurotrophic factor (BDNF), which is the neurotrophic factor that influences the plasticity of synapses in the central nervous system. We sought to determine whether BDNF influences the network of dopaminergic amacrine cells in the axotomized rat retina, by immunocytochemistry with an anti-tyrosine hydroxylase (TH) antiserum. In the control retina, we found two types of TH-immunoreactive amacrine cells, type I and type II, in the inner nuclear layer adjacent to the inner plexiform layer (IPL). The type I amacrine cell varicosities formed ring-like structures in contact with AII amacrine cell somata in stratum 1 of the IPL. In the axotomized retinas, TH-labeled processes formed loose networks of fibers, unlike the dense networks in the control retina, and the ring-like structures were disrupted. In the axotomized retinas treated with BDNF, strong TH-immunoreactive varicosities were present in stratum 1 of the IPL and formed ring-like structures. Our data suggest that BDNF affects the expression of TH immunoreactivity in the axotomized rat retina and may therefore influence the retinal dopaminergic system.

  2. Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration.

    PubMed Central

    Ling, B N; Seal, E E; Eaton, D C

    1993-01-01

    We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin

  3. Structural relation of phase A to ringwoodite: predicted possible low-pressure polymorph of Mg 7Si 2H 6O 14 (phase AII) derived as recombination structure from forsterite

    NASA Astrophysics Data System (ADS)

    Kudoh, Y.

    2004-06-01

    A possible low-pressure polymorph of Mg 7Si 2H 6O 14 (phase AII) was derived from forsterite as a recombination structure. The present study suggests that phase A, high-pressure polymorph of Mg 7Si 2H 6O 14, can be regarded as one member of a homologous series of hypothetical post-humite high-pressure phases derived from ringwoodite as a parent structure. This implies the possible significance of post-humite high-pressure phases whose stability field might be close to phase A as candidates for the hosts of water in the mantle.

  4. Mechanisms of cGMP-dependent mesangial-cell relaxation: a role for myosin light-chain phosphatase activation.

    PubMed Central

    Torrecillas, G; Díez-Marqués, M L; García-Escribano, C; Bosch, R J; Rodríguez-Puyol, D; Rodríguez-Puyol, M

    2000-01-01

    Although the cGMP-dependent relaxation of contractile cells seems to depend on the ability of the cyclic nucleotide to interfere with intracellular calcium, this does not appear to be the only mechanism involved. The present experiments were designed to analyse alternative mechanisms, trying to test the hypothesis that cGMP could relax rat mesangial cells by activating myosin light-chain phosphatase (MLC-PP), with the subsequent dephosphorylation of myosin light chain (MLC). The effect of a cGMP analogue, dibutyryl cGMP (dbcGMP), on angiotensin II-(AII) and PMA-induced MLC phosphorylation (MLCP) was tested, in the presence of calyculin A (CA), an inhibitor of MLC-PP. MLCP was measured, after cell labelling with (32)P, by immunoprecipitation. dbcGMP prevented the increased MLCP induced by AII or PMA, and this inhibition was blocked by CA. dbcGMP also increased the MLC dephosphorylation observed in cells incubated with AII and in which MLC kinase and protein kinase C activities were blocked. The AII-elicited increased intracellular calcium concentration was only partially inhibited by dbcGMP. These results suggest that the cGMP-induced mesangial-cell relaxation could be due, at least partially, to the stimulation of MLC-PP. PMID:10657260

  5. Structure, infrared and Raman spectroscopic studies of newly synthetic AII(SbV0.50FeIII0.50)(PO4)2 (Adbnd Ba, Sr, Pb) phosphates with yavapaiite structure

    NASA Astrophysics Data System (ADS)

    Aatiq, Abderrahim; Tigha, My Rachid; Fakhreddine, Rachid; Bregiroux, Damien; Wallez, Gilles

    2016-08-01

    The synthesis and structural study of three new AII(SbV0.5FeIII0.5)(PO4)2 (Adbnd Ba, Sr, Pb) phosphates belonging to the Asbnd Sbsbnd Fesbnd Psbnd O system were reported here for the first time. Structures of [Ba], [Sr] and [Pb] compounds, obtained by solid state reaction in air atmosphere, were determined at room temperature from X-ray powder diffraction using the Rietveld method. BaII(SbV0.5FeIII0.5)(PO4)2 features the yavapaiite-type structure, with space group C2/m, Z = 2 and a = 8.1568(4) Å; b = 5.1996(3) Å c = 7.8290(4) Å; β = 94.53(1)°. AII(SbV0.5FeIII0.5)(PO4)2 (Adbnd Sr, Pb) compounds have a distorted yavapaiite structure with space group C2/c, Z = 4 and a = 16.5215(2) Å; b = 5.1891(1) Å c = 8.0489(1) Å; β = 115.70(1)° for [Sr]; a = 16.6925(2) Å; b = 5.1832(1) Å c = 8.1215(1) Å; β = 115.03(1)° for [Pb]. Raman and Infrared spectroscopic study was used to obtain further structural information about the nature of bonding in selected compositions.

  6. Neurotrophins and development of the rod pathway: an elementary deduction.

    PubMed

    Rickman, D W

    2000-07-15

    The rodent retina is a particularly attractive model for the study of neuronal developmental processes since considerable neurogenesis, cellular migration, phenotypic differentiation of retinal cell types and synaptogenesis occurs postnatally. In addition, the retina is readily accessible to surgical intervention, pharmacological manipulation, and local suppression of gene expression-tools that can be utilized to study mechanisms underlying the development of retinal neurons and their interconnections that form distinct functional circuits. Here, I review our studies describing the ontogeny of a specific retinal interneuron, the AII amacrine cell, an integral element in the rod (scotopic) pathway. Specifically, we used a number of approaches to examine the potential role of neurotrophic factors on the morphological and neurochemical differentiation of the AII.

  7. Complexin 3 Increases the Fidelity of Signaling in a Retinal Circuit by Regulating Exocytosis at Ribbon Synapses.

    PubMed

    Mortensen, Lena S; Park, Silvia J H; Ke, Jiang-Bin; Cooper, Benjamin H; Zhang, Lei; Imig, Cordelia; Löwel, Siegrid; Reim, Kerstin; Brose, Nils; Demb, Jonathan B; Rhee, Jeong-Seop; Singer, Joshua H

    2016-06-07

    Complexin (Cplx) proteins modulate the core SNARE complex to regulate exocytosis. To understand the contributions of Cplx to signaling in a well-characterized neural circuit, we investigated how Cplx3, a retina-specific paralog, shapes transmission at rod bipolar (RB)→AII amacrine cell synapses in the mouse retina. Knockout of Cplx3 strongly attenuated fast, phasic Ca(2+)-dependent transmission, dependent on local [Ca(2+)] nanodomains, but enhanced slower Ca(2+)-dependent transmission, dependent on global intraterminal [Ca(2+)] ([Ca(2+)]I). Surprisingly, coordinated multivesicular release persisted at Cplx3(-/-) synapses, although its onset was slowed. Light-dependent signaling at Cplx3(-/-) RB→AII synapses was sluggish, owing largely to increased asynchronous release at light offset. Consequently, propagation of RB output to retinal ganglion cells was suppressed dramatically. Our study links Cplx3 expression with synapse and circuit function in a specific retinal pathway and reveals a role for asynchronous release in circuit gain control.

  8. Retinal light trapping in textured photovoltaic cells

    NASA Astrophysics Data System (ADS)

    Kravets, V. G.; Grigorenko, A. N.

    2010-10-01

    We suggest a new structure for light trapping in solar cells which is loosely based on retina of human eye. In this design, the incident light is scattered by noble metal nanoparticles acting as amacrine retinal cells and then is guided and concentrated by conelike structures. We show that the proposed textured structure should lead to a significant enhancement of optical path of trapped light resulting in a higher degree of light conversion into electric current. The proposed design should work efficiently in direct sunlight and in cloudy weather.

  9. Melanopsin‐expressing ganglion cells on macaque and human retinas form two morphologically distinct populations

    PubMed Central

    Liao, Hsi‐Wen; Ren, Xiaozhi; Peterson, Beth B.; Marshak, David W.; Yau, King‐Wai; Gamlin, Paul D.

    2016-01-01

    ABSTRACT The long‐term goal of this research is to understand how retinal ganglion cells that express the photopigment melanopsin, also known as OPN4, contribute to vision in humans and other primates. Here we report the results of anatomical studies using our polyclonal antibody specifically against human melanopsin that confirm and extend previous descriptions of melanopsin cells in primates. In macaque and human retina, two distinct populations of melanopsin cells were identified based on dendritic stratification in either the inner or the outer portion of the inner plexiform layer (IPL). Variation in dendritic field size and cell density with eccentricity was confirmed, and dendritic spines, a new feature of melanopsin cells, were described. The spines were the sites of input from DB6 diffuse bipolar cell axon terminals to the inner stratifying type of melanopsin cells. The outer stratifying melanopsin type received inputs from DB6 bipolar cells via a sparse outer axonal arbor. Outer stratifying melanopsin cells also received inputs from axon terminals of dopaminergic amacrine cells. On the outer stratifying melanopsin cells, ribbon synapses from bipolar cells and conventional synapses from amacrine cells were identified in electron microscopic immunolabeling experiments. Both inner and outer stratifying melanopsin cell types were retrogradely labeled following tracer injection in the lateral geniculate nucleus (LGN). In addition, a method for targeting melanopsin cells for intracellular injection using their intrinsic fluorescence was developed. This technique was used to demonstrate that melanopsin cells were tracer coupled to amacrine cells and would be applicable to electrophysiological experiments in the future. J. Comp. Neurol. 524:2845–2872, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26972791

  10. Melanopsin-expressing ganglion cells on macaque and human retinas form two morphologically distinct populations.

    PubMed

    Liao, Hsi-Wen; Ren, Xiaozhi; Peterson, Beth B; Marshak, David W; Yau, King-Wai; Gamlin, Paul D; Dacey, Dennis M

    2016-10-01

    The long-term goal of this research is to understand how retinal ganglion cells that express the photopigment melanopsin, also known as OPN4, contribute to vision in humans and other primates. Here we report the results of anatomical studies using our polyclonal antibody specifically against human melanopsin that confirm and extend previous descriptions of melanopsin cells in primates. In macaque and human retina, two distinct populations of melanopsin cells were identified based on dendritic stratification in either the inner or the outer portion of the inner plexiform layer (IPL). Variation in dendritic field size and cell density with eccentricity was confirmed, and dendritic spines, a new feature of melanopsin cells, were described. The spines were the sites of input from DB6 diffuse bipolar cell axon terminals to the inner stratifying type of melanopsin cells. The outer stratifying melanopsin type received inputs from DB6 bipolar cells via a sparse outer axonal arbor. Outer stratifying melanopsin cells also received inputs from axon terminals of dopaminergic amacrine cells. On the outer stratifying melanopsin cells, ribbon synapses from bipolar cells and conventional synapses from amacrine cells were identified in electron microscopic immunolabeling experiments. Both inner and outer stratifying melanopsin cell types were retrogradely labeled following tracer injection in the lateral geniculate nucleus (LGN). In addition, a method for targeting melanopsin cells for intracellular injection using their intrinsic fluorescence was developed. This technique was used to demonstrate that melanopsin cells were tracer coupled to amacrine cells and would be applicable to electrophysiological experiments in the future. J. Comp. Neurol. 524:2845-2872, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

  11. Resveratrol inhibits the intracellular calcium increase and angiotensin/endothelin system activation induced by soluble uric acid in mesangial cells.

    PubMed

    Albertoni, G; Schor, N

    2014-10-24

    Resveratrol (Resv) is natural polyphenol found in grapes. This study evaluated the protective effect of Resv against the effects of uric acid (UA) in immortalized human mesangial cells (ihMCs). ihMCs were preincubated with Resv (12.5 µM) for 1 h and treated with UA (10 mg/dL) for 6 or 12 h. The intracellular calcium concentration [Ca2+]i was quantified by fluorescence using flow cytometry. Angiotensinogen (AGT) and pre-pro endothelin-1 (ppET-1) mRNA were assayed by quantitative real-time RT-PCR. Angiotensin II (AII) and endothelin-1 (ET-1) were assayed by ELISA. UA significantly increased [Ca2+]i. Pre-incubation with Resv significantly reduced the change in [Ca2+]i induced by UA. Incubation with UA for 6 or 12 h also increased AGT mRNA expression and AII protein synthesis. Resv blunted these increases in AGT mRNA expression and AII protein. Incubation with UA in the ihMCs increased ppET-1 expression and ET-1 protein synthesis at 6 and 12 h. When ihMCs were pre-incubated with Resv, UA had a significantly diminished effect on ppET-1 mRNA expression and ET-1 protein synthesis at 6 and 12 h, respectively. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. The renin-angiotensin system may contribute to the pathogenesis of renal function and chronic kidney disease. Resv can minimize the impact of UA on AII, ET-1 and the increase of [Ca2+]i in mesangial cells, suggesting that, at least in part, Resv can prevent the effects of soluble UA in mesangial cells.

  12. Resveratrol inhibits the intracellular calcium increase and angiotensin/endothelin system activation induced by soluble uric acid in mesangial cells.

    PubMed

    Albertoni, G; Schor, N

    2015-01-01

    Resveratrol (Resv) is natural polyphenol found in grapes. This study evaluated the protective effect of Resv against the effects of uric acid (UA) in immortalized human mesangial cells (ihMCs). ihMCs were preincubated with Resv (12.5 µM) for 1 h and treated with UA (10 mg/dL) for 6 or 12 h. The intracellular calcium concentration [Ca2+]i was quantified by fluorescence using flow cytometry. Angiotensinogen (AGT) and pre-pro endothelin-1 (ppET-1) mRNA were assayed by quantitative real-time RT-PCR. Angiotensin II (AII) and endothelin-1 (ET-1) were assayed by ELISA. UA significantly increased [Ca2+]i. Pre-incubation with Resv significantly reduced the change in [Ca2+]i induced by UA. Incubation with UA for 6 or 12 h also increased AGT mRNA expression and AII protein synthesis. Resv blunted these increases in AGT mRNA expression and AII protein. Incubation with UA in the ihMCs increased ppET-1 expression and ET-1 protein synthesis at 6 and 12 h. When ihMCs were pre-incubated with Resv, UA had a significantly diminished effect on ppET-1 mRNA expression and ET-1 protein synthesis at 6 and 12 h, respectively. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. The renin-angiotensin system may contribute to the pathogenesis of renal function and chronic kidney disease. Resv can minimize the impact of UA on AII, ET-1 and the increase of [Ca2+]i in mesangial cells, suggesting that, at least in part, Resv can prevent the effects of soluble UA in mesangial cells.

  13. Twelve chromatically opponent ganglion cell types in turtle retina.

    PubMed

    Rocha, F A F; Saito, C A; Silveira, L C L; de Souza, J M; Ventura, D F

    2008-01-01

    The turtle retina has been extensively used for the study of chromatic processing mechanisms. Color opponency has been previously investigated with trichromatic paradigms, but behavioral studies show that the turtle has an ultraviolet (UV) channel and a tetrachromatic visual system. Our laboratory has been working in the characterization of neuronal responses in the retina of vertebrates using stimuli in the UV-visible range of the electromagnetic spectrum. In the present investigation, we recorded color-opponent responses from turtle amacrine and ganglion cells to UV and visible stimuli and extended our previous results that UV color-opponency is present at the level of the inner nuclear layer. We recorded from 181 neurons, 36 of which were spectrally opponent. Among these, there were 10 amacrine (5%), and 26 ganglion cells (15%). Morphological identification of color-opponent neurons was possible for two ganglion cell classes (G17 and G22) and two amacrine cell classes (A22 and A23b). There was a variety of cell response types and a potential for complex processing of chromatic stimuli, with intensity- and wavelength-dependent response components. Ten types of color opponency were found in ganglion cells and by adding previous results from our laboratory, 12 types of opponent responses have been found. The majority of the ganglion cells were R+UVBG- and RG+UVB-color-opponents but there were other less frequent types of chromatic opponency. This study confirms the participation of a UV channel in the processing of color opponency in the turtle inner retina and shows that the turtle visual system has the retinal mechanisms to allow many possible chromatic combinations.

  14. Purification and characterization of two new cell-bound bioactive compounds produced by wild Lactococcus lactis strain.

    PubMed

    Saraiva, Margarete Alice Fontes; Brede, Dag Anders; Nes, Ingolf Figved; Baracat-Pereira, Maria Cristina; de Queiroz, Marisa Vieira; de Moraes, Célia Alencar

    2017-07-03

    Novel compounds and innovative methods are required considering that antibiotic resistance has reached a crisis point. In the study, two cell-bound antimicrobial compounds produced by Lactococcus lactis ID1.5 were isolated and partially characterized. Following purification by cationic exchange and a solid-phase C18 column, antimicrobial activity was recovered after three runs of RPC using 60% (v/v) and 100% (v/v) of 2-propanol for elution, suggesting that more than one antimicrobial compound were produced by L. lactis ID1.5, which were in this study called compounds AI and AII. The mass spectrum of AI and AII showed major intensity ions at m/z 1070.05 and 955.9 Da, respectively. The compound AI showed a spectrum of antimicrobial activity mainly against L. lactis species, while the organisms most sensitive to compound AII were Bacillus subtilis, Listeria innocua, Streptococcus pneumoniae and Pseudomonas aeruginosa. The antimicrobial activity of both compounds was suppressed by treatment with Tween 80. Nevertheless, both compounds showed high stability to heat and proteases treatments. The isolated compounds, AI and AII, showed distinct properties from other antimicrobial substances already reported as produced by L. lactis, and have a significant inhibitory effect against two clinically important respiratory pathogens. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Heat Shock Protein 70 and CHIP Promote Nox4 Ubiquitination and Degradation within the Losartan Antioxidative Effect in Proximal Tubule Cells.

    PubMed

    Gil Lorenzo, Andrea F; Costantino, Valeria V; Appiolaza, Martin López; Cacciamani, Valeria; Benardon, Maria E; Bocanegra, Victoria; Vallés, Patricia G

    2015-01-01

    Angiotensin II/Angiotensin II type 1 receptor (AT1R) effects are dependent on ROS production stimulated by NADPH oxidase activation. Hsp70 regulates a diverse set of signaling pathways through their interactions with proteins. CHIP is a E3 ubiquitin ligase that targets proteins for polyubiquitination and degradation. We study whether Hsp70/CHIP contribute to the negative regulation of Nox4 after AT1R blockage. Primary culture of proximal tubule epithelial cells (PTCs) from SHR and WKY were stimulated with Angiotensin II (AII) or treated with Losartan (L) or Losartan plus Angiotensin II (L+AII). Losartan decreased AT1R and Nox4 while enhancing caveolin-1 and Hsp70 protein expression in SHR PTCs. Immunoprecipitation and immunofluorescence proved interaction and colocalization of increased Hsp70/CHIP with decreased Nox4 in SHR PTCs (L) vs (All). Hsp72 knockdown resulted in enhanced Nox4 protein levels, NADPH oxidase activity and ROS generation in (L+AII) revealing that Losartan was unable to abrogate AII effects on Nox4 expression and oxidative activity. Moreover, MG132 exposed PTCs (L) demostrated blocked ubiquitinated Nox4 degradation and increased colocalization of Nox4/Ubiquitin by inmunofluorescence. Conversely, Hsp72 depletion reduced Nox4/Ubiquitin colocalization causing Nox4 upregulation due to proteosomal degradation inhibition, although Losartan treatment. Our study demonstrates that Hsp70 and CHIP mediates the ubiquitination and proteasomal degradation of Nox4 as part of the antioxidative effect of Losartan in SHR. © 2015 S. Karger AG, Basel.

  16. Endemicity and phylogeny of the human T cell lymphotropic virus type II subtype A from the Kayapo Indians of Brazil: evidence for limited regional dissemination.

    PubMed

    Switzer, W M; Black, F L; Pieniazek, D; Biggar, R J; Lal, R B; Heneine, W

    1996-05-01

    Long terminal repeat (LTR)-based restriction fragment length polymorphism (RFLP) analysis of human T cell lymphotropic virus type II (HTLV-II) from 17 seropositive Kayapo Indians from Brazil showed that all 17 samples contained a unique HTLV-IIa subtype (A-II). Additional RFLP screening demonstrated the presence of this subtype in two of three Brazilian blood donors and a Mexican prostitute and her child. In contrast, 129 samples from blood donors and intravenous drug users (IDUs) from the United States, two Pueblo Indian samples, five samples from Norwegian IDUs, and two samples from blood donors from Denmark were all found to be a different HTLV-IIa subtype (A-III). Phylogenetic analysis of two Kayapo and one Mexican LTR sequences showed that they cluster with a subtype A-II sequence from a Brazilian blood donor and with sequences from two prostitutes from Ghana and Cameroon. These results demonstrate that infection with the A-II subtype is endemic among the Kayapo Amerindians, has disseminated to non-Indian populations in Brazil, and is also present in Mexico. Furthermore, the A-II subtype does not appear to represent an origin for the HTLV-IIa infection in urban areas of the United States and Europe. This study provides evidence that HTLV-IIa may be a Paleo-Indian subtype as previously suggested for HTLV-IIb.

  17. Aberrant activity in retinal degeneration impairs central visual processing and relies on Cx36-containing gap junctions.

    PubMed

    Ivanova, Elena; Yee, Christopher W; Baldoni, Robert; Sagdullaev, Botir T

    2016-09-01

    In retinal degenerative disease (RD), the diminished light signal from dying photoreceptors has been considered the sole cause of visual impairment. Recent studies show a 10-fold increase in spontaneous activity in the RD network, challenging this paradigm. This aberrant activity forms a new barrier for the light signal, and not only exacerbates the loss of vision, but also may stand in the way of visual restoration. This activity originates in AII amacrine cells and relies on excessive activation of gap junctions. However, it remains unclear whether aberrant activity affects central visual processing and what mechanisms lead to this excessive activation of gap junctions. By combining genetic manipulation with electrophysiological recordings of light-induced activity in both living mice and isolated wholemount retina, we demonstrate that aberrant activity extends along retinotectal projections to alter activity in higher brain centers. Next, to selectively eliminate Cx36-containing gap junctions, which are the primary type expressed by AII amacrine cells, we crossed rd10 mice, a slow-degenerating model of RD, with Cx36 knockout mice. We found that retinal aberrant activity was reduced in the rd10/Cx36KO mice compared to rd10 controls, a direct evidence for involvement of Cx36-containing gap junctions in generating aberrant activity in RD. These data provide an essential support for future experiments to determine if selectively targeting these gap junctions could be a valid strategy for reducing aberrant activity and restoring light responses in RD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Cell-Specific Expression of Plasma Membrane Calcium ATPase Isoforms in Retinal Neurons

    PubMed Central

    Krizaj, David; Demarco, Steven J.; Johnson, Juliette; Strehler, Emanuel E.; Copenhagen, David R.

    2007-01-01

    Ca2+ extrusion by high-affinity plasma membrane calcium ATPases (PMCAs) is a principal mechanism for the clearance of Ca2+ from the cytosol. The PMCA family consists of four isoforms (PMCA1–4). Little is known about the selective expression of these isoforms in brain tissues or about the physiological function conferred upon neurons by any given isoform. We investigated the cellular and subcellular distribution of PMCA isoforms in a mammalian retina. Mouse photoreceptors, cone bipolar cells and horizontal cells, which respond to light with a graded polarization, express isoform 1 (PMCA1) of the PMCA family. PMCA2 is localized to rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and PMCA3 is predominantly expressed in spiking neurons, including both amacrine and ganglion cells but is also found in horizontal cells. PMCA4 was found to be selectively expressed in both synaptic layers. Optical measurements of Ca2+ clearance showed that PMCAs mediate Ca2+ extrusion in both rod and cone bipolar cells. In addition, we found that rod bipolar cells, but not cone bipolar cells possess a prominent Na+/Ca2+ exchange mechanism. We conclude that PMCA isoforms are selectively expressed in retinal neurons and that processes of Ca2+ clearance are different in rod and cone bipolar cells. PMID:12209837

  19. Dendritic morphology of indoleamine cells revealed by intracellular injection of lucifer yellow in fixed carp retina.

    PubMed

    Teranishi, T; Negishi, K; Hidaka, S; Naka, K I

    1987-07-01

    The dendritic morphology of indoleamine amacrine cells in carp retina was investigated by identifying their fluorescent cell bodies by preloading with noradrenaline followed by iontophoretic injection of Lucifer Yellow in isolated and aldehyde-fixed preparations under microscopic control. Although two subpopulations of serotonin-like immunoreactive amacrine cells (small and large in soma size) were found, small cells were not seen in aldehyde-fixed preparations. Cells preloaded with noradrenaline corresponded to large immunoreactive cells and were labeled with Lucifer Yellow. The cell bodies labeled were located at the innermost level of the inner nuclear layer, and gave rise to three to five primary dendrites which branched frequently and were found mainly in sublamina a of the inner plexiform layer. These cells examined in an intermediate region between the optic disc and the retinal periphery were pyriform in soma shape while dendritic fields were found or oval covering an area of 0.18 +/- 0.05 mm2 (510 +/- 80 microns in diameter). Cell density in this region was about 32 cells/mm2 and, therefore, their dendritic field coverage was approximately 6.0.

  20. Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer.

    PubMed

    Arellano, Rogelio O; Robles-Martínez, Leticia; Serrano-Flores, Bárbara; Vázquez-Cuevas, Francisco; Garay, Edith

    2012-10-01

    Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion-current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage-clamp technique. All agonists elicited Cl(-) currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca(2+) concentration ([Ca(2+) ](i)), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl(-) (F(Cl)) currents, while AII activated an oscillatory response; a robust Ca(2+) influx linked specifically to F(Cl) activation elicited an inward current (I(iw,Ca)) which was carried mainly by Cl(-) ions, through channels with a sequence of permeability of SCN(-)  > I(-)  > Br(-)  > Cl(-). Like F(Cl), I(iw,Ca) was not dependent on oocyte [Ca(2+) ](i) ; instead both were eliminated by preventing [Ca(2+) ](i) increase in the follicular cells, and also by U73122 and 2-APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F(Cl) and I(iw,Ca) were produced by the expected, PLC-stimulated Ca(2+) -release and Ca(2+) -influx, respectively, and by the opening of I(Cl(Ca)) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca(2+) -influx appeared to be driven through store-operated, calcium-like channels. The AII response, which is also known to require PLC activation, did not activate I(iw,Ca) and was strictly dependent on oocyte [Ca(2+) ](i) increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap-junction channels.

  1. Interstitial deletion of chromosome 1q [del(1)(q24q25.3)] identified by fluorescence in situ hybridization and gene dosage analysis of apolipoprotein A-II, coagulation factor V, and antithrombin III

    SciTech Connect

    Takano, Takako; Yamanouchi, Yasuko; Mori, Yosuke

    1997-01-20

    We report on a 12-month-old Japanese boy with an interstitial deletion of the long-arm of chromosome 1 and meningomyelocele, hydrocephalus, anal atresia, atrial septal defect, left renal agenesis, bilateral cryptorchidism, talipes equinovarus, low birth weight, growth/developmental retardation, and many minor anomalies. By conventional GTG-banding, his karyotype was first interpreted as 46,XY,de1(1)(q23q24), but it was corrected as 46,XY.ish del(1)(q24q25.3) by fluorescence in situ hybridization using 11 known cosmid clones as probes. His serum levels of apolipoprotein A-II (gene symbol: APOA2, previously assigned to 1q21-q23) and coagulation factor V (F5, 1q21-q25) were normal, while serum concentration and activity of antithrombin III (AT3, 1q23-q25.1) was low. The results indicated that localization of APOA2 and F5 are proximal to the deleted region and AT3 is located within the deletion extent in the patient. 16 refs., 4 figs.

  2. Apelin mediates the induction of profibrogenic genes in human hepatic stellate cells.

    PubMed

    Melgar-Lesmes, Pedro; Casals, Gregori; Pauta, Montserrat; Ros, Josefa; Reichenbach, Vedrana; Bataller, Ramon; Morales-Ruiz, Manuel; Jimenez, Wladimiro

    2010-11-01

    Apelin is a peptide with relevant functions in angiogenesis and inflammation. Recent studies have demonstrated that apelin is overexpressed in hepatic stellate cells (HSCs) of cirrhotic rats. Moreover, patients with cirrhosis show high circulating levels of this peptide. We evaluated the role of endogenous apelin system in fibrogenesis-related gene induction in human HSCs. Messenger expression and immunolocalization of apelin were analyzed in human cirrhotic liver and in control samples. Apelin expression was analyzed in a human HSC line (LX-2) under hypoxic conditions or in the presence of proinflammatory or profibrogenic stimuli. LX-2 cells were stimulated with apelin, and a selected profile of fibrogenesis-related genes was quantified. In vivo inactivation of apelin was analyzed in the liver of fibrotic rats after administrating specific blockers of the molecules triggering apelin induction. Apelin was overexpressed in HSCs from human cirrhotic liver. Neither hypoxia nor proinflammatory substances induced the expression of apelin in LX-2. By contrast, both profibrogenic molecules angiotensin II (AII) and endothelin-1 (ET-1) enhanced apelin expression in these cells. Apelin increased the synthesis of collagen-I and platelet-derived growth factor receptor β (PDGFRβ) in LX-2. AII and ET-1 stimulated collagen-I and PDGFRβ expression, and this induction was drastically reduced when apelin receptor was blocked in these cells. In accordance, AII or ET-1 receptor antagonists reduced the hepatic synthesis of apelin, collagen-I, and PDGFRβ in fibrotic rats. apelin mediates some of the fibrogenic effects triggered by AII and ET-1, thus suggesting that apelin could be an important mediator of fibrogenesis in human liver disease.

  3. Soluble uric acid increases intracellular calcium through an angiotensin II-dependent mechanism in immortalized human mesangial cells.

    PubMed

    Albertoni, Guilherme; Maquigussa, Edgar; Pessoa, Edson; Barreto, Jose Augusto; Borges, Fernanda; Schor, Nestor

    2010-07-01

    Hyperuricemia is associated with increases in cardiovascular risk and renal disease. Mesangial cells regulate glomerular filtration rates through the release of hormones and vasoactive substances. This study evaluates the signaling pathway of uric acid (UA) in immortalized human mesangial cells (ihMCs). To evaluate cell proliferation, ihMCs were exposed to UA (6-10 mg/dL) for 24-144 h. In further experiments, ihMCs were treated with UA (6-10 mg/dL) for 12 and 24 h simultaneously with losartan (10(-7) mmol/L). Angiotensin II (AII) and endothelin-1 (ET-1) were assessed using the enzyme-linked immunosorbent assay (ELISA) technique. Pre-pro-ET mRNA was evaluated by the real-time PCR technique. It was observed that soluble UA (8 and 10 mg/dL) stimulated cellular proliferation. UA (10 mg/dL) for 12 h significantly increased AII protein synthesis and ET-1 expression and protein production was increased after 24 h. Furthermore, UA increased [Ca(2+)](i), and this effect was significantly blocked when ihMCs were preincubated with losartan. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. In addition, UA can potentially affect glomerular function due to UA-induced proliferation and contraction of mesangial cells. The latter mechanism could be related to the long-term effects of UA on renal function and chronic kidney disease.

  4. Characterization of insulin-like growth factor I and insulin receptors on cultured bovine adrenal fasciculata cells. Role of these peptides on adrenal cell function

    SciTech Connect

    Penhoat, A.; Chatelain, P.G.; Jaillard, C.; Saez, J.M.

    1988-06-01

    We have characterized insulin-like growth factor I (IGF-I) and insulin receptors in cultured bovine adrenal cells by binding and cross-linking affinity experiments. At equilibrium the dissociation constant and the number of binding sites per cell for IGF-I were 1.4 +/- (SE) 0.3 x 10(-9) M and 19,200 +/- 2,100, respectively. Under reduction conditions, disuccinimidyl suberate cross-linked (/sup 125/I)iodo-IGF-I to one receptor complex with an Mr of 125,000. Adrenal cells also contain specific insulin receptors with an apparent dissociation constant (Kd) of 10(-9) M. Under reduction conditions (/sup 125/I)iodo-insulin binds to one band with an approximate Mr of 125,000. IGF-I and insulin at micromolar concentrations, but not at nanomolar concentrations, slightly stimulated DNA synthesis, but markedly potentiated the mitogenic action of fibroblast growth factor. Adrenal cells cultured in a serum-free medium containing transferrin, ascorbic acid, and insulin (5 micrograms/ml) maintained fairly constant angiotensin-II (A-II) receptor concentration per cell and increased cAMP release on response to ACTH and their steroidogenic response to both ACTH and A-II. When the cells were cultured in the same medium without insulin, the number of A-II receptors significantly decreased to 65% and the increased responsiveness was blunted. Treatment of such cells for 3 days with increasing concentrations of IGF-I (1-100 ng/ml) produced a 2- to 3-fold increase in A-II receptors and enhanced the cAMP response (3- to 4-fold) to ACTH and the steroidogenic response (4- to 6-fold) to ACTH and A-II. These effects were time and dose dependent (ED50 approximately equal to 10(-9) M). Insulin at micromolar concentrations produced an effect similar to that of IGF-I, but at nanomolar concentrations the effect was far less.

  5. Neuronal cell death in the inner retina and the influence of vascular endothelial growth factor inhibition in a diabetic rat model.

    PubMed

    Park, Hae-Young Lopilly; Kim, Jie Hyun; Park, Chan Kee

    2014-06-01

    To inhibit vascular changes in diabetic retinopathy, inhibiting vascular endothelial growth factor (VEGF) has become a mainstay of the treatment of diabetic retinopathy. However, its effects on neuronal cells remain to be elucidated. We aimed to evaluate the effect of VEGF inhibition on neuronal cells in a streptozotocin-induced diabetic rat retina. VEGF inhibition was performed by intravitreal VEGF-A antibody injection. After anti-VEGF treatment, apoptosis in retinal ganglion cells (RGCs) increased, and novel apoptosis in amacrine and bipolar cells of the inner nuclear layer was observed by TUNEL staining. Phosphorylated Akt expression was significantly higher in RGCs but was decreased in neuronal cells of the inner nuclear layer after anti-VEGF treatment by Western blot analysis and immunohistochemical staining. These results demonstrate that VEGF inhibition significantly increased RGC apoptosis and neuronal cell apoptosis in the inner nuclear layer of a diabetic retina, which seems to consist primarily of amacrine and bipolar cells. The phosphorylated Akt pathway, which plays a neuroprotective role via VEGF, was significantly affected by VEGF inhibition in the inner nuclear layer, suggesting that neurotrophic factor deprivation is the main mechanism for neuronal cell death after inhibiting VEGF. The results of this study show that inhibiting VEGF may have detrimental effects on the apoptosis of neuronal cells in the inner layers of the diabetic retina.

  6. Organizational motifs for ground squirrel cone bipolar cells

    PubMed Central

    Light, Adam C.; Zhu, Yongling; Shi, Jun; Saszik, Shannon; Lindstrom, Sarah; Davidson, Laura; Li, Xiaoyu; Chiodo, Vince A.; Hauswirth, William W.; Li, Wei; DeVries, Steven H.

    2012-01-01

    In daylight vision, parallel processing starts at the cone synapse. Cone signals flow to On and Off bipolar cells, which are further divided into types according to morphology, immunocytochemistry, and function. The axons of the bipolar cell types stratify at different levels in the inner plexiform layer (IPL), and can interact with costratifying amacrine and ganglion cells. These interactions endow the ganglion cell types with unique functional properties. The wiring that underlies the interactions between bipolar, amacrine, and ganglion cells is poorly understood. It may be easier to elucidate this wiring if organizational rules can be established. We identify 13 types of cone bipolar cells in the ground squirrel, 11 of which contact contiguous cones with the possible exception of short-wavelength sensitive cones. Cells were identified by antibody labeling, tracer filling, and Golgi-like filling following transduction with an adeno-associated virus encoding for GFP. The 11 bipolar cell types displayed two organizational patterns. In the first pattern, 8-10 of the 11 types came in pairs with partially overlapping axonal stratification. Pairs shared morphological, immunocytochemical, and functional properties. The existence of similar pairs is a new motif that may have implications for how signals first diverge from a cone to bipolar cells, and then re-converge onto a costratifying ganglion cell. The second pattern is a mirror symmetric organization about the middle of the IPL involving at least 7 bipolar cell types. This anatomical symmetry may be associated with a functional symmetry in On and Off ganglion cell responses. PMID:22778006

  7. Organizational motifs for ground squirrel cone bipolar cells.

    PubMed

    Light, Adam C; Zhu, Yongling; Shi, Jun; Saszik, Shannon; Lindstrom, Sarah; Davidson, Laura; Li, Xiaoyu; Chiodo, Vince A; Hauswirth, William W; Li, Wei; DeVries, Steven H

    2012-09-01

    In daylight vision, parallel processing starts at the cone synapse. Cone signals flow to On and Off bipolar cells, which are further divided into types according to morphology, immunocytochemistry, and function. The axons of the bipolar cell types stratify at different levels in the inner plexiform layer (IPL) and can interact with costratifying amacrine and ganglion cells. These interactions endow the ganglion cell types with unique functional properties. The wiring that underlies the interactions among bipolar, amacrine, and ganglion cells is poorly understood. It may be easier to elucidate this wiring if organizational rules can be established. We identify 13 types of cone bipolar cells in the ground squirrel, 11 of which contact contiguous cones, with the possible exception of short-wavelength-sensitive cones. Cells were identified by antibody labeling, tracer filling, and Golgi-like filling following transduction with an adeno-associated virus encoding for green fluorescent protein. The 11 bipolar cell types displayed two organizational patterns. In the first pattern, eight to 10 of the 11 types came in pairs with partially overlapping axonal stratification. Pairs shared morphological, immunocytochemical, and functional properties. The existence of similar pairs is a new motif that might have implications for how signals first diverge from a cone to bipolar cells and then reconverge onto a costratifying ganglion cell. The second pattern is a mirror symmetric organization about the middle of the IPL involving at least seven bipolar cell types. This anatomical symmetry may be associated with a functional symmetry in On and Off ganglion cell responses. Copyright © 2012 Wiley Periodicals, Inc.

  8. Sip1 regulates the generation of the inner nuclear layer retinal cell lineages in mammals.

    PubMed

    Menuchin-Lasowski, Yotam; Oren-Giladi, Pazit; Xie, Qing; Ezra-Elia, Raaya; Ofri, Ron; Peled-Hajaj, Shany; Farhy, Chen; Higashi, Yujiro; Van de Putte, Tom; Kondoh, Hisato; Huylebroeck, Danny; Cvekl, Ales; Ashery-Padan, Ruth

    2016-08-01

    The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals. © 2016. Published by The Company of Biologists Ltd.

  9. Identification of molecular markers of bipolar cells in the murine retina

    PubMed Central

    Kim, Douglas S; Ross, Sarah E; Trimarchi, Jeffrey M; Aach, John; Greenberg, Michael E; Cepko, Constance L

    2008-01-01

    Retinal bipolar neurons serve as relay interneurons that connect rod and cone photoreceptor cells to amacrine and ganglion cells. They exhibit diverse morphologies essential for correct routing of photoreceptor cell signals to specific postsynaptic amacrine and ganglion cells. The development and physiology of these interneurons have not been completely defined molecularly. Despite previous identification of genes expressed in several bipolar cell subtypes, molecules that mark each bipolar cell type still await discovery. In this report, novel genetic markers of murine bipolar cells were found. Candidates were initially generated by using microarray analysis of single bipolar cells and mining of retinal serial analysis of gene expression (SAGE) data. These candidates were subsequently tested for expression in bipolar cells by RNA in situ hybridization. Ten new molecular markers were identified, five of which are highly enriched in their expression in bipolar cells within the adult retina. Double-labeling experiments using probes for previously characterized subsets of bipolar cells were performed to identify the subtypes of bipolar cells that express the novel markers. Additionally, the expression of bipolar cell genes was analyzed in Bhlhb4 knockout retinas, in which rod bipolar cells degenerate postnatally, to delineate further the identity of bipolar cells in which novel markers are found. From the analysis of Bhlhb4 mutant retinas, cone bipolar cell gene expression appears to be relatively unaffected by the degeneration of rod bipolar cells. Identification of molecular markers for the various subtypes of bipolar cells will lead to greater insights into the development and function of these diverse interneurons. PMID:18260140

  10. Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination

    PubMed Central

    Eldred, Megan K.; Charlton-Perkins, Mark; Muresan, Leila

    2017-01-01

    ABSTRACT To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process. PMID:28174240

  11. Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination.

    PubMed

    Eldred, Megan K; Charlton-Perkins, Mark; Muresan, Leila; Harris, William A

    2017-03-15

    To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.

  12. Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development.

    PubMed

    Nakamoto, Chizu; Kuan, Soh-Leh; Findlay, Amy S; Durward, Elaine; Ouyang, Zhufeng; Zakrzewska, Ewa D; Endo, Takuma; Nakamoto, Masaru

    2014-01-01

    For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1-like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.

  13. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration.

    PubMed

    Li, Yiqing; Andereggen, Lukas; Yuki, Kenya; Omura, Kumiko; Yin, Yuqin; Gilbert, Hui-Ya; Erdogan, Burcu; Asdourian, Maria S; Shrock, Christine; de Lima, Silmara; Apfel, Ulf-Peter; Zhuo, Yehong; Hershfinkel, Michal; Lippard, Stephen J; Rosenberg, Paul A; Benowitz, Larry

    2017-01-10

    Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn(2+)) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn(2+) increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn(2+) accumulation in amacrine cell processes involves the Zn(2+) transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn(2+) chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn(2+) chelation extends for several days after nerve injury. These results show that retinal Zn(2+) dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn(2+) chelation as a strategy to promote long-term RGC protection and enhance axon regeneration.

  14. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration

    PubMed Central

    Li, Yiqing; Andereggen, Lukas; Yuki, Kenya; Omura, Kumiko; Yin, Yuqin; Gilbert, Hui-Ya; Erdogan, Burcu; Asdourian, Maria S.; Shrock, Christine; de Lima, Silmara; Apfel, Ulf-Peter; Zhuo, Yehong; Hershfinkel, Michal; Lippard, Stephen J.; Benowitz, Larry

    2017-01-01

    Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn2+) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn2+ increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn2+ accumulation in amacrine cell processes involves the Zn2+ transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn2+ chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn2+ chelation extends for several days after nerve injury. These results show that retinal Zn2+ dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn2+ chelation as a strategy to promote long-term RGC protection and enhance axon regeneration. PMID:28049831

  15. Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression.

    PubMed

    Ortiz-Sánchez, Elizabeth; Santiago-López, Luz; Cruz-Domínguez, Verónica B; Toledo-Guzmán, Mariel E; Hernández-Cueto, Daniel; Muñiz-Hernández, Saé; Garrido, Efraín; Cantú De León, David; García-Carrancá, Alejandro

    2016-05-31

    Cancer stem cells (CSC) exhibit high tumorigenic capacity in several tumor models. We have now determined an extended phenotype for cervical cancer stem cells. Our results showed increased CK-17, p63+, AII+, CD49f+ expression in these cells, together with higher Aldehyde dehydrogenase (ALDHbright)activity in Cervical CSC (CCSC) enriched in cervospheres. An increase in stem cell markers, represented by OCT-4, Nanog, and β-catenin proteins, was also observed, indicating that under our culture conditions, CCSC are enriched in cervospheres, as compared to monolayer cultures. In addition, we were able to show that an increased ALDHbright activity correlated with higher tumorigenic activity. Flow cytometry and immunflorescence assays demonstrated that CCSC in cervosphere cultures contain a sub-population of cells that contain Annexin II, a Human papillomavirus (HPV) co-receptor. Taken together, under our conditions there is an increase in the number of CCSC in cervosphere cultures which exhibit the following phenotype: CK-17, p63+, AII+, CD49f+ and high ALDH activity, which in turn correlates with higher tumorigenicity. The presence of Annexin II and CD49f in CCSC opens the possibility that normal cervical stem cells could be the initial target of infection by high risk HPV.

  16. Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression

    PubMed Central

    Ortiz-Sánchez, Elizabeth; Santiago-López, Luz; Cruz-Domínguez, Verónica B.; Toledo-Guzmán, Mariel E.; Hernández-Cueto, Daniel; Muñiz-Hernández, Saé; Garrido, Efraín; De León, David Cantú; García-Carrancá, Alejandro

    2016-01-01

    Cancer stem cells (CSC) exhibit high tumorigenic capacity in several tumor models. We have now determined an extended phenotype for cervical cancer stem cells. Our results showed increased CK-17, p63+, AII+, CD49f+ expression in these cells, together with higher Aldehyde dehydrogenase (ALDHbright)activity in Cervical CSC (CCSC) enriched in cervospheres. An increase in stem cell markers, represented by OCT-4, Nanog, and β-catenin proteins, was also observed, indicating that under our culture conditions, CCSC are enriched in cervospheres, as compared to monolayer cultures. In addition, we were able to show that an increased ALDHbright activity correlated with higher tumorigenic activity. Flow cytometry and immunflorescence assays demonstrated that CCSC in cervosphere cultures contain a sub-population of cells that contain Annexin II, a Human papillomavirus (HPV) co-receptor. Taken together, under our conditions there is an increase in the number of CCSC in cervosphere cultures which exhibit the following phenotype: CK-17, p63+, AII+, CD49f+ and high ALDH activity, which in turn correlates with higher tumorigenicity. The presence of Annexin II and CD49f in CCSC opens the possibility that normal cervical stem cells could be the initial target of infection by high risk HPV. PMID:27008711

  17. New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives.

    PubMed Central

    Bonnafous, J C; Tence, M; Seyer, R; Marie, J; Aumelas, A; Jard, S

    1988-01-01

    The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed. PMID:3415650

  18. New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives.

    PubMed

    Bonnafous, J C; Tence, M; Seyer, R; Marie, J; Aumelas, A; Jard, S

    1988-05-01

    The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed.

  19. Parvalbumin immunoreactivity is enhanced by brain-derived neurotrophic factor in organotypic cultures of rat retina.

    PubMed

    Rickman, D W

    1999-11-15

    The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand-receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk(B), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway-the parvalbumin (PV)-immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV-immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well-immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit.

  20. Nicotinic Antagonists Enhance Process Outgrowth by Rat Retinal Ganglion Cells in Culture

    NASA Astrophysics Data System (ADS)

    Lipton, Stuart A.; Frosch, Matthew P.; Phillips, Micheal D.; Tauck, David L.; Aizenman, Elias

    1988-03-01

    Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.

  1. Directional Excitatory Input to Direction-Selective Ganglion Cells in the Rabbit Retina.

    PubMed

    Percival, Kumiko A; Venkataramani, Sowmya; Smith, Robert G; Rowland Taylor, W

    2017-03-14

    Directional responses in retinal ganglion cells are generated in large part by direction-selective release of GABA from starburst amacrine cells onto direction-selective ganglion cells (DSGCs). The excitatory inputs to DSGCs are also widely reported to be direction-selective, however, recent evidence suggests that glutamate release from bipolar cells is not directional, and directional excitation seen in patch-clamp analyses may be an artifact resulting from incomplete voltage control. Here we test this voltage-clamp-artifact hypothesis in recordings from 62 On-Off DSGCs in the rabbit retina. The strength of the directional excitatory signal varies considerably across the sample of cells, but is not correlated with the strength of directional inhibition, as required for a voltage-clamp artifact. These results implicate additional mechanisms in generating directional excitatory inputs to DSGCs. This article is protected by copyright. All rights reserved.

  2. Synaptic Input of ON-Bipolar Cells onto the Dopaminergic Neurons of the Mouse Retina

    PubMed Central

    Contini, Massimo; Lin, Bin; Kobayashi, Kazuto; Okano, Hideyuki; Masland, Richard H.; Raviola, Elio

    2010-01-01

    In the retina, dopamine fulfills a crucial role in neural adaptation to photopic illumination, but the pathway that carries cone signals to the dopaminergic amacrine (DA) cells was not known. We identified the site of ON-cone bipolar input onto DA cells in transgenic mice in which both types of catecholaminergic amacrine (CA) cells were labeled with green fluorescent protein or human placental alkaline phosphatase (PLAP). In confocal Z series of retinal whole mounts stained with antibodies to tyrosine hydroxylase (TH), DA cells gave rise to varicose processes that descended obliquely through the scleral half of the inner plexiform layer (IPL) and formed a loose, tangential plexus in the middle of this layer. Comparison with the distribution of the dendrites of type 2 CA cells and examination of neurobiotin-injected DA cells proved that their vitreal processes were situated in stratum S3 of the IPL. Electron microscope demonstration of PLAP activity showed that bipolar cell endings in S3 established ribbon synapses onto a postsynaptic dyad in which one or both processes were labeled by a precipitate of lead phosphate and therefore belonged to DA cells. In places, the postsynaptic DA cell processes returned a reciprocal synapse onto the bipolar endings. Confocal images of sections stained with antibodies to TH, kinesin Kif3a, which labels synaptic ribbons, and glutamate or GABAA receptors, confirmed that ribbon-containing endings made glutamatergic synapses onto DA cells processes in S3 and received from them GABAergic synapses. The presynaptic ON-bipolar cells most likely belonged to the CB3 (type 5) variety. PMID:20394057

  3. Cyclin-dependent kinase inhibitor, p21Waf1, regulates vascular smooth muscle cell hypertrophy.

    PubMed

    Okamoto, Kenichi; Kato, Seiya; Arima, Nobuyuki; Fujii, Teruhiko; Morimatsu, Minoru; Imaizumi, Tsutomu

    2004-04-01

    In the process of vascular diseases, smooth muscle cells (SMC) undergo not only hyperplasia but also hypertrophy, resulting in vascular remodeling. A cyclin-dependent kinase inhibitor (CDKI), p21Waf1, has been shown to play an important role in SMC hyperplasia. Here we investigated a potential role of p21Waf1 in SMC hypertrophy. An exposure of cultured rat SMC to serum drove the cell cycle progression with up-regulation of various cell cycle markers and increased activities of cyclin-dependent kinases, but did not cause SMC hypertrophy. In contrast, incubation of SMC for 48 h with angiotensin II (AII, 100 nmol/l) resulted in a significant increase in the cell size measured by flowcytometric forward-angle light scatter assay, in association with an increase in the ratio of [3H]leucine/[3H]thymidine uptake, indicating SMC hypertrophy. At 48 h, p21Waf1 expression was up-regulated in SMC exposed to AII but not in those exposed to serum. These results suggest that p21Waf1 may be involved in hypertrophy. To further investigate this issue, two manipulations of the p21Waf1 gene were performed. Adenovirus-mediated over-expression of p21Waf1 not only reduced S-phasic cells but also caused hypertrophy, despite the exposure to serum. Antisense oligodeoxynucleotide for p21Waf1 inhibited the hypertrophy of SMC exposed to AII. Our data suggest that p21Waf1 may play a role in SMC hypertrophy as well.

  4. Evolution of eyes and photoreceptor cell types.

    PubMed

    Arendt, Detlev

    2003-01-01

    The evolution of the eye is a matter of debate ever since Darwin's Origin of Species. While morphological comparisons of eye anatomy and photoreceptor cell types led to the view that animal eyes evolved multiple times independently, the molecular conservation of the pax6 eye-specifying cascade has indicated the contrary - that animal eyes evolved from a common, simple precursor, the proto-eye. Morphological and molecular comparative approaches are combined here in a novel Evo-Devo approach, the molecular comparison of cell types ("comparative molecular cell biology"). In the eye, the various types of photoreceptor cells, as well as pigment and lens cells, each require distinct combinations of specifying transcription factors that control their particular differentiation programmes, such as opsin expression in photoreceptors, specific neurotransmitter metabolism, or axonal outgrowth. Comparing the molecular combinatorial codes of cell types of animal extant eyes, their evolutionary histories can be reconstructed. This is exemplified here on the evolution of ciliary and rhabdomeric photoreceptor cells in bilaterian eyes and on the evolution of cell type diversity in the vertebrate retina. I propose that the retinal ganglion, amacrine and horizontal cells are evolutionary sister cell types that evolved from a common rhabdomeric photoreceptor cell precursor.

  5. A Thy1-CFP DBA/2J mouse line with cyan fluorescent protein expression in retinal ganglion cells

    PubMed Central

    RAYMOND, IONA D.; POOL, ANGELA L.; VILA, ALEJANDRO; BRECHA, NICHOLAS C.

    2013-01-01

    A DBA/2J (D2) transgenic mouse line with cyan fluorescent protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1-CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1-CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1-CFP transgene. CFP-fluorescent ganglion cells, 6–20 μm in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma. PMID:19930759

  6. NO signaling in retinal bipolar cells.

    PubMed

    Agurto, A; Vielma, A H; Cadiz, B; Couve, E; Schmachtenberg, O

    2017-08-01

    Nitric oxide (NO) is a neuromodulator involved in physiological and pathological processes in the retina. In the inner retina, a subgroup of amacrine cells have been shown to synthesize NO, but bipolar cells remain controversial as NO sources. This study correlates NO synthesis in dark-adapted retinas, through labeling with the NO marker DAF-FM, with neuronal nitric oxide synthase (nNOS) and inducible NOS expression, and presence of the NO receptor soluble guanylate cyclase in bipolar cells. NO containing bipolar cells were morphologically identified by dialysis of DAF fluorescent cells with intracellular dyes, or by DAF labeling followed by immunohistochemistry for nNOS and other cellular markers. DAF fluorescence was observed in all types of bipolar cells that could be identified, but the most intense DAF fluorescence was observed in bipolar cells with severed processes, supporting pathological NO signaling. Among nNOS expressing bipolar cells, type 9 was confirmed unequivocally, while types 2, 3a, 3b, 4, 5, 7, 8 and the rod bipolar cell were devoid of this enzyme. These results establish specific bipolar cell types as NO sources in the inner retina, and support the involvement of NO signaling in physiological and pathological processes in the inner retina. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Synthesis of hydroxyeicosatetraenoic acids (HETE's) by adrenal glomerulosa cells and incorporation into cellular lipids

    SciTech Connect

    Campbell, W.B.; Richards, C.F.; Brady, M.T.; Falck, J.R.

    1986-03-05

    The role of lipoxygenase metabolites of arachidonic acid (AA) in the regulation of aldosterone secretion was studied in isolated rat adrenal glomerulosa cells. Cells were incubated with /sup 14/C-AA in the presence of angiotensin (AII). The media was extracted, metabolites isolated by HPLC, and structures of the metabolites determined by UV absorbance and mass spectrometry. The major products were 12- and 15-HETE with lesser amounts of 11- and 5-HETE. When adrenal cells were incubated with 15-, 12- or 5-HPETE or their respective HETE's (0.03-300nM), there was no significant change in basal or AII-stimulated aldosterone release. Cells were incubated with (/sup 3/H)-AA, -5-HETE, -15-HETE, -12-HETE or -LTB. The cellular lipids were extracted and analyzed by TLC. AA was incorporated into phospholipids (22%), cholesterol esters (50%) and triglycerides (21%). Neither the HETE's or LTB/sub 4/ were incorporated into phospholipids. 5-HETE was taken up into di- and mono-glycerides. The rates of incorporation of AA and 5-HETE were similar (+ 1/2 = 10 min). The incorporation of 5-HETE into glycerol esters did not modify the release of aldosterone by the cells. Thus, while adrenal cells synthesize HETE's, these eicosanoids do not appear to alter the synthesis of aldosterone.

  8. Ectopic photoreceptors and cone bipolar cells in the developing and mature retina.

    PubMed

    Günhan, Emine; van der List, Deborah; Chalupa, Leo M

    2003-02-15

    An antibody against recoverin, the calcium-binding protein, labels photoreceptors, cone bipolar cells, and a subpopulation of cells in the ganglion cell layer. In the present study, we sought to establish the origin and identity of the cells expressing recoverin in the ganglion cell layer of the rat retina. By double labeling with rhodopsin, we demonstrate that early in development some of the recoverin-positive cells in the ganglion cell layer are photoreceptors. During the first postnatal week, these rhodopsin-positive cells are eliminated from the ganglion cell layer, but such neurons remain in the inner nuclear layer well into the first postnatal month. Another contingent of recoverin-positive cells, with morphological features equivalent to those of bipolar cells, is present in the postnatal retina, and approximately 50% of these neurons survive to maturity. The incidence of such cells in the ganglion cell layer was not affected by early transection of the optic nerve, a manipulation that causes rapid loss of retinal ganglion cells. These recoverin-positive cells were not double-labeled by cell-specific markers expressed by photoreceptors, rod bipolar cells, or horizontal and amacrine cells. Based on their staining with recoverin and salient morphological features, these ectopic profiles in the ganglion cell layer are most likely cone bipolar cells. Collectively, the results provide evidence for photoreceptors in the ganglion cell and inner nuclear layers of the developing retina, and a more permanent subpopulation of cone bipolar cells displaced to the ganglion cell layer.

  9. Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina

    PubMed Central

    Liu, Xiaoqin; Zhang, Zhijing; Ribelayga, Christophe P.

    2012-01-01

    Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue. PMID:23189207

  10. Activity-dependent transport of GABA analogues into specific cell types demonstrated at high resolution using a novel immunocytochemical strategy.

    PubMed

    Pow, D V; Baldridge, W; Crook, D K

    1996-08-01

    We have raised antisera against the GABA analogues gamma-vinyl GABA, diaminobutyric acid and gabaculine. These analogues are thought to be substrates for high-affinity GABA transporters. Retinae were exposed to micromolar concentrations of these analogues in the presence or absence of uptake inhibitors and then fixed and processed for immunocytochemistry at the light and electron microscopic levels. Immunolabelling for gamma-vinyl GABA revealed specific labelling of GABAergic amacrine cells and displaced amacrine cells in retinae of rabbits, cats, chickens, fish and a monkey. GABA-containing horizontal cells of cat and monkey retinae failed to exhibit labelling for gamma-vinyl GABA, suggesting that they lacked an uptake system for this molecule. In light-adapted fish, gamma-vinyl GABA was readily detected in H1 horizontal cells; similar labelling was also observed in light-adapted chicken retinae. The pattern of labelling in the fish and chicken retinae was modified by dark adaptation, when labelling was greatly reduced in the horizontal cells, indicating the activity dependence of GABA (analogue) transport. Intraperitoneal injection of gamma-vinyl GABA into rats resulted in its transport across the blood-brain barrier and subsequent uptake into populations of GABAergic neurons. The other analogues investigated in this study exhibited different patterns of transport; gabaculine was taken up into glial cells, whilst diaminobutyric acid was taken up into neurons, glial cells and retinal pigment epithelia. Thus, these analogues are probably substrates for different GABA transporters. We conclude that immunocytochemical detection of the high-affinity uptake of gamma-vinyl GABA permits the identification of GABAergic neurons which are actively transporting GABA, and suggest that this novel methodology will be a useful tool in rapidly assessing the recent activity of GABAergic neurons at the cellular level.

  11. Transcription Factors CTCF and Pax6 Are Segregated to Different Cell Types During Retinal Cell Differentiation

    PubMed Central

    Canto-Soler, M. Valeria; Huang, Hu; Romero, M. Soledad; Adler, Ruben

    2008-01-01

    We have hypothesized that the transcription factor CTCF may influence retinal cell differentiation by controlling Pax6 expression, because (1) CTCF has been shown to repress Pax6 expression in some tissues, and (2) Pax6 blocks the differentiation of retinal progenitor cells as photoreceptors and promotes their differentiation as nonphotoreceptor neurons. Our results show that, as predicted by this hypothesis, CTCF and Pax6 become segregated to different retinal cell types. The factors are initially coexpressed in the undifferentiated neuroepithelium, but already at that time they show complementary periphery-to-fundus gradients of distribution. As the retina laminates, Pax6 becomes restricted to ganglion and amacrine cells, and CTCF to the bipolar/Muller cell layer and the outer nuclear layer. Polymerase chain reaction analysis of laser capture microdissection samples and dissociated cells showed that both immature and differentiated photoreceptors are CTCF (+)/Pax6 (−). Functional studies are now under way to further analyze the role of CTCF in retinal cell differentiation. PMID:18224715

  12. Cell autonomy of DSCAM function in retinal development

    PubMed Central

    Fuerst, Peter G.; Bruce, Freyja; Rounds, Ryan P.; Erskine, Lynda; Burgess, Robert W.

    2011-01-01

    Cell adhesion molecules (CAMs) provide identifying cues by which neural architecture is sculpted. The Down Syndrome Cell Adhesion Molecule (DSCAM) is required for many neurodevelopmental processes in different species and also has several potential mechanisms of activity, including homophilic adhesion, homophilic repulsion and heterophilic interactions. In the mouse retina, Dscam is expressed in many, but not all neuronal subtypes. Mutations in Dscam cause the fasciculation of dendrites of neighboring homotypic neurons, indicating a role in self-avoidance among cells of a given type, a disruption of the non-random patterning of their cell bodies, and a decrease in developmental cell death in affected cell populations. In order to address how DSCAM facilitates retinal pattering, we developed a conditional allele of Dscam to use alongside existing Dscam mutant mouse strains. Conditional deletion of Dscam reproduces cell spacing, cell number and dendrite arborization defects. Inducible deletion of Dscam and retinal ganglion cell depletion in Brn3b mutant retinas both indicate that these DSCAM-mediated phenotypes can occur independently. In chimeric retinas, in which wild type and Dscam mutant cells are comingled, Dscam mutant cells entangle adjacent wild type cells of the same type, as if both cells were lacking Dscam, consistent with DSCAM-dependent cell spacing and neurite arborization being mediated through homophilic binding cell-to-cell. Deletion of Dscam in specific cell types causes cell-type-autonomous cell body spacing defects, indicating that DSCAM mediates arborization and spacing by acting within given cell types. We also examine the cell autonomy of DSCAM in laminar stratification and find that laminar disorganization can be caused in a non-cell autonomous fashion. Finally, we find Dscam dosage-dependent defects in developmental cell death and amacrine cell spacing, relevant to the increased cell death and other disorders observed in Down syndrome mouse

  13. Embryonic stem cell-derived microvesicles induce gene expression changes in Müller cells of the retina.

    PubMed

    Katsman, Diana; Stackpole, Emma J; Domin, Daniel R; Farber, Debora B

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  14. Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina

    PubMed Central

    Katsman, Diana; Stackpole, Emma J.; Domin, Daniel R.; Farber, Debora B.

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation. PMID:23226281

  15. Broad Thorny Ganglion Cells: A Candidate for Visual Pursuit Error Signaling in the Primate Retina

    PubMed Central

    Manookin, Michael B.; Neitz, Jay; Rieke, Fred

    2015-01-01

    Functional analyses exist only for a few of the morphologically described primate ganglion cell types, and their correlates in other mammalian species remain elusive. Here, we recorded light responses of broad thorny cells in the whole-mounted macaque retina. They showed ON-OFF-center light responses that were strongly suppressed by stimulation of the receptive field surround. Spike responses were delayed compared with parasol ganglion cells and other ON-OFF cells, including recursive bistratified ganglion cells and A1 amacrine cells. The receptive field structure was shaped by direct excitatory synaptic input and strong presynaptic and postsynaptic inhibition in both ON and OFF pathways. The cells responded strongly to dark or bright stimuli moving either in or out of the receptive field, independent of the direction of motion. However, they did not show a maintained spike response either to a uniform background or to a drifting plaid pattern. These properties could be ideally suited for guiding movements involved in visual pursuit. The functional characteristics reported here permit the first direct cross-species comparison of putative homologous ganglion cell types. Based on morphological similarities, broad thorny ganglion cells have been proposed to be homologs of rabbit local edge detector ganglion cells, but we now show that the two cells have quite distinct physiological properties. Thus, our data argue against broad thorny cells as the homologs of local edge detector cells. PMID:25834063

  16. Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina

    PubMed Central

    Tachibana, Nobuhiko; Zinyk, Dawn; Ringuette, Randy; Wallace, Valerie; Schuurmans, Carol

    2017-01-01

    All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7–10 days in vitro. During this time frame, RPCs will differentiate, and the fate of the BrdU+ RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabe et al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuette et al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, and Pten as a modulator of the Tgfβ2 response (Ma et al., 2007; Tachibana et al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.

  17. Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina.

    PubMed

    Goebel, D J; Aurelia, J L; Tai, Q; Jojich, L; Poosch, M S

    1998-10-19

    Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2

  18. Bipolar Cell-Photoreceptor Connectivity in the Zebrafish (Danio rerio) Retina

    PubMed Central

    Li, Yong N.; Tsujimura, Taro; Kawamura, Shoji; Dowling, John E.

    2013-01-01

    Bipolar cells convey luminance, spatial and color information from photoreceptors to amacrine and ganglion cells. We studied the photoreceptor connectivity of 321 bipolar cells in the adult zebrafish retina. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was inserted into whole-mounted transgenic zebrafish retinas to label bipolar cells. The photoreceptors that connect to these DiI-labeled cells were identified by transgenic fluorescence or their positions relative to the fluorescent cones, as cones are arranged in a highly-ordered mosaic: rows of alternating blue- (B) and ultraviolet-sensitive (UV) single cones alternate with rows of red- (R) and green-sensitive (G) double cones. Rod terminals intersperse among cone terminals. As many as 18 connectivity subtypes were observed, 9 of which – G, GBUV, RG, RGB, RGBUV, RGRod, RGBRod, RGBUVRod and RRod bipolar cells – accounted for 96% of the population. Based on their axon terminal stratification, these bipolar cells could be further sub-divided into ON, OFF, and ON-OFF cells. The dendritic spread size, soma depth and size, and photoreceptor connections of the 308 bipolar cells within the 9 common connectivity subtypes were determined, and their dendritic tree morphologies and axonal stratification patterns compared. We found that bipolar cells with the same axonal stratification patterns could have heterogeneous photoreceptor connectivity whereas bipolar cells with the same dendritic tree morphology usually had the same photoreceptor connectivity, although their axons might stratify on different levels. PMID:22907678

  19. Calcium buffer proteins are specific markers of human retinal neurons.

    PubMed

    Kántor, Orsolya; Mezey, Szilvia; Adeghate, Jennifer; Naumann, Angela; Nitschke, Roland; Énzsöly, Anna; Szabó, Arnold; Lukáts, Ákos; Németh, János; Somogyvári, Zoltán; Völgyi, Béla

    2016-07-01

    Ca(2+)-buffer proteins (CaBPs) modulate the temporal and spatial characteristics of transient intracellular Ca(2+)-concentration changes in neurons in order to fine-tune the strength and duration of the output signal. CaBPs have been used as neurochemical markers to identify and trace neurons of several brain loci including the mammalian retina. The CaBP content of retinal neurons, however, varies between species and, thus, the results inferred from animal models cannot be utilised directly by clinical ophthalmologists. Moreover, the shortage of well-preserved human samples greatly impedes human retina studies at the cellular and network level. Our purpose has therefore been to examine the distribution of major CaBPs, including calretinin, calbindin-D28, parvalbumin and the recently discovered secretagogin in exceptionally well-preserved human retinal samples. Based on a combination of immunohistochemistry, Neurolucida tracing and Lucifer yellow injections, we have established a database in which the CaBP marker composition can be defined for morphologically identified cell types of the human retina. Hence, we describe the full CaBP make-up for a number of human retinal neurons, including HII horizontal cells, AII amacrine cells, type-1 tyrosine-hydroxylase-expressing amacrine cells and other lesser known neurons. We have also found a number of unidentified cells whose morphology remains to be characterised. We present several examples of the colocalisation of two or three CaBPs with slightly different subcellular distributions in the same cell strongly suggesting a compartment-specific division of labour of Ca(2+)-buffering by CaBPs. Our work thus provides a neurochemical framework for future ophthalmological studies and renders new information concerning the cellular and subcellular distribution of CaBPs for experimental neuroscience.

  20. Quorum quenching activity in cell-free lysate of endophytic bacteria isolated from Pterocarpus santalinus Linn., and its effect on quorum sensing regulated biofilm in Pseudomonas aeruginosa PAO1.

    PubMed

    Rajesh, P S; Ravishankar Rai, V

    2014-01-01

    Quorum sensing mechanism allows the microorganisms to resist the antibiotic treatment by forming biofilms. Quorum quenching is one of the mechanisms to control the development of drug resistance in microbes. Endophyte bacteria are beneficial to plant growth as they support the immune system against the pathogen attack. The endophytic bacteria present in Pterocarpus santalinus were screened for the presence of N-acyl homoserine lactones (AHLs) degrading bacteria using biosensor strains and further confirmed by quantifying the violacein production. Cell-free lysate of endophytic bacteria, Bacillus firmus PT18 and Enterobacter asburiae PT39 exhibited potent AHL degrading ability by inhibiting about 80% violacein production in biosensor strain. Furthermore, when the cell-free lysate was applied to Pseudomonas aeruginosa PAO1 and PAO1-JP2 biofilm it resulted in significant (p<0.01) inhibition of biofilm formation. The biofilm inhibition was confirmed by visualization of biofilm slides under fluorescence microscopy, which showed decrease in total biomass formation in treated slides. Isolation and amplification of the gene (aiiA) indicated that the presence of AHL lactonase in cell-free lysate and sequence alignment indicated that AiiA contains a "HXHXDH" zinc-binding motif that is being conserved in several groups of metallohydrolases. Therefore, the study shows the potential of AHLs degradation by AHL lactonase present in cell-free lysate of isolated endophytic bacteria and inhibition of quorum sensing regulated biofilm formation in P. aeruginosa PAO1. Copyright © 2013 Elsevier GmbH. All rights reserved.

  1. Morphologic maturation of tachykinin peptide-expressing cells in the postnatal rabbit retina.

    PubMed

    Casini, G; Trasarti, L; Andolfi, L; Bagnoli, P

    1997-04-18

    Tachykinin (TK) peptides, which include substance P, neurokinin A, two neurokinin A-related peptides and neurokinin B, are widely present in the nervous system, including the retina, where they act as neurotransmitters/modulators as well as growth factors. In the present study, we investigated the maturation of TK-immunoreactive (IR) cells in the rabbit retina with the aim of further contributing to the knowledge of the development of transmitter-identified retinal cell populations. In the adult retina, the pattern of TK immunostaining is consistent with the presence of TK peptides in amacrine, displaced amacrine, interplexiform and ganglion cells. In the newborn retina, intensely immunostained TK-IR somata are located in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL). They are characterized by an oval-shaped cell body originating a single process without ramifications. TK-IR processes are occasionally observed in the IPL and in the outer plexiform layer (OPL). Long TK-IR fiber bundles are observed in the ganglion cell axon layer. TK-IR profiles resembling small somata are rarely observed in the INL adjacent to the OPL. At postnatal day (PND) 2, some TK-IR cells display more complex morphologic features, including processes with secondary ramifications. Long TK-IR processes in the IPL are often seen to terminate with growth cones. Between PND 6 and PND 11 (eye opening), there is a dramatic increase in the number of immunolabeled processes with growth cones both in the IPL and in the OPL and the mature lamination of TK-IR fibers in laminae 1, 3 and 5 of the IPL is established. TK-IR cells attain mature morphological characteristics and the rare, putative TK-IR somata in the distal INL are no longer observed. After eye opening, growth cones are not present and the pattern typical of the adult is reached. These observations indicate that the development of TK-IR cells can be divided into an early phase

  2. Differential Expression of Three T-Type Calcium Channels in Retinal Bipolar Cells in Rat

    PubMed Central

    Hu, Caiping; Bi, Anding; Pan, Zhuo-Hua

    2009-01-01

    Retinal bipolar cells convey visual information from photoreceptors to retinal third-order neurons, amacrine and ganglion cells, with graded potentials through diversified cell types. To understand the possible role of voltage-dependent T-type Ca2+ currents in retinal bipolar cells, we investigated the pharmacological and biophysical properties of T-type Ca2+ currents in acutely dissociated retinal cone bipolar cells from rat using whole-cell patch-clamp recordings. We observed a broad group of cone bipolar cells with prominent T-type Ca2+ currents (T-rich) and another group with prominent L-type Ca2+ currents (L-rich). Based on the pharmacological and biophysical properties of the T-type Ca2+ currents, T-rich cone bipolar cells could be divided into three subgroups. Each subgroup appeared to express a single dominant T-type Ca2+ channel subunit. The T-type calcium currents could generate low threshold regenerative potentials or spikes. Our results suggest that T-type Ca2+ channels may play an active and distinct signaling role in second-order neurons of the visual system, in contrast to the common signaling by L-rich bipolar cells. PMID:19275782

  3. Cone and rod cells have different target preferences in vitro as revealed by optical tweezers

    PubMed Central

    Clarke, Robert J.; Högnason, Kormákur; Brimacombe, Michael

    2008-01-01

    Purpose When neural circuits are damaged in adulthood, regenerating and sprouting processes must distinguish appropriate targets to recreate the normal circuitry. We tested the ability of adult nerve cells to target specific cells in culture using the retina as a model system. Methods Under sterile culture conditions, retinal cells, isolated from tiger salamander retina, were micromanipulated with optical tweezers to create pairs of first-order photoreceptor cells with second- or third-order retinal neurons. The development of cell contact and presynaptic varicosities, the direction and amount of neuritic growth, and nerve cell polarity were assessed after seven days in vitro. Cultures were labeled for rod opsin to distinguish rod from cone cells and for the alpha subunit of the trimeric G protein Go (Goα) to identify cone-dominated and mixed rod-cone ON bipolar cells. Results Quantitative analysis of growth demonstrated that target preferences were cell-specific: Cone cells preferred second-order bipolar cells, whereas rod cells grew toward third-order neurons, which include amacrine and ganglion cells. In addition, when rod cells grew toward bipolar cells, they chose an abnormally high number of Goα-positive bipolar cells. These growth patterns were not affected by tweezers manipulation or the amount of growth. Cell orientation of the photoreceptor also did not affect preferences: Cells oriented away from dendritic processes could reorient their axonal pole toward the target cell. Conclusions Cone cells preferred normal partners, and rod cells preferred novel partners. These intrinsic preferences indicate that adult nerve cells can have differing capacities for targeting even if they come from the same cell class. Further, these differences may help explain the patterns of photoreceptor sprouting seen in retinal degeneration in which rod, but not cone, cells invade the inner retinal layers where third-order neurons are located. PMID:18432315

  4. Engineering Retina from Human Retinal Progenitors (Cell Lines)

    PubMed Central

    Cao, Yang

    2009-01-01

    Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell–cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr2e3, expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription–polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell

  5. Characteristics of photosensors based on solid solutions of AII BVIcompounds

    NASA Astrophysics Data System (ADS)

    Lubegin, Gennady V.; Gusliannikov, Vladimir V.

    1999-10-01

    In the work there are submitted the results of the research of photosensors on a base of solid solutions of A(superscript II)B(superscript VI) compounds for measurement and control of intensity of low-level light in narrow areas of spectrum. The basic principles of technological process of manufacturing of injection photo diodes are described. The results of measurements of voltage-current characteristics and spectral characteristics of photo diodes, received in laboratory technological process with the various contents of cadmy and zinc in ZnCd(subscript 1-x)S(subscript x) solid solutions, and also sulfur and selen in CdS(subscript 1-x)Se(subscript x) solid solutions are submitted. The investigation results have shown, that photosensors work at low positive bias voltage, do not require cooling, have high sensitivity in a maximum and narrow selectivity. In CdS(subscript 1-x)Se(subscript x)- photosensors the photosensitive protecting coverage of transparent films on the base of As(subscript 2)S(subscript 3) compounds is applied. The opportunity of creation of a wide discrete range of photo diodes with the sensitivity in range from near ultraviolet up to near infrared area of spectrum is shown.

  6. Neets'aii Gwiindaii: Living in the Chandalar Country.

    ERIC Educational Resources Information Center

    Peter, Katherine

    The book is Katherine Peter's own account, in her native Gwich'in Athabaskan language, of life in 1936-47 in Arctic Village, Alaska. An introductory section offers background information on the author's life and the origins of this work. The text that follows is in Gwich'in Athabaskan on the left page, and in English on the right page. It tells of…

  7. DSCAM promotes refinement in the mouse retina through cell death and restriction of exploring dendrites.

    PubMed

    Li, Shuai; Sukeena, Joshua M; Simmons, Aaron B; Hansen, Ethan J; Nuhn, Renee E; Samuels, Ivy S; Fuerst, Peter G

    2015-04-08

    In this study we develop and use a gain-of-function mouse allele of the Down syndrome cell adhesion molecule (Dscam) to complement loss-of-function models. We assay the role of Dscam in promoting cell death, spacing, and laminar targeting of neurons in the developing mouse retina. We find that ectopic or overexpression of Dscam is sufficient to drive cell death. Gain-of-function studies indicate that Dscam is not sufficient to increase spatial organization, prevent cell-to-cell pairing, or promote active avoidance in the mouse retina, despite the similarity of the Dscam loss-of-function phenotype in the mouse retina to phenotypes observed in Drosophila Dscam1 mutants. Both gain- and loss-of-function studies support a role for Dscam in targeting neurites; DSCAM is necessary for precise dendrite lamination, and is sufficient to retarget neurites of outer retinal cells after ectopic expression. We further demonstrate that DSCAM guides dendrite targeting in type 2 dopaminergic amacrine cells, by restricting the stratum in which exploring retinal dendrites stabilize, in a Dscam dosage-dependent manner. Based on these results we propose a single model to account for the numerous Dscam gain- and loss-of-function phenotypes reported in the mouse retina whereby DSCAM eliminates inappropriately placed cells and connections.

  8. Response to change is facilitated by a three-neuron disinhibitory pathway in the tiger salamander retina.

    PubMed

    Roska, B; Nemeth, E; Werblin, F S

    1998-05-01

    Most retinal ganglion cells respond only transiently, for approximately 150 msec at the onset and termination of a light flash. The responses are transient because it has been shown that bipolar-to-ganglion cell transmission is truncated after 150 msec by a feedback inhibition to bipolar cell terminals. The feedback inhibition itself must be delayed by approximately 150 msec to allow the initial bipolar-ganglion cell transmission. This study identifies a three-component serial synaptic pathway from glycinergic amacrine cells to GABAergic amacrine cells to bipolar cell terminals as one source of this delay. We used perforated and whole-cell patch-clamp recordings to measure the timing of light responses in amacrine, bipolar, and ganglion cells under control and glycine and GABA receptor-blocked conditions. Our results suggest that, after a light flash, a population of glycinergic amacrine cells responds first, inhibiting a population of GABAergic amacrine cells for approximately 150 msec. The GABAergic amacrine cells feed back to bipolar terminals, but only after the 150 msec delay, allowing the bipolar terminals to excite ganglion cells for the first 150 msec. Blocking the glycinergic amacrine cell activity with strychnine allows the GABAergic system to become active earlier. GABAergic amacrine cells then inhibit release from bipolar cells earlier. Under these conditions, the ganglion cell response to change would be decreased.

  9. Synaptotagmin-7 Is Essential for Ca2+-Triggered Delayed Asynchronous Release But Not for Ca2+-Dependent Vesicle Priming in Retinal Ribbon Synapses

    PubMed Central

    Bacaj, Taulant

    2015-01-01

    Most synapses release neurotransmitters in two phases: (1) a fast synchronous phase lasting a few milliseconds; and (2) a delayed “asynchronous” phase lasting hundreds of milliseconds. Ca2+ triggers fast synchronous neurotransmitter release by binding to synaptotagmin-1, synaptotagmin-2, or synaptotagmin-9, but how Ca2+ triggers delayed asynchronous release has long remained enigmatic. Recent results suggested that consistent with the Ca2+-sensor function of synaptotagmin-7 in neuroendocrine exocytosis, synaptotagmin-7 also functions as a Ca2+ sensor for synaptic vesicle exocytosis but operates during delayed asynchronous release. Puzzlingly, a subsequent study postulated that synaptotagmin-7 is not a Ca2+ sensor for release but mediates Ca2+-dependent vesicle repriming after intense stimulation. To address these issues, we here analyzed synaptic transmission at rod bipolar neuron–AII amacrine cell synapses in acute mouse retina slices as a model system. Using paired recordings, we show that knock-out of synaptotagmin-7 selectively impairs delayed asynchronous release but not fast synchronous release. Delayed asynchronous release was blocked in wild-type synapses by intracellular addition of high concentrations of the slow Ca2+-chelator EGTA, but EGTA had no effect in synaptotagmin-7 knock-out neurons because delayed asynchronous release was already impaired. Moreover, direct measurements of vesicle repriming failed to uncover an effect of the synaptotagmin-7 knock-out on vesicle repriming. Our data demonstrate that synaptotagmin-7 is selectively essential for Ca2+-dependent delayed asynchronous release in retinal rod bipolar cell synapses, that its function can be blocked by simply introducing a slow Ca2+ buffer into the cells, and that synaptotagmin-7 is not required for normal vesicle repriming. SIGNIFICANCE STATEMENT How Ca2+ triggers delayed asynchronous release has long remained enigmatic. Synaptotagmin-7 has been implicated recently as Ca2+ sensor in

  10. Synaptotagmin-7 Is Essential for Ca2+-Triggered Delayed Asynchronous Release But Not for Ca2+-Dependent Vesicle Priming in Retinal Ribbon Synapses.

    PubMed

    Luo, Fujun; Bacaj, Taulant; Südhof, Thomas C

    2015-08-05

    Most synapses release neurotransmitters in two phases: (1) a fast synchronous phase lasting a few milliseconds; and (2) a delayed "asynchronous" phase lasting hundreds of milliseconds. Ca(2+) triggers fast synchronous neurotransmitter release by binding to synaptotagmin-1, synaptotagmin-2, or synaptotagmin-9, but how Ca(2+) triggers delayed asynchronous release has long remained enigmatic. Recent results suggested that consistent with the Ca(2+)-sensor function of synaptotagmin-7 in neuroendocrine exocytosis, synaptotagmin-7 also functions as a Ca(2+) sensor for synaptic vesicle exocytosis but operates during delayed asynchronous release. Puzzlingly, a subsequent study postulated that synaptotagmin-7 is not a Ca(2+) sensor for release but mediates Ca(2+)-dependent vesicle repriming after intense stimulation. To address these issues, we here analyzed synaptic transmission at rod bipolar neuron-AII amacrine cell synapses in acute mouse retina slices as a model system. Using paired recordings, we show that knock-out of synaptotagmin-7 selectively impairs delayed asynchronous release but not fast synchronous release. Delayed asynchronous release was blocked in wild-type synapses by intracellular addition of high concentrations of the slow Ca(2+)-chelator EGTA, but EGTA had no effect in synaptotagmin-7 knock-out neurons because delayed asynchronous release was already impaired. Moreover, direct measurements of vesicle repriming failed to uncover an effect of the synaptotagmin-7 knock-out on vesicle repriming. Our data demonstrate that synaptotagmin-7 is selectively essential for Ca(2+)-dependent delayed asynchronous release in retinal rod bipolar cell synapses, that its function can be blocked by simply introducing a slow Ca(2+) buffer into the cells, and that synaptotagmin-7 is not required for normal vesicle repriming. How Ca(2+) triggers delayed asynchronous release has long remained enigmatic. Synaptotagmin-7 has been implicated recently as Ca(2+) sensor in

  11. A synaptic mechanism for retinal adaptation to luminance and contrast

    PubMed Central

    Jarsky, Tim; Cembrowski, Mark; Logan, Stephen M.; Kath, William L.; Riecke, Hermann; Demb, Jonathan B.; Singer, Joshua H.

    2011-01-01

    The gain of signaling in primary sensory circuits is matched to the stimulus intensity by the process of adaptation. Retinal neural circuits adapt to visual scene statistics, including the mean (background adaptation) and the temporal variance (contrast adaptation) of the light stimulus. The intrinsic properties of retinal bipolar cells and synapses contribute to background and contrast adaptation, but it is unclear whether both forms of adaptation depend on the same cellular mechanisms. Studies of bipolar cell synapses identified synaptic mechanisms of gain control, but the relevance of these mechanisms to visual processing is uncertain owing to the historical focus on fast, phasic transmission rather than the tonic transmission evoked by ambient light. Here, we studied use-dependent regulation of bipolar cell synaptic transmission evoked by small, ongoing modulations of membrane potential (VM) in the physiological range. We made paired whole-cell recordings from rod bipolar (RB) and AII amacrine cells in a mouse retinal slice preparation. Quasi-white noise voltage commands modulated RB VM and evoked EPSCs in the AII. We mimicked changes in background luminance or contrast, respectively, by depolarizing the VM or increasing its variance. A linear systems analysis of synaptic transmission showed that increasing either the mean or the variance of the presynaptic VM reduced gain. Further electrophysiological and computational analyses demonstrated that adaptation to mean potential resulted from both Ca channel inactivation and vesicle depletion, whereas adaptation to variance resulted from vesicle depletion alone. Thus, background and contrast adaptation apparently depend in part on a common synaptic mechanism. PMID:21795549

  12. Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

    2010-01-01

    The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

  13. Glycinergic synaptic inputs to bipolar cells in the salamander retina

    PubMed Central

    Maple, Bruce R; Wu, Samuel M

    1998-01-01

    Glycine activated strychnine-sensitive chloride conductances at both the dendrites and the axonal telodendria of most bipolar cells in the salamander retina. The chloride equilibrium potential of bipolar cells was found to be negative to -50 mV, indicating that glycinergic synapses on bipolar cells are inhibitory. Some bipolar cells exhibited discrete, strychnine-sensitive, chloride-mediated inhibitory postsynaptic currents (IPSCs). These were elicited by focal application of glutamate at the inner plexiform layer (IPL). Glycinergic synapses were localized using simultaneous focal application of calcium to retinal slices bathed in calcium-free media. Both dendritic and telodendritic glycinergic IPSCs were observed. The decay of the telodendritic IPSCs was well fitted by a single exponential with a time constant of 17.7 ± 8.7 ms. Similar kinetics were observed for dendritic IPSCs in some cells, but in one class of on-centre bipolar cell the decay of the dendritic IPSCs was better fitted by a sum of two exponentials with time constants 9.9 ± 4.3 and 51.3 ± 24.3 ms. The dendritic IPSCs were best driven by application of glutamate at the distal IPL (the off sublamina), while the telodendritic IPSCs were driven best by application near the telodendria. These results suggest that bipolar cell dendrites receive inhibitory glycinergic inputs from interplexiform cells that are excited by off-centre bipolar cells, whereas bipolar cell telodendria receive glycinergic amacrine cell inputs that are antagonistic to the photoreceptor inputs. Both inputs could be elicited in the presence of tetrodotoxin (TTX), but the dendritic IPSCs were sometimes abolished by TTX, suggesting that sodium-dependent spikes play an important role in the transmission of interplexiform cell signals to the outer plexiform layer. PMID:9503334

  14. GAP-43 expression is upregulated in retinal ganglion cells after ischemia/reperfusion-induced damage.

    PubMed

    Dijk, Frederike; Bergen, Arthur A B; Kamphuis, Willem

    2007-05-01

    In response to injury, the adult mammalian retina shows signs of structural remodeling, possibly in an attempt to preserve or regain some of its functional neural connections. In order to study the mechanisms involved in injury-induced plasticity, we have studied changes in growth associated protein 43 (GAP-43) after retinal ischemia/reperfusion in the rat. GAP-43 is a marker for neuronal remodeling and is involved in synapse formation. Ischemic injury of the rat retina was induced by 60 min of ischemia followed by reperfusion times varying from 2h up to 4 weeks. GAP-43 mRNA levels were significantly increased between 12h and 72 h reperfusion with a peak around 24h. GAP-43 specific antibodies showed that the total amount of GAP-43 labeling in the inner plexiform layer was diminished after 12h of reperfusion by approximately 35% and remained at this level up to 1 week postischemia despite the reduction in thickness of this layer during this period resulting from the ischemia-induced cell loss. At 2 and 4 weeks reperfusion, the amount of labeling was reduced by 70%, simultaneously with a decrease of GAP-43 transcript level. Between 72 h up to 2 weeks postischemia, the induction of intense GAP-43 labeling was observed in NeuN- and beta-tubulin-positive ganglion cell somata and in horizontally and vertically oriented processes in the inner plexiform layer. Ischemia also induced GAP-43 expression in some GFAP-positive Müller cells. Double-labeling showed that in controls and after ischemia GAP-43 was expressed by some amacrine cells of the glycinergic (glycine transporter 1), calretinin-positive, and dopaminergic (tyrosine hydroxylase) subpopulations. No increase of GAP-43 expression levels was found in these amacrine cells. The results demonstrate that ganglion cells show an elevated expression of GAP-43 after ischemia-inflicted damage. These findings suggest a temporal window during which ganglion cells may remodel their neuronal network in the damaged retina.

  15. Ionotropic glutamate receptors mediate OFF responses in light-adapted ON bipolar cells

    PubMed Central

    Pang, Ji-Jie; Gao, Fan; Wu, Samuel M.

    2013-01-01

    Previous studies have suggested that photoreceptor synaptic inputs to depolarizing bipolar cells (DBCs or ON bipolar cells) are mediated by mGluR6 receptors and those to hyperpolarizing bipolar cells (HBCs or OFF bipolar cells) are mediated by AMPA/kainate receptors. Here we show that in addition to mGluR6 receptors which mediate the sign-inverting, depolarizing light responses, subpopulations of cone-dominated and rod/cone mixed DBCs use GluR4 AMPA receptors to generate a transient sign-preserving OFF response under light adapted conditions. These AMPA receptors are located at the basal junctions postsynaptic to rods and they are silent under dark-adapted conditions, as tonic glutamate release in darkness desensitizes these receptors. Light adaptation enhances rod-cone coupling and thus allows cone photocurrents with an abrupt OFF depolarization to enter the rods. The abrupt rod depolarization triggers glutamate activation of unoccupied AMPA receptors, resulting in a transient OFF response in DBCs. It has been widely accepted that the DNQX-sensitive, OFF transient responses in retinal amacrine cells and ganglion cells are mediated exclusively by HBCs. Our results suggests that this view needs revision as AMPA receptors in subpopulations of DBCs are likely to significantly contribute to the DNQX-sensitive OFF transient responses in light-adapted third- and higher-order visual neurons. PMID:22842089

  16. Endothelial cell injury initiates glomerular sclerosis in the rat remnant kidney.

    PubMed Central

    Lee, L K; Meyer, T W; Pollock, A S; Lovett, D H

    1995-01-01

    The development of progressive glomerulosclerosis in the renal ablation model has been ascribed to a number of humoral and hemodynamic events, including the peptide growth factor, transforming growth factor-beta 1 (TGF-beta 1). An important role has also been attributed to angiotensin II (AII), which, in addition to its hemodynamic effects, can stimulate transcription of TGF-beta 1. We postulated that increased glomerular production of AII, resulting from enhanced intrinsic angiotensinogen expression, stimulates local TGF-beta 1 synthesis, activating glomerular matrix protein synthesis, and leads to sclerosis. Using in situ reverse transcription, the glomerular cell sites of alpha-1 (IV) collagen, fibronectin, laminin B1, angiotensinogen, and TGF-beta 1 mRNA synthesis were determined at sequential periods following renal ablation. The early hypertrophic phase was associated with global, but transient, increases in the mRNA for alpha-1 (IV) collagen. No changes were noted for fibronectin, TGF-beta 1, and angiotensinogen mRNAs. At 24 d after ablation, at which time sclerosis is not evident, endothelial cells, particularly in the dilated capillaries at the vascular pole, expressed angiotensinogen and TGF-beta 1 mRNAs, as well as fibronectin and laminin B1 RNA transcripts. By 74 d after ablation angiotensinogen and TGF-beta 1 mRNAs were widely distributed among endothelial and mesangial cells, and were particularly prominent in regions of evolving sclerosis. These same regions were also notable for enhanced expression of matrix protein mRNAs, particularly fibronectin. All receptor blockade inhibited angiotensinogen, TGF-beta 1, fibronectin, and laminin B1 mRNA expression by the endothelium. We conclude that, as a result of hemodynamic changes, injured or activated endothelium synthesizes angiotensinogen, triggering a cascade of TGF-beta 1 and matrix protein gene expression with resultant development of the segmental glomerular sclerotic lesion. Images PMID:7635988

  17. Rbpj cell autonomous regulation of retinal ganglion cell and cone photoreceptor fates in the mouse retina.

    PubMed

    Riesenberg, Amy N; Liu, Zhenyi; Kopan, Raphael; Brown, Nadean L

    2009-10-14

    Vertebrate retinal progenitor cells (RPCs) are pluripotent, but pass through competence states that progressively restrict their developmental potential (Cepko et al., 1996; Livesey and Cepko, 2001; Cayouette et al., 2006). In the rodent eye, seven retinal cell classes differentiate in overlapping waves, with RGCs, cone photoreceptors, horizontals, and amacrines forming predominantly before birth, and rod photoreceptors, bipolars, and Müller glia differentiating postnatally. Both intrinsic and extrinsic factors regulate each retinal cell type (for review, see Livesey and Cepko, 2001). Here, we conditionally deleted the transcription factor Rbpj, a critical integrator of multiple Notch signals (Jarriault et al., 1995; Honjo, 1996; Kato et al., 1997; Han et al., 2002), during prenatal mouse retinal neurogenesis. Removal of Rbpj caused reduced proliferation, premature neuronal differentiation, apoptosis, and profound mispatterning. To determine the cell autonomous requirements for Rbpj during RGC and cone formation, we marked Cre-generated retinal lineages with GFP expression, which showed that Rbpj autonomously promotes RPC mitotic activity, and suppresses RGC and cone fates. In addition, the progressive loss of Rbpj-/- RPCs resulted in a diminished progenitor pool available for rod photoreceptor formation. This circumstance, along with the overproduction of Rbpj-/- cones, revealed that photoreceptor development is under homeostatic regulation. Finally, to understand how the Notch pathway regulates the simultaneous formation of multiple cell types, we compared the RGC and cone phenotypes of Rbpj to Notch1 (Jadhav et al., 2006b; Yaron et al., 2006), Notch3, and Hes1 mutants. We found particular combinations of Notch pathway genes regulate the development of each retinal cell type.

  18. Kainate Receptors Mediate Signaling in Both Transient and Sustained OFF Bipolar Cell Pathways in Mouse Retina

    PubMed Central

    Looger, Loren L.; Tomita, Susumu

    2014-01-01

    A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors. PMID:24790183

  19. Receptive fields of retinal bipolar cells are mediated by heterogeneous synaptic circuitry

    PubMed Central

    Zhang, Ai-Jun; Wu, Samuel M.

    2009-01-01

    Center-surround antagonistic receptive field (CSARF) organization is the basic synaptic circuit that serves as elementary building blocks for spatial information processing in the visual system. Cells with such receptive fields converge into higher-order visual neurons to form more complex receptive fields. Retinal bipolar cells (BCs) are the first neurons along the visual pathway that exhibit CSARF organization. BCs have been classified according to their response polarities and rod/cone inputs, and they project signals to target cells at different sublaminae of the inner plexiform layer. On the other hand, CSARFs of various types of BCs have been assumed be organized the same way. Here we examined center and surround responses of over 250 salamander BCs, and demonstrated that different types of BCs exhibit different patterns of dye coupling, receptive field center size, surround response strength, and conductance changes associated with center and surround responses. We show that BC receptive field center sizes varied with the degree of BC-BC coupling, and that surround responses of different BCs are mediated by different combinations of five lateral synaptic pathways mediated by the horizontal cells and amacrine cells. The finding of heterogeneous receptive field circuitry fundamentally challenges the common assumption that CSARFs of different subtypes of visual neurons are mediated by the same synaptic pathways. BCs carrying different visual signals use different synaptic circuits to process spatial information, allowing shape and contrast computation be differentially modulated by various lighting and adaptation conditions. PMID:19158304

  20. Membrane currents of spiking cells isolated from turtle retina.

    PubMed

    Lasater, E M; Witkovsky, P

    1990-05-01

    We examined the membrane properties of spiking neurons isolated from the turtle (Pseudemys scripta) retina. The cells were maintained in culture for 1-7 days and were studied with the whole cell patch clamp technique. We utilized cells whose perikaryal diameters were greater than 15 microns since Kolb (1982) reported that ganglion cell perikarya in Pseudemys retina are 13-25 microns, whereas amacrine perikarya are less than 14 microns in diameter. We identified 5 currents in the studied cells: (1) a transient sodium current (INa) blocked by TTX, (2) a sustained calcium current (ICa) blocked by cobalt and enhanced by Bay-K 8644, (3) a calcium-dependent potassium current (IK(Ca)), (4) an A-type transient potassium current (IA) somewhat more sensitive to 4-AP than TEA, (5) a sustained potassium current (IK) more sensitive to TEA than 4-AP. The estimated average input resistance of the cells at -70 mV was 720 +/- 440 M omega. When all active currents were blocked, the membrane resistance between -130 and +20 mV was 2.5 G omega. When examined under current clamp, some cells produced multiple spikes to depolarizing steps of 0.1-0.3 nA, whereas other cells produced only a single spike irrespective of the strength of the current pulse. Most single spikers had an outward current that rose to a peak relatively slowly, whereas multiple spikers tend to have a more rapidly activating outward current. Under current clamp, 4-AP slowed the repolarization phase of the spike thus broadening it, but did not always abolish the ability to produce multiple spikes. TEA induced a depolarized plateau following the initial spike which precluded further spikes. It thus appears that the spiking patterns of the retinal cells are shaped primarily by the kinetics of INa, IK and IA and to a lesser extent by IK(Ca).

  1. Identification of protein interaction partners in mammalian cells using SILAC-immunoprecipitation quantitative proteomics.

    PubMed

    Emmott, Edward; Goodfellow, Ian

    2014-07-06

    Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

  2. Distribution and morphology of retinal ganglion cells in the Japanese quail.

    PubMed

    Ikushima, M; Watanabe, M; Ito, H

    1986-06-25

    A ganglion cell density map was produced from the Nissl-stained retinal whole mount of the Japanese quail. Ganglion cell density diminished nearly concentrically from the central area toward the retinal periphery. The mean soma area of ganglion cells in isodensity zones increased as the cell density decreased. The histograms of soma areas in each zone indicated that a population of small-sized ganglion cells persists into the peripheral retina. The total number of ganglion cells was estimated at about 2.0 million. Electron microscopic examination of the optic nerve revealed thin unmyelinated axons to comprise 69% of the total fiber count (about 2.0 million). Since there was no discrepancy between both the total numbers of neurons in the ganglion cell layer and optic nerve fibers, it is inferred that displaced amacrine cells are few, if any. The spectrum in optic nerve fiber diameter showed a unimodal skewed distribution quite similar to the histogram of soma areas of ganglion cells in the whole retina. This suggests a close correlation between soma areas and axon diameters. Retinal ganglion cells filled from the optic nerve with horseradish peroxidase were classified into 7 types according to such morphological characteristics as size, shape and location of the soma, as well as dendritic arborization pattern. Taking into account areal ranges of somata of each cell type, it can be assumed that most of the ganglion cells in the whole retinal ganglion cell layer are composed of type I, II and III cells, and that the population of uniformly small-sized ganglion cells corresponds to type I cells and is an origin of unmyelinated axons in the optic nerve.

  3. Purification of an angiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA

    SciTech Connect

    Elton, T.S.; Dion, L.D.; Bost, K.L.; Oparil, S.; Blalock, J.E.

    1988-04-01

    The authors have generated a monospecific antibody to a synthetic peptide encoded by an RNA complementary to the mRNA for angiotensin II (AII) and determined whether this antibody recognizes the AII receptor. They demonstrate that the antibody competes specifically with /sup 125/I-labeled AII for the same binding site on rat adrenal membranes. Furthermore, they show this antibody inhibits the secretion of aldosterone from cultured rat adrenal cells, suggesting that the antibody recognizes the biologically relevant AII receptor. Finally, they demonstrate that antibody to the complementary peptide can be used to immunoaffinity-purify a protein of M/sub r/ 66,000 that specifically binds radiolabeled AII.

  4. A high frequency resonance in the responses of retinal ganglion cells to rapidly modulated stimuli: A computer model

    PubMed Central

    MILLER, J.A.; DENNING, K.S.; GEORGE, J.S.; MARSHAK, D.W.; KENYON, G.T.

    2012-01-01

    Brisk Y-type ganglion cells in the cat retina exhibit a high frequency resonance (HFR) in their responses to large, rapidly modulated stimuli. We used a computer model to test whether negative feedback mediated by axon-bearing amacrine cells onto ganglion cells could account for the experimentally observed properties of HFRs. Temporal modulation transfer functions (tMTFs) recorded from model ganglion cells exhibited HFR peaks whose amplitude, width, and locations were qualitatively consistent with experimental data. Moreover, the wide spatial distribution of axon-mediated feedback accounted for the observed increase in HFR amplitude with stimulus size. Model phase plots were qualitatively similar to those recorded from Y ganglion cells, including an anomalous phase advance that in our model coincided with the amplification of low-order harmonics that overlapped the HFR peak. When axon-mediated feedback in the model was directed primarily to bipolar cells, whose synaptic output was graded, or else when the model was replaced with a simple cascade of linear filters, it was possible to produce large HFR peaks but the region of anomalous phase advance was always eliminated, suggesting the critical involvement of strongly non-linear feedback loops. To investigate whether HFRs might contribute to visual processing, we simulated high frequency ocular tremor by rapidly modulating a naturalistic image. Visual signals riding on top of the imposed jitter conveyed an enhanced representation of large objects. We conclude that by amplifying responses to ocular tremor, HFRs may selectively enhance the processing of large image features. PMID:17020633

  5. Excitatory synaptic inputs to mouse on-off direction-selective retinal ganglion cells lack direction tuning.

    PubMed

    Park, Silvia J H; Kim, In-Jung; Looger, Loren L; Demb, Jonathan B; Borghuis, Bart G

    2014-03-12

    Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.

  6. KUS121, a VCP modulator, attenuates ischemic retinal cell death via suppressing endoplasmic reticulum stress

    PubMed Central

    Hata, Masayuki; Ikeda, Hanako O.; Kikkawa, Chinami; Iwai, Sachiko; Muraoka, Yuki; Hasegawa, Tomoko; Kakizuka, Akira; Yoshimura, Nagahisa

    2017-01-01

    Ischemic neural damages cause several devastating diseases, including brain stroke and ischemic retinopathies, and endoplasmic reticulum (ER) stress has been proposed to be the underlying mechanism of the neuronal cell death of these conditions. We previously synthesized Kyoto University substances (KUSs) as modulators of valosin-containing protein (VCP); KUSs inhibit VCP ATPase activity and protect cells from different cell death-inducing insults. Here, we examined the efficacy of KUS121 in a rat model of retinal ischemic injury. Systemic administration of KUS121 to rats with ischemic retinal injury significantly suppressed inner retinal thinning and death of retinal ganglion and amacrine cells, with a significant functional maintenance of visual functions, as judged by electroretinography. Furthermore, intravitreal injection of KUS121, which is the clinically preferred route of drug administration for retinal diseases, appeared to show an equal or better neuroprotective efficacy in the ischemic retina compared with systemic administration. Indeed, induction of the ER stress marker C/EBP homologous protein (CHOP) after the ischemic insult was significantly suppressed by KUS121 administration. Our study suggests VCP modulation by KUS as a promising novel therapeutic strategy for ischemic neuronal diseases. PMID:28317920

  7. Multiple components of ganglion cell desensitization in response to prosthetic stimulation

    NASA Astrophysics Data System (ADS)

    Freeman, Daniel K.; Fried, Shelley I.

    2011-02-01

    Retinal prostheses aim to restore functional vision to those blinded by outer retinal diseases using electric stimulation of surviving neurons. Previous work indicates that repetitive stimulation with stimuli that activate the synaptic network reduces the sensitivity of retinal neurons to further stimulation. Such desensitization may contribute to the fading of visual percepts over time reported by human subjects. Here, we show that desensitization may be more complex than previously considered. We recorded spike trains from rabbit retinal ganglion cells and found that desensitization persists in the presence of inhibitory blockers (strychnine and picrotoxin), indicating amacrine cell inhibition is not solely responsible for reducing sensitivity in response to electric stimulation. The threshold for direct activation of the ganglion cell changes little during the simultaneous desensitization of the synaptically mediated response, indicating that desensitization likely occurs upstream of the spike generator. In addition to rapid desensitization acting over hundreds of milliseconds (τ = 176.4 ± 8.8 ms), we report the presence of slow acting desensitization with a time course of seconds (τ = 14.0 ± 1.1 s). The time courses of the two components of desensitization that we found are similar to the two phases of brightness fading seen in human subjects. This suggests that the reduction in ganglion cell firing due to desensitization may be responsible for the fading of visual percepts over time in response to prosthetic stimulation.

  8. Retinal Connectomics: Towards Complete, Accurate Networks

    PubMed Central

    Marc, Robert E.; Jones, Bryan W.; Watt, Carl B.; Anderson, James R.; Sigulinsky, Crystal; Lauritzen, Scott

    2013-01-01

    Connectomics is a strategy for mapping complex neural networks based on high-speed automated electron optical imaging, computational assembly of neural data volumes, web-based navigational tools to explore 1012–1015 byte (terabyte to petabyte) image volumes, and annotation and markup tools to convert images into rich networks with cellular metadata. These collections of network data and associated metadata, analyzed using tools from graph theory and classification theory, can be merged with classical systems theory, giving a more completely parameterized view of how biologic information processing systems are implemented in retina and brain. Networks have two separable features: topology and connection attributes. The first findings from connectomics strongly validate the idea that the topologies complete retinal networks are far more complex than the simple schematics that emerged from classical anatomy. In particular, connectomics has permitted an aggressive refactoring of the retinal inner plexiform layer, demonstrating that network function cannot be simply inferred from stratification; exposing the complex geometric rules for inserting different cells into a shared network; revealing unexpected bidirectional signaling pathways between mammalian rod and cone systems; documenting selective feedforward systems, novel candidate signaling architectures, new coupling motifs, and the highly complex architecture of the mammalian AII amacrine cell. This is but the beginning, as the underlying principles of connectomics are readily transferrable to non-neural cell complexes and provide new contexts for assessing intercellular communication. PMID:24016532

  9. Dual requirement for Pax6 in retinal progenitor cells

    PubMed Central

    Elgart, Michael; Marquardt, Till; Remizova, Lena; Yaron, Orly; Xie, Qing; Cvekl, Ales; Ashery-Padan, Ruth

    2014-01-01

    Throughout the developing central nervous system, pre-patterning of the ventricular zone into discrete neural progenitor domains is one of the predominant strategies used to produce neuronal diversity in a spatially coordinated manner. In the retina, neurogenesis proceeds in an intricate chronological and spatial sequence, yet it remains unclear whether retinal progenitor cells (RPCs) display intrinsic heterogeneity at any given time point. Here, we performed a detailed study of RPC fate upon temporally and spatially confined inactivation of Pax6. Timed genetic removal of Pax6 appeared to unmask a cryptic divergence of RPCs into qualitatively divergent progenitor pools. In the more peripheral RPCs under normal circumstances, Pax6 seemed to prevent premature activation of a photoreceptor-differentiation pathway by suppressing expression of the transcription factor Crx. More centrally, Pax6 contributed to the execution of the comprehensive potential of RPCs: Pax6 ablation resulted in the exclusive generation of amacrine interneurons. Together, these data suggest an intricate dual role for Pax6 in retinal neurogenesis, while pointing to the cryptic divergence of RPCs into distinct progenitor pools. PMID:19004853

  10. Independent control of reciprocal and lateral inhibition at the axon terminal of retinal bipolar cells

    PubMed Central

    Tanaka, Masashi; Tachibana, Masao

    2013-01-01

    Bipolar cells (BCs), the second order neurons in the vertebrate retina, receive two types of GABAergic feedback inhibition at their axon terminal: reciprocal and lateral inhibition. It has been suggested that two types of inhibition may be mediated by different pathways. However, how each inhibition is controlled by excitatory BC output remains to be clarified. Here, we applied single/dual whole cell recording techniques to the axon terminal of electrically coupled BCs in slice preparation of the goldfish retina, and found that each inhibition was regulated independently. Activation voltage of each inhibition was different: strong output from a single BC activated reciprocal inhibition, but could not activate lateral inhibition. Outputs from multiple BCs were essential for activation of lateral inhibition. Pharmacological examinations revealed that composition of transmitter receptors and localization of Na+ channels were different between two inhibitory pathways, suggesting that different amacrine cells may mediate each inhibition. Depending on visual inputs, each inhibition could be driven independently. Model simulation showed that reciprocal and lateral inhibition cooperatively reduced BC outputs as well as background noise, thereby preserving high signal-to-noise ratio. Therefore, we conclude that excitatory BC output is efficiently regulated by the dual operating mechanisms of feedback inhibition without deteriorating the quality of visual signals. PMID:23690563

  11. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    PubMed

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists.

  12. Automated computation of arbor densities: a step toward identifying neuronal cell types

    PubMed Central

    Sümbül, Uygar; Zlateski, Aleksandar; Vishwanathan, Ashwin; Masland, Richard H.; Seung, H. Sebastian

    2014-01-01

    The shape and position of a neuron convey information regarding its molecular and functional identity. The identification of cell types from structure, a classic method, relies on the time-consuming step of arbor tracing. However, as genetic tools and imaging methods make data-driven approaches to neuronal circuit analysis feasible, the need for automated processing increases. Here, we first establish that mouse retinal ganglion cell types can be as precise about distributing their arbor volumes across the inner plexiform layer as they are about distributing the skeletons of the arbors. Then, we describe an automated approach to computing the spatial distribution of the dendritic arbors, or arbor density, with respect to a global depth coordinate based on this observation. Our method involves three-dimensional reconstruction of neuronal arbors by a supervised machine learning algorithm, post-processing of the enhanced stacks to remove somata and isolate the neuron of interest, and registration of neurons to each other using automatically detected arbors of the starburst amacrine interneurons as fiducial markers. In principle, this method could be generalizable to other structures of the CNS, provided that they allow sparse labeling of the cells and contain a reliable axis of spatial reference. PMID:25505389

  13. Photoresponse diversity among the five types of intrinsically photosensitive retinal ganglion cells.

    PubMed

    Zhao, Xiwu; Stafford, Ben K; Godin, Ashley L; King, W Michael; Wong, Kwoon Y

    2014-04-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate non-image-forming visual responses, including pupillary constriction, circadian photoentrainment and suppression of pineal melatonin secretion. Five morphological types of ipRGCs, M1-M5, have been identified in mice. In order to understand their functions better, we studied the photoresponses of all five cell types, by whole-cell recording from fluorescently labelled ipRGCs visualized using multiphoton microscopy. All ipRGC types generated melanopsin-based ('intrinsic') as well as synaptically driven ('extrinsic') light responses. The intrinsic photoresponses of M1 cells were lower threshold, higher amplitude and faster than those of M2-M5. The peak amplitudes of extrinsic light responses differed among the ipRGC types; however, the responses of all cell types had comparable thresholds, kinetics and waveforms, and all cells received rod input. While all five types exhibited inhibitory amacrine-cell and excitatory bipolar-cell inputs from the 'on' channel, M1 and M3 received additional 'off'-channel inhibition, possibly through their 'off'-sublamina dendrites. The M2-M5 ipRGCs had centre-surround-organized receptive fields, implicating a capacity to detect spatial contrast. In contrast, the receptive fields of M1 cells lacked surround antagonism, which might be caused by the surround of the inhibitory input nullifying the surround of the excitatory input. All ipRGCs responded robustly to a wide range of motion speeds, and M1-M4 cells appeared tuned to different speeds, suggesting that they might analyse the speed of motion. Retrograde labelling revealed that M1-M4 cells project to the superior colliculus, suggesting that the contrast and motion information signalled by these cells could be used by this sensorimotor area to detect novel objects and motion in the visual field.

  14. Pharmacological Analysis of Intrinsic Neuronal Oscillations in rd10 Retina

    PubMed Central

    Biswas, Sonia; Haselier, Christine; Mataruga, Anja; Thumann, Gabriele; Walter, Peter; Müller, Frank

    2014-01-01

    In the widely used mouse model of retinal degeneration, rd1, the loss of photoreceptors leads to rhythmic electrical activity of around 10–16 Hz in the remaining retinal network. Recent studies suggest that this oscillation is formed within the electrically coupled network of AII amacrine cells and ON-bipolar cells. A second mouse model, rd10, displays a delayed onset and slower progression of degeneration, making this mouse strain a better model for human retinitis pigmentosa. In rd10, oscillations occur at a frequency of 3–7 Hz, raising the question whether oscillations have the same origin in the two mouse models. As rd10 is increasingly being used as a model to develop experimental therapies, it is important to understand the mechanisms underlying the spontaneous rhythmic activity. To study the properties of oscillations in rd10 retina we combined multi electrode recordings with pharmacological manipulation of the retinal network. Oscillations were abolished by blockers for ionotropic glutamate receptors and gap junctions. Frequency and amplitude of oscillations were modulated strongly by blockers of inhibitory receptors and to a lesser extent by blockers of HCN channels. In summary, although we found certain differences in the pharmacological modulation of rhythmic activity in rd10 compared to rd1, the overall pattern looked similar. This suggests that the generation of rhythmic activity may underlie similar mechanisms in rd1 and rd10 retina. PMID:24918437

  15. Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca(2+) channels.

    PubMed

    Luo, Fujun; Liu, Xinran; Südhof, Thomas C; Acuna, Claudio

    2017-09-05

    Fast neurotransmitter release from ribbon synapses via Ca(2+)-triggered exocytosis requires tight coupling of L-type Ca(2+) channels to release-ready synaptic vesicles at the presynaptic active zone, which is localized at the base of the ribbon. Here, we used genetic, electrophysiological, and ultrastructural analyses to probe the architecture of ribbon synapses by perturbing the function of RIM-binding proteins (RBPs) as central active-zone scaffolding molecules. We found that genetic deletion of RBP1 and RBP2 did not impair synapse ultrastructure of ribbon-type synapses formed between rod bipolar cells (RBCs) and amacrine type-2 (AII) cells in the mouse retina but dramatically reduced the density of presynaptic Ca(2+) channels, decreased and desynchronized evoked neurotransmitter release, and rendered evoked and spontaneous neurotransmitter release sensitive to the slow Ca(2+) buffer EGTA. These findings suggest that RBPs tether L-type Ca(2+) channels to the active zones of ribbon synapses, thereby synchronizing vesicle exocytosis and promoting high-fidelity information transfer in retinal circuits.

  16. Mechanisms of allele-selective down-regulation of HLA class I in Burkitt's lymphoma.

    PubMed

    Imreh, M P; Zhang, Q J; de Campos-Lima, P O; Imreh, S; Krausa, P; Browning, M; Klein, G; Masucci, M G

    1995-07-04

    Burkitt lymphomas (BL) that arise in HLA-AII-positive individuals are characterized by selective loss/down-regulation of the HLA AII polypeptide. We have investigated the molecular basis of such down-regulation by comparing 5 pairs of BL lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The presence of apparently intact HLA AII genes was confirmed in all 5 BL/LCL pairs by polymerase chain reaction (PCR) typing and by Southern-blot hybridization with HLA A locus-specific probes. Northern-blot analysis with locus- and allele-specific probes revealed a significantly lower expression or absence of AII-specific mRNA in all 5 BL lines compared to the corresponding LCLs. Up-regulation of AII-specific mRNA was achieved by IFN alpha treatment of 2 BL lines with low HLA AII expression (BL-28 and BL-72) while the treatment had no effect in 3 BL lines (WWI-BL, WW2-BL and BL41) that did not express the endogenous gene. HLA AII expression was restored by transfection of the gene in WWI-BL whereas transfectants of BL-41 remained AII-negative. An HLA-AII-promoter-driven chloramphenicol acetyl transferase reporter gene (pAIICAT) was active in WWI-BL but not in BL-41. HLA-AII was expressed in hybrids of BL-41 with an AII-positive LCL, while expression of the endogenous HLA AII gene could not be restored by fusion of BL-41 with an AII-negative LCL, although an adequate set of transcription factors was present in the hybrid. Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down-regulation of HLA AII in BL cells.

  17. Histone demethylase Jmjd3 is required for the development of subsets of retinal bipolar cells.

    PubMed

    Iida, Atsumi; Iwagawa, Toshiro; Kuribayashi, Hiroshi; Satoh, Shinya; Mochizuki, Yujin; Baba, Yukihiro; Nakauchi, Hiromitsu; Furukawa, Takahisa; Koseki, Haruhiko; Murakami, Akira; Watanabe, Sumiko

    2014-03-11

    Di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) is an important gene repression mechanism. H3K27me2/3-specific demethylase, Jmjd3, was expressed in the inner nuclear layer during late retinal development. In contrast, H3K27 methyltransferase, Ezh2, was highly expressed in the embryonic retina but its expression decreased rapidly after birth. Jmjd3 loss of function in the developing retina resulted in failed differentiation of PKC-positive bipolar cell subsets (rod-ON-BP) and reduced transcription factor Bhlhb4 expression, which is critical for the differentiation of rod-ON-BP cells. Overexpression of Bhlhb4, but not of other BP cell-related genes, such as transcription factors Neurod and Chx10, in Jmjd3-knockdown retina rescued loss of PKC-positive BP cells. Populations of other retinal cell subsets were not significantly affected. In addition, proliferation activity and apoptotic cell number during retinal development were not affected by the loss of Jmjd3. Levels of histone H3 trimethyl Lys27 (H3K27me3) in the Bhlhb4 locus were lower in Islet-1-positive BP cells and amacrine cells than in the Islet-1-negative cell fraction. The Islet-1-negative cell fraction consisted mainly of photoreceptors, suggestive of lineage-specific demethylation of H3K27me3 in the Bhlhb4 locus. We propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal cells.

  18. Inhibitory input to the direction-selective ganglion cell is saturated at low contrast

    PubMed Central

    Taylor, W. Rowland; Smith, Robert G.

    2015-01-01

    Direction-selective ganglion cells (DSGCs) respond selectively to motion toward a “preferred” direction, but much less to motion toward the opposite “null” direction. Directional signals in the DSGC depend on GABAergic inhibition and are observed over a wide range of speeds, which precludes motion detection based on a fixed temporal correlation. A voltage-clamp analysis, using narrow bar stimuli similar in width to the receptive field center, demonstrated that inhibition to DSGCs saturates rapidly above a threshold contrast. However, for wide bar stimuli that activate both the center and surround, inhibition depends more linearly on contrast. Excitation for both wide and narrow bars was also more linear. We propose that positive feedback, likely within the starburst amacrine cell or its network, produces steep saturation of inhibition at relatively low contrast. This mechanism renders GABA release essentially contrast and speed invariant, which enhances directional signals for small objects and thereby increases the signal-to-noise ratio for direction-selective signals in the spike train over a wide range of stimulus conditions. The steep saturation of inhibition confers to a neuron immunity to noise in its spike train, because when inhibition is strong no spikes are initiated. PMID:26063782

  19. Gap Junctions Are Essential for Generating the Correlated Spike Activity of Neighboring Retinal Ganglion Cells

    PubMed Central

    Paul, David L.; Wang, Jack T.; Huberman, Andrew D.; Bloomfield, Stewart A.

    2013-01-01

    Neurons throughout the brain show spike activity that is temporally correlated to that expressed by their neighbors, yet the generating mechanism(s) remains unclear. In the retina, ganglion cells (GCs) show robust, concerted spiking that shapes the information transmitted to central targets. Here we report the synaptic circuits responsible for generating the different types of concerted spiking of GC neighbors in the mouse retina. The most precise concerted spiking was generated by reciprocal electrical coupling of GC neighbors via gap junctions, whereas indirect electrical coupling to a common cohort of amacrine cells generated the correlated activity with medium precision. In contrast, the correlated spiking with the lowest temporal precision was produced by shared synaptic inputs carrying photoreceptor noise. Overall, our results demonstrate that different synaptic circuits generate the discrete types of GC correlated activity. Moreover, our findings expand our understanding of the roles of gap junctions in the retina, showing that they are essential for generating all forms of concerted GC activity transmitted to central brain targets. PMID:23936012

  20. Differential distribution of glycine transporters in Müller cells and neurons in amphibian retinas.

    PubMed

    Jiang, Zheng; Li, Baoqin; Jursky, Frantisek; Shen, Wen

    2007-01-01

    Amphibian retinas are commonly used for electrophysiological studies on neural function and transduction because they share the same general properties as higher vertebrate retinas. Glycinergic synapses have been well described in amphibian retinas. However, the role of glycine transporters in the synapses is largely unknown. We studied the distribution and function of glycine transporters in the retinas from tiger salamanders, mudpuppies, and leopard frogs by immunofluorescence labeling and whole-cell recording methods. Our results indicated that GlyT1- and GlyT2-like transporters were present in Müller cells and neurons, respectively. GlyT1 labeling was present in Müller glial cells and co-localized with Glial fibrillary acidic protein (GFAP), a Müller cell marker, whereas the GlyT2 immunoreactivity was present in the somas of amacrine cells (ACs) and processes in the inner plexiform layer (IPL) and the outer plexiform layer (OPL). Because the axon processes of glycinergic interplexiform cells (IPCs) are the only source of glycine input in the OPL, GlyT2 staining revealed a spatial pattern of the axon processes of IPCs in the OPL. The function of GlyT2 in the IPCs was studied in tiger salamander retinal horizontal cells (HCs) by whole-cell gramicidin perforated recording. The results demonstrated that inhibition of GlyT2 by a specific inhibitor, amoxapine, increased a tonic glycine input to HCs. Thus, the GlyT2 transporter is responsible for uptake of synaptic glycine in the outer retina. We also compared the distribution of glycine transporters in other amphibian species: salamander, mudpuppy, and frog. The results are consistent with the general pattern that GlyT1-like transporters are present in Müller cells and GlyT2-like transporters in neurons in amphibian retinas.

  1. Intricate paths of cells and networks becoming "Cholinergic" in the embryonic chicken retina.

    PubMed

    Thangaraj, Gopenath; Greif, Alexander; Bachmann, Gesine; Layer, Paul G

    2012-10-01

    Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are the decisive enzymatic activities regulating the availability of acetylcholine (ACh) at a given synaptic or nonsynaptic locus. The only cholinergic cells of the mature inner retina are the so-called starburst amacrine cells (SACs). A type-I SAC, found at the outer border of the inner plexiform layer (IPL), forms a synaptic subband "a" within the IPL, while a type-II SAC located at the inner IPL border projects into subband "d." Applying immunohistochemistry for ChAT and AChE on sections of the chicken retina, we here have revealed intricate relationships of how retinal networks became dominated by AChE or by ChAT reactivities. AChE+ cells were first detectable in an embryonic day (E)4 retina, while ChAT appeared 1 day later in the very same cells; at this stage all are Brn3a+, a marker for ganglion cells (GCs). On either side of a faint AChE+ band, indicating the future IPL, pairs of ChAT+ /AChE- /Brn3a- cells appeared between E7/8. Type-I cells had increased ChAT and lost AChE; type-II cells presented less ChAT, but some AChE on their surfaces. Direct neighbors of SACs tended to express much AChE. Along with maturation, subband "a" presented more ChAT but less AChE; in subband "d" this pattern was reversed. In conclusion, the two retinal cholinergic networks segregate out from one cell pool, become locally opposed to each other, and become dominated by either synthesis or degradation of ACh. These "cholinergic developmental divergences" may also have significant physiologic consequences.

  2. The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina

    PubMed Central

    Rodriguez, Allen R.; de Sevilla Müller, Luis Pérez; Brecha, Nicholas C.

    2014-01-01

    There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity purified guinea pig and rabbit antibodies against RNA-binding protein with multiple splicing (RBPMS). On Western blots these antibodies recognize a single band at ~24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit and monkey retina. RBPMS immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semi-quantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI-, DRAQ5-, NeuroTrace- and NeuN-stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC-1) immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at three weeks, and all Brn3a, SMI-32 and melanopsin immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to CFP-fluorescent RGCs in the B6.Cg-Tg(Thy1-CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs. PMID:24318667

  3. Generation of a KOR-Cre Knockin Mouse Strain to Study Cells Involved in Kappa Opioid Signaling

    PubMed Central

    Kuzirian, Marissa S.; Snyder, Lindsey M.; Matsushita, Megumi; Lee, Michael C.; Ferguson, Carolyn; Homanics, Gregg E.; Barth, Alison L.; Ross, Sarah E.

    2015-01-01

    The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non-neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR-Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR-Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR-Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR-Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR-Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR. PMID:26575788

  4. Angiotensin II receptors in the gonads

    SciTech Connect

    Aguilera, G.; Millan, M.A.; Harwood, J.P.

    1989-05-01

    The presence of components of the renin-angiotensin system in ovaries and testes suggests that angiotensin II (AII) is involved in gonadal function, and thus we sought to characterize receptors for AII in rat and primate gonads. In the testes, autoradiographic studies showed receptors in the interstitium in all species. In rat interstitial cells fractionated by Percoll gradient, AII receptors coincided with hCG receptors indicating that AII receptors are located on the Leydig cells. In Leydig cells and membranes from rat and rhesus monkey prepuberal testes, AII receptors were specific for AII analogues and of high affinity (Kd=nM). During development, AII receptor content in rat testes decreases with age parallel to a fall in the ratio of interstitial to tubular tissue. In the ovary, the distribution of AII receptors was dependent on the stage of development, being high in the germinal epithelium and stromal tissue between five and 15 days, and becoming localized in secondary follicles in 20-and 40-day-old rats. No binding was found in primordial or primary follicles. In rhesus monkey ovary, AII receptors were higher in stromal tissue and lower in granulosa and luteal cells of the follicles. Characterization of the binding in rat and monkey ovarian membranes showed a single class of sites with a Kd in the nmol/L range and specificity similar to that of the adrenal glomerulosa and testicular AII receptors. Receptors for AII were also present in membrane fractions from PMSG/hCG primed rat ovaries. Infusion of AII (25 ng/min) or captopril (1.4 micrograms/min) during the PMSG/hCG induction period had no effect on ovarian weight or AII receptor concentration in the ovaries.

  5. ECIL guidelines for the diagnosis of Pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients.

    PubMed

    Alanio, Alexandre; Hauser, Philippe M; Lagrou, Katrien; Melchers, Willem J G; Helweg-Larsen, Jannik; Matos, Olga; Cesaro, Simone; Maschmeyer, Georg; Einsele, Hermann; Donnelly, J Peter; Cordonnier, Catherine; Maertens, Johan; Bretagne, Stéphane

    2016-09-01

    The Fifth European Conference on Infections in Leukaemia (ECIL-5) convened a meeting to establish evidence-based recommendations for using tests to diagnose Pneumocystis jirovecii pneumonia (PCP) in adult patients with haematological malignancies. Immunofluorescence assays are recommended as the most sensitive microscopic method (recommendation A-II: ). Real-time PCR is recommended for the routine diagnosis of PCP ( A-II: ). Bronchoalveolar lavage (BAL) fluid is recommended as the best specimen as it yields good negative predictive value ( A-II: ). Non-invasive specimens can be suitable alternatives ( B-II: ), acknowledging that PCP cannot be ruled out in case of a negative PCR result ( A-II: ). Detecting β-d-glucan in serum can contribute to the diagnosis but not the follow-up of PCP ( A-II: ). A negative serum β-d-glucan result can exclude PCP in a patient at risk ( A-II: ), whereas a positive test result may indicate other fungal infections. Genotyping using multilocus sequence markers can be used to investigate suspected outbreaks ( A-II: ). The routine detection of dihydropteroate synthase mutations in cases of treatment failure is not recommended ( B-II: ) since these mutations do not affect response to high-dose co-trimoxazole. The clinical utility of these diagnostic tests for the early management of PCP should be further assessed in prospective, randomized interventional studies.

  6. Lazy eyes zebrafish mutation affects Müller glial cells, compromising photoreceptor function and causing partial blindness.

    PubMed

    Kainz, Pamela M; Adolph, Alan R; Wong, Kwoon Y; Dowling, John E

    2003-08-25

    A behavioral assay based on the optokinetic reflex was used to screen chemically mutagenized zebrafish larvae for deficits in visual function. A homozygous recessive mutation, lazy eyes (lze), was isolated based on the observation that 5-day postfertilization (dpf) mutants displayed weaker and less frequent eye movements than wild-type fish in response to moving stripes. Electroretinographic (ERG) recordings revealed that mutants had severely reduced a- and b-wave amplitudes relative to wild-type fish, indicating outer retinal dysfunction. Retinal lamination and cellular differentiation were normal in the lze retina; however, mutant photoreceptor cells had small outer segments and pyknotic nuclei were occasionally observed in the outer retina and the marginal zone of lze. Cone, rod, amacrine, bipolar, and Müller cell marker analyses indicated that the typical lze retina contained fewer rod photoreceptors and fewer Müller cells than wild-type fish at 5 dpf. At 3 dpf, however, mutant retinas had normal numbers of rod photoreceptors and Müller cells, suggesting that the initial differentiation of these cell types occurred normally. Rod photoreceptor histology was normal at this early stage, but Müller cells were often hypertrophied, suggesting that they were unhealthy. Constant light rearing of mutant animals accelerated the Müller cell degeneration, severely worsened the visual deficit, but had no obvious affect on the photoreceptors. When ERG responses and Müller cell degeneration from the same mutant animals were analyzed, the extent of the Müller cell loss matched closely the degree to which ERG responses were reduced. In summary, the lze gene appears to be required for Müller cell viability and normal visual function. The lze mutant may be a model for the study of the involvement of Müller cells in photoreceptor development and function. Copyright 2003 Wiley-Liss, Inc.

  7. The directed differentiation of human iPS cells into kidney podocytes.

    PubMed

    Song, Bi; Smink, Alexandra M; Jones, Christina V; Callaghan, Judy M; Firth, Stephen D; Bernard, Claude A; Laslett, Andrew L; Kerr, Peter G; Ricardo, Sharon D

    2012-01-01

    The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.

  8. PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress.

    PubMed

    Salabei, Joshua K; Cummins, Timothy D; Singh, Mahavir; Jones, Steven P; Bhatnagar, Aruni; Hill, Bradford G

    2013-05-01

    Vascular injury and chronic arterial diseases result in exposure of VSMCs (vascular smooth muscle cells) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation, but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with PDGF (platelet-derived growth factor)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and up-regulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3 (microtubule-associated protein light chain 3 α; also known as MAP1LC3A)-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1 or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 stabilized α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix, and abrogated VSMC hyperproliferation and migration. Treatment of cells with PDGF prevented protein damage and cell death caused by exposure to the lipid peroxidation product 4-hydroxynonenal. The results of the present study demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under the conditions of high oxidative stress found to occur in vascular lesions.

  9. The Directed Differentiation of Human iPS Cells into Kidney Podocytes

    PubMed Central

    Song, Bi; Smink, Alexandra M.; Jones, Christina V.; Callaghan, Judy M.; Firth, Stephen D.; Bernard, Claude A.; Laslett, Andrew L.; Kerr, Peter G.; Ricardo, Sharon D.

    2012-01-01

    The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm’s tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine. PMID:23029522

  10. Wnt Regulates Proliferation and Neurogenic Potential of Müller Glial Cells via a Lin28/let-7 miRNA-Dependent Pathway in Adult Mammalian Retinas.

    PubMed

    Yao, Kai; Qiu, Suo; Tian, Lin; Snider, William D; Flannery, John G; Schaffer, David V; Chen, Bo

    2016-09-27

    In cold-blooded vertebrates such as zebrafish, Müller glial cells (MGs) readily proliferate to replenish lost retinal neurons. In mammals, however, MGs lack regenerative capability as they do not spontaneously re-enter the cell cycle unless the retina is injured. Here, we show that gene transfer of β-catenin in adult mouse retinas activates Wnt signaling and MG proliferation without retinal injury. Upstream of Wnt, deletion of GSK3β stabilizes β-catenin and activates MG proliferation. Downstream of Wnt, β-catenin binds to the Lin28 promoter and activates transcription. Deletion of Lin28 abolishes β-catenin-mediated effects on MG proliferation, and Lin28 gene transfer stimulates MG proliferation. We further demonstrate that let-7 miRNAs are critically involved in Wnt/Lin28-regulated MG proliferation. Intriguingly, a subset of cell-cycle-reactivated MGs express markers for amacrine cells. Together, these results reveal a key role of Wnt-Lin28-let7 miRNA signaling in regulating proliferation and neurogenic potential of MGs in the adult mammalian retina. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins

    PubMed Central

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Jeon, Mili; Birnbaum, Michael E.; Bellen, Hugo J.; Garcia, K. Christopher; Zinn, Kai

    2015-01-01

    Summary We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a 9-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the 6 dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity. PMID:26687361

  12. Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins.

    PubMed

    Carrillo, Robert A; Özkan, Engin; Menon, Kaushiki P; Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Jeon, Mili; Birnbaum, Michael E; Bellen, Hugo J; Garcia, K Christopher; Zinn, Kai

    2015-12-17

    We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

  13. Muller glia, vision-guided ocular growth, retinal stem cells, and a little serendipity: the Cogan lecture.

    PubMed

    Fischer, Andy J

    2011-09-29

    Hypothesis-driven science is expected to result in a continuum of studies and findings along a discrete path. By comparison, serendipity can lead to new directions that branch into different paths. Herein, I describe a diverse series of findings that were motivated by hypotheses, but driven by serendipity. I summarize how investigations into vision-guided ocular growth in the chick eye led to the identification of glucagonergic amacrine cells as key regulators of ocular elongation. Studies designed to assess the impact of the ablation of different types of neurons on vision-guided ocular growth led to the finding of numerous proliferating cells within damaged retinas. These proliferating cells were Müller glia-derived retinal progenitors with a capacity to produce new neurons. Studies designed to investigate Müller glia-derived progenitors led to the identification of a domain of neural stem cells that form a circumferential marginal zone (CMZ) that lines the periphery of the retina. Accelerated ocular growth, caused by visual deprivation, stimulated the proliferation of CMZ progenitors. We formulated a hypothesis that growth-regulating glucagonergic cells may regulate both overall eye size (scleral growth) and the growth of the retina (proliferation of CMZ cells). Subsequent studies identified unusual types of glucagonergic neurons with terminals that ramify within the CMZ; these cells use visual cues to control equatorial ocular growth and the proliferation of CMZ cells. Finally, while studying the signaling pathways that stimulate CMZ and Müller glia-derived progenitors, serendipity led to the discovery of a novel type of glial cell that is scattered across the inner retinal layers.

  14. Inhibition of Müller Glial Cell Division Blocks Regeneration of the Light-Damaged Zebrafish Retina

    PubMed Central

    Thummel, Ryan; Kassen, Sean C.; Montgomery, Jacob E.; Enright, Jennifer M.; Hyde, David R.

    2013-01-01

    The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas. PMID:18161852

  15. Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

    PubMed Central

    Singh, Ratnesh K.; Mallela, Ramya K.; Cornuet, Pamela K.; Reifler, Aaron N.; Chervenak, Andrew P.; West, Michael D.; Wong, Kwoon Y.; Nasonkin, Igor O.

    2015-01-01

    Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for

  16. ON Bipolar Cells in Macaque Retina: Type-Specific Synaptic Connectivity with Special Reference to OFF Counterparts

    PubMed Central

    Tsukamoto, Yoshihiko; Omi, Naoko

    2016-01-01

    To date, 12 macaque bipolar cell types have been described. This list includes all morphology types first outlined by Polyak (1941) using the Golgi method in the primate retina and subsequently identified by other researchers using electron microscopy (EM) combined with the Golgi method, serial section transmission EM (SSTEM), and immunohistochemical imaging. We used SSTEM for the rod-dense perifoveal area of macaque retina, reconfirmed ON (cone) bipolar cells to be classified as invaginating midget bipolar (IMB), diffuse bipolar (DB)4, DB5, DB6, giant bipolar (GB), and blue bipolar (BB) types, and clarified their type-specific connectivity. DB4 cells made reciprocal synapses with a kind of ON-OFF lateral amacrine cell, similar to OFF DB2 cells. GB cells contacted rods and cones, similar to OFF DB3b cells. Retinal circuits formed by GB and DB3b cells are thought to substantiate the psychophysical finding of fast rod signals in mesopic vision. DB6 cell output synapses were directed to ON midget ganglion (MG) cells at 70% of ribbon contacts, similar to OFF DB1 cells that directed 60% of ribbon contacts to OFF MG cells. IMB cells contacted medium- or long-wavelength sensitive (M/L-) cones but not short-wavelength sensitive (S-) cones, while BB cells contacted S-cones but not M/L-cones. However, IMB and BB dendrites had similar morphological architectures, and a BB cell contacting a single S-cone resembled an IMB cell. Thus, both IMB and BB may be the ON bipolar counterparts of the OFF flat midget bipolar (FMB) type, likewise DB4 of DB2, DB5 of DB3a, DB6 of DB1, and GB of DB3b OFF bipolar type. The ON DB plus GB, and OFF DB cells predominantly contacted M/L-cones and their outputs were directed mainly to parasol ganglion (PG) cells but also moderately to MG cells. BB cells directed S-cone-driven outputs almost exclusively to small bistratified ganglion (SBG) cells. Some FMB cells predominantly contacted S-cones and their outputs were directed to OFF MG cells. Thus, two

  17. The effect of GABA and the GABA-uptake-blocker NO-711 on the b-wave of the ERG and the responses of horizontal cells to light.

    PubMed

    Hanitzsch, Renate; Küppers, Lea; Flade, Andreas

    2004-09-01

    The effects of GABA in the retina have now become of special interest because the anti-epileptic drug vigabatrin, a GABA analogue, can cause visual field loss in humans. Vigabatrin inhibits the GABA-aminotransferase, which finally results in GABA accumulation in the extracellular space. The b-wave of the electroretinogram (ERG), which originates partly in on-bipolar cells, is influenced by both GABAergic horizontal cells (HCs) and GABAergic amacrine cells (ACs). Their influences, however, are difficult to separate. In an attempt to isolate the effect of GABAergic ACs, use has been made of the specific effect of the GABA-uptake-blocker NO-711, which blocks only the GABA transporter GAT1 of GABAergic ACs. The ERG and the intracellular responses of HCs to light were recorded in the isolated rabbit retina, and the effects of GABA and NO-711, when added separately to the superfusate, were determined. GABA reduced significantly both the light responses of HCs and the b-wave. NO-711 enlarged the b-wave drastically, but did not affect the responses of HCs to light. An increase in the extracellular GABA concentration decreases the b-wave; an impairment of the function of ACs increases the b-wave. These conditions are discussed in the context of the lack of consistent changes to the b-wave during therapy with vigabatrin.

  18. Arginase: A Novel Proliferative Determinant in Prostate Cancer

    DTIC Science & Technology

    2006-04-01

    Figure 6. Tissue microarray spots stained with AII; (A) normal prostate with low levels of AII staining, (B) malignant prostate with high AII...laboratory, we unexpectedly discovered that is expressed at high levels in the normal prostate and even higher in neoplastic prostate samples. The...pivotal role in the synthesis of polyamines, chemicals involved in cell growth and regulation that are found in high levels in normal prostate tissue

  19. Opposing motion inhibits responses of direction-selective ganglion cells in the fish retina.

    PubMed

    Damjanović, Ilija; Maximova, Elena; Aliper, Alexey; Maximov, Paul; Maximov, Vadim

    2015-03-01

    Inhibitory influences in receptive fields (RFs) of the fish retinal direction-selective ganglion cells (DS GCs) were investigated. Responses of the fast retinal DS GCs were recorded extracellularly from their axon terminals in the superficial layer of tectum opticum of immobilized fish. The data were collected from two cyprinid species - Carassius gibelio, a wild form of the goldfish, and the barbel fish Labeobarbus intermedius. Visual stimuli were presented to the fish on the monitor screen within a square area of stimulation occupying approximately 11 × 11° of the visual field. DS GCs were stimulated by pairs of narrow stripes moving in opposing directions. One of them entered central (responsive) area of cell receptive field (RRF) from the preferred, and the other one from the null side. Stimuli merged at center of stimulation area, and subsequently moved away from each other. It was shown that the cell response evoked by the stripe coming from the preferred side of RF was inhibited by the stimulus coming from the opposite direction. In the majority of units recorded inhibitory effect induced by the null-side stimulus was initiated in the RF periphery. As a rule, inhibitory influences sent from the RF periphery were spread across the entire central area of RF. Modifications of the inhibitory influences were investigated throughout the whole motion of paired stimuli. Evident inhibitory effects mediated from the null direction were recorded during the approach of stimuli. When stripes crossed each other and moved apart inhibition was terminated, and cell response appeared again. Null-side inhibition observed in fish DS GCs is most likely induced by starburst-like amacrine cells described in morphological studies of different fish species. Possible mechanisms underlying direction selectivity in fish DS GCs are discussed.

  20. Properties of the ON bistratified ganglion cell in the rabbit retina.

    PubMed

    Hoshi, Hideo; Tian, Lian-Ming; Massey, Stephen C; Mills, Stephen L

    2013-05-01

    The identity of the types of different neurons in mammalian retinae is now close to being completely known for a few mammalian species; comparison reveals strong homologies for many neurons across the order. Still, there remain some cell types rarely encountered and inadequately described, despite not being rare in relative frequency. Here we describe in detail an additional ganglion cell type in rabbit that is bistratified with dendrites in both sublaminae, yet spikes only at light onset and has no response bias to the direction of moving bars. This ON bistratified ganglion cell type is most easily distinguished by the unusual behavior of its dendritic arbors. While dendrites that arborize in sublamina b terminate at that level, those that ascend to arborize in sublamina a do not normally terminate there. Instead, when they reach the approximate radius of the dendrites in sublamina b, they dive sharply back down to ramify in sublamina b. Here they continue to course even further away from the soma at the same level as the branches wholly contained in sublamina b, thereby forming an annulus of secondary ON dendrites in sublamina b. This pattern of branching creates a bistratified dendritic field of approximately equal area in the two sublaminae initially, to which is then added an external annulus of dendrites only in sublamina b whose origin is entirely from processes descending from sublamina a. It is coupled to a population of wide-field amacrine cells upon which the dendrites of the ganglion cell often terminate. Copyright © 2012 Wiley Periodicals, Inc.

  1. Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors

    PubMed Central

    2013-01-01

    Background Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. Results We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. Conclusions We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells. PMID:23870169

  2. Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

    PubMed

    Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

    2013-07-19

    Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

  3. A mammalian retinal bipolar cell uses both graded changes in membrane voltage and all-or-nothing Na+ spikes to encode light.

    PubMed

    Saszik, Shannon; DeVries, Steven H

    2012-01-04

    Barlow (1953) studied summation in ganglion cell receptive fields and observed a fine discrimination of spatial information from which he inferred that retinal interneurons use analog signals to process images. Subsequent intracellular recordings confirmed that the interneurons of the outer retina, including photoreceptors, horizontal cells, and bipolar cells, respond to light with slow, graded changes in membrane potential. Analog processing may enable interneurons to discriminate fine gradations in light intensity and spatiotemporal pattern, but at the expense of the speed, temporal precision, and threshold discrimination that are characteristic of all-or-nothing Na(+) spikes. We show that one type of mammalian On bipolar cell, the ground squirrel cb5b, has a large tetrodotoxin (TTX)-sensitive Na(+) current. When recorded from in the perforated patch configuration, cb5b cells can signal the onset of a light step with 1-3 all-or-nothing action potentials that attain a peak amplitude of -10 to -20 mV (peak width at half-height equals 2-3 ms). When exposed to a continuous, temporally fluctuating stimulus, cb5b cells generate both graded and spiking responses. Cb5b cells spike with millisecond precision, selecting for stimulus sequences in which transitions to light are preceded by a period of darkness. The axon terminals of cb5b bipolar cells costratify with the dendrites of amacrine and ganglion cells that encode light onset with a short latency burst of spikes. The results support the idea that a spiking On bipolar cell is part of a dedicated retinal pathway for rapidly and reliably signaling dark to light transitions.

  4. Expression of Nicotinic Acetylcholine Receptor α4 and β2 Subunits on Direction-Selective Retinal Ganglion Cells in the Rabbit

    PubMed Central

    Lee, Jun-Seok; Kim, Hyun-Jin; Ahn, Chang-Hyun; Jeon, Chang-Jin

    2017-01-01

    The direction selectivity of the retina is a distinct mechanism that is critical function of eyes for survival. The direction-selective retinal ganglion cells (DS RGCs) strongly respond to a preferred direction, but rarely respond to opposite direction or null directional visual stimuli. The DS RGCs are sensitive to acetylcholine, which is secreted from starburst amacrine cells (SACs) to the DS RGCs. Here, we investigated the existence and distribution of the nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits on the dendritic arbors of the DS RGCs in adult rabbit retina using immunocytochemistry. The DS RGCs were injected with Lucifer yellow to identify their dendritic morphology. The double-labeled images of dendrites and nAChR subunits were visualized for reconstruction using high-resolution confocal microscopy. Although our results revealed that the distributional pattern of the nAChR subunits on the dendritic arbors of the DS RGCs was not asymmetric in the adult rabbit retina, the distribution of nAChR α4 and β2 subunits and molecular profiles of cholinergic inputs to DS RGCs in adult rabbit retina provide anatomical evidence for direction selectivity. PMID:28386148

  5. Physiological and morphological characterization of ganglion cells in the salamander retina

    PubMed Central

    Wang, Jing; Jacoby, Roy; Wu, Samuel M.

    2016-01-01

    Retinal ganglion cells (RGCs) integrate visual information from the retina and transmit collective signals to the brain. A systematic investigation of functional and morphological characteristics of various types of RGCs is important to comprehensively understand how the visual system encodes and transmits information via various RGC pathways. This study evaluated both physiological and morphological properties of 67 RGCs in dark-adapted flat-mounted salamander retina by examining light-evoked cation and chloride current responses via voltage-clamp recordings and visualizing morphology by Lucifer yellow fluorescence with a confocal microscope. Six groups of RGCs were described: asymmetrical ON–OFF RGCs, symmetrical ON RGCs, OFF RGCs, and narrow-, medium- and wide-field ON–OFF RGCs. Dendritic field diameters of RGCs ranged 102–490 µm: narrow field (<200 µm, 31% of RGCs), medium field (200–300 µm, 45%) and wide field (>300 µm, 24%). Dendritic ramification patterns of RGCs agree with the sub-lamina A/B rule. 34% of RGCs were monostratified, 24% bistratified and 42% diffusely stratified. 70% of ON RGCs and OFF RGCs were monostratified. Wide-field RGCs were diffusely stratified. 82% of RGCs generated light-evoked ON–OFF responses, while 11% generated ON responses and 7% OFF responses. Response sensitivity analysis suggested that some RGCs obtained separated rod/cone bipolar cell inputs whereas others obtained mixed bipolar cell inputs. 25% of neurons in the RGC layer were displaced amacrine cells. Although more types may be defined by more refined classification criteria, this report is to incorporate more physiological properties into RGC classification. PMID:26731645

  6. Physiological and morphological characterization of ganglion cells in the salamander retina.

    PubMed

    Wang, Jing; Jacoby, Roy; Wu, Samuel M

    2016-02-01

    Retinal ganglion cells (RGCs) integrate visual information from the retina and transmit collective signals to the brain. A systematic investigation of functional and morphological characteristics of various types of RGCs is important to comprehensively understand how the visual system encodes and transmits information via various RGC pathways. This study evaluated both physiological and morphological properties of 67 RGCs in dark-adapted flat-mounted salamander retina by examining light-evoked cation and chloride current responses via voltage-clamp recordings and visualizing morphology by Lucifer yellow fluorescence with a confocal microscope. Six groups of RGCs were described: asymmetrical ON-OFF RGCs, symmetrical ON RGCs, OFF RGCs, and narrow-, medium- and wide-field ON-OFF RGCs. Dendritic field diameters of RGCs ranged 102-490 μm: narrow field (<200 μm, 31% of RGCs), medium field (200-300 μm, 45%) and wide field (>300 μm, 24%). Dendritic ramification patterns of RGCs agree with the sublamina A/B rule. 34% of RGCs were monostratified, 24% bistratified and 42% diffusely stratified. 70% of ON RGCs and OFF RGCs were monostratified. Wide-field RGCs were diffusely stratified. 82% of RGCs generated light-evoked ON-OFF responses, while 11% generated ON responses and 7% OFF responses. Response sensitivity analysis suggested that some RGCs obtained separated rod/cone bipolar cell inputs whereas others obtained mixed bipolar cell inputs. 25% of neurons in the RGC layer were displaced amacrine cells. Although more types may be defined by more refined classification criteria, this report is to incorporate more physiological properties into RGC classification.

  7. Gene expression and protein distribution of orexins and orexin receptors in rat retina.

    PubMed

    Liu, F; Xu, G Z; Wang, L; Jiang, S X; Yang, X L; Zhong, Y M

    2011-08-25

    Orexins, composed of orexin A and orexin B, are identified as endogenous ligands of two orphan G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). Orexins are implicated in regulating wake/sleep states, feeding behaviors, etc. Using reverse transcription-polymerase chain reactive (RT-PCR) analysis and immunofluorescence double labeling, we investigated the distributions of orexin A, orexin B, OX1R and OX2R in rat retina. RT-PCR analysis revealed the presence of mRNAs of prepro-orexin, OX1R and OX2R in rat retina. Immunostaining for orexin A and orexin B was observed in many cells in the inner nuclear layer and the ganglion cell layer. In the outer retina, horizontal cells, labeled by calbindin, and bipolar cells, labeled by homeobox protein Chx10, were orexin A- and orexin B-positive. In the inner retina, two orexins were both found in GABAergic amacrine cells (ACs), including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase and choline acetyltransferase respectively. Glycinergic ACs, including AII ACs, also expressed orexins. Weak to moderate labeling for orexin A and orexin B was diffusely distributed in the inner plexiform layer. Additionally, orexins were expressed in almost all ganglion cells (GCs) retrogradely labeled by cholera toxin B subunit. Specifically, double-labeling experiments demonstrated that melanopsin-positive GCs (intrinsically photosensitive retinal GCs, ipRGCs) were labeled by two orexins. Morever, OX1R immunoreactivity was observed in most of GCs and all dopaminergic ACs, as well as in both outer and inner plexiform layers. In contrast, no obvious OX2R immunostaining was detectable in the rat retina. These results suggest that orexins may modulate the function of neurons, especially in the inner retina. We further hypothesize that the orexin signaling via ipRGCs may be involved in setting the suprachiasmatic nucleus (SCN) circadian clock.

  8. Screening of gap junction antagonists on dye coupling in the rabbit retina

    PubMed Central

    PAN, FENG; MILLS, STEPHEN L.; MASSEY, STEPHEN C.

    2008-01-01

    Many cell types in the retina are coupled via gap junctions and so there is a pressing need for a potent and reversible gap junction antagonist. We screened a series of potential gap junction antagonists by evaluating their effects on dye coupling in the network of A-type horizontal cells. We evaluated the following compounds: meclofenamic acid (MFA), mefloquine, 2-aminoethyldiphenyl borate (2-APB), 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid (18-β-GA), retinoic acid, flufenamic acid, niflumic acid, and carbenoxolone. The efficacy of each drug was determined by measuring the diffusion coefficient for Neurobiotin (Mills & Massey, 1998). MFA, 18-β-GA, 2-APB and mefloquine were the most effective antagonists, completely eliminating A-type horizontal cell coupling at a concentration of 200 μM. Niflumic acid, flufenamic acid, and carbenoxolone were less potent. Additionally, carbenoxolone was difficult to wash out and also may be harmful, as the retina became opaque and swollen. MFA, 18-β-GA, 2-APB and mefloquine also blocked coupling in B-type horizontal cells and AII amacrine cells. Because these cell types express different connexins, this suggests that the antagonists were relatively non-selective across several different types of gap junction. It should be emphasized that MFA was water-soluble and its effects on dye coupling were easily reversible. In contrast, the other gap junction antagonists, except carbenoxolone, required DMSO to make stock solutions and were difficult to wash out of the preparation at the doses required to block coupling in A-type HCs. The combination of potency, water solubility and reversibility suggest that MFA may be a useful compound to manipulate gap junction coupling. PMID:17711600

  9. A mammalian retinal bipolar cell uses both graded changes in membrane voltage and all-or-nothing Na+ spikes to encode light

    PubMed Central

    Saszik, Shannon; DeVries, Steven H.

    2012-01-01

    Barlow studied summation in ganglion cell receptive fields and observed a fine discrimination of spatial information from which he inferred that retinal interneurons use analog signals to process images. Subsequent intracellular recordings confirmed that the interneurons of the outer retina, including photoreceptors, horizontal cells, and bipolar cells, respond to light with slow, graded changes in membrane potential. Analog processing may enable interneurons to discriminate fine gradations in light intensity and spatiotemporal pattern, but at the expense of the speed, temporal precision, and threshold discrimination that are characteristic of all-or-nothing Na+ spikes. We show that one type of mammalian On bipolar cell, the ground squirrel cb5b, has a large tetrodotoxin (TTX)-sensitive Na+ current. When recorded from in the perforated patch configuration, cb5b cells can signal the onset of a light step with 1–3 all-or-nothing action potentials that attain a peak amplitude of −10 to −20 mV (peak width at half-height equals 2 – 3 ms). When exposed to a continuous, temporally fluctuating stimulus, cb5b generate both graded and spiking responses. cb5b cells spike with millisecond precision, selecting for stimulus sequences in which transitions to light are preceded by a period of darkness. The axon terminals of cb5b bipolar cells co-stratify with the dendrites of amacrine and ganglion cells that encode light onset with a short latency burst of spikes. The results support the idea that a spiking On bipolar cell is part of a dedicated retinal pathway for rapidly and reliably signaling dark to light transitions. PMID:22219291

  10. Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells.

    PubMed

    Portugal, Camila Cabral; Miya, Vivian Sayuri; Calaza, Karin da Costa; Santos, Rochelle Alberto Martins; Paes-de-Carvalho, Roberto

    2009-01-01

    Vitamin C is transported in the brain by sodium vitamin C co-transporter 2 (SVCT-2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT-2 and the uptake and release of [(14)C] ascorbate in chick retinal cells. SVCT-2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT-2 was expressed in cultured retinal neurons, but not in glial cells. [(14)C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium-free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate-stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l-beta-threo-benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [(3)H] D-aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2-Carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7-initroquinoxaline-2,3-dione (DNQX) or (5R,2S)-(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). However, DNQX, but not MK-801 or 2-amino-5-phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non-NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2-bis (2-aminophenoxy) ethane-N',N',N',N',-tetraacetic acid tetrakis (acetoxy-methyl ester) (BAPTA-AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT-2, and the release can be stimulated by NMDA or non-NMDA glutamate receptors.

  11. High-density lipoprotein 3 physicochemical modifications induced by interaction with human polymorphonuclear leucocytes affect their ability to remove cholesterol from cells.

    PubMed Central

    Cogny, A; Atger, V; Paul, J L; Soni, T; Moatti, N

    1996-01-01

    1. We have recently reported that a short incubation (60 min) in vitro of high-density lipoprotein (HDL) 3 with human polymorphonuclear leucocytes (PMNs) leads to a proteolytic cleavage of apolipoprotein (apo) AII and to a change in the distribution of apo AI isoforms [Cogny, Paul, Atger, Soni and Moatti (1994) Eur. J. Biochem. 222, 965-973]. Since PMNs have been observed to be present in the earliest atherosclerotic lesions for a number of days, we investigated the HDL3 physiochemical modifications induced by in vitro interaction for a long period of time (24 h) with PMNs and the consequences of the changes on the ability of HDL3 to remove cholesterol from cells. 2. The stimulated PMN modification of HDL3 over 24 h resulted in a partial loss of protein with no variation in lipid molar ratio and a loss of 50% of HDL alpha-tocopherol content. The decrease in total protein was due first to a complete degradation of apo AII, and secondly to a partial loss of apo AI. The apo AI remaining on the particles was in part hydrolysed and the apo AI-1 isoform was completely shifted to the apo AI-2 isoform. These apo changes were accompanied by a displacement of the native HDL3 apparent size toward predominantly larger particles. 3. The ability of PMN-modified HDL3 to remove 3H-labelled free cholesterol from cells was measured in two cell lines: Fu5AH rat hepatoma cells and J774 mouse macrophages. HDL3 which had only a limited contact with PMNs (60 min) showed only a small non-significant reduction in the efficiency of cholesterol efflux. On the other hand, compared with native HDL3, HDL3 modified by PMNs for 24 h had a markedly reduced ability to remove cholesterol from cells, regardless of the type of cell. 4. The results suggest that PMN-modified HDL3, if occurring in vivo, could contribute to acceleration of the atherogenic process by decreasing the cholesterol efflux from cells. PMID:8660296

  12. Elevated intraocular pressure decreases response sensitivity of inner retinal neurons in experimental glaucoma mice

    PubMed Central

    Pang, Ji-Jie; Frankfort, Benjamin J.; Gross, Ronald L.; Wu, Samuel M.

    2015-01-01

    Glaucoma is the second leading cause of blindness in the United States and the world, characterized by progressive degeneration of the optic nerve and retinal ganglion cells (RGCs). Glaucoma patients exhibit an early diffuse loss of retinal sensitivity followed by focal loss of RGCs in sectored patterns. Recent evidence has suggested that this early sensitivity loss may be associated with dysfunctions in the inner retina, but detailed cellular and synaptic mechanisms underlying such sensitivity changes are largely unknown. In this study, we use whole-cell voltage-clamp techniques to analyze light responses of individual bipolar cells (BCs), AII amacrine cells (AIIACs), and ON and sustained OFF alpha-ganglion cells (ONαGCs and sOFFαGCs) in dark-adapted mouse retinas with elevated intraocular pressure (IOP). We present evidence showing that elevated IOP suppresses the rod ON BC inputs to AIIACs, resulting in less sensitive AIIACs, which alter AIIAC inputs to ONαGCs via the AIIAC→cone ON BC→ONαGC pathway, resulting in lower ONαGC sensitivity. The altered AIIAC response also reduces sOFFαGC sensitivity via the AIIAC→sOFFαGC chemical synapses. These sensitivity decreases in αGCs and AIIACs were found in mice with elevated IOP for 3–7 wk, a stage when little RGC or optic nerve degeneration was observed. Our finding that elevated IOP alters neuronal function in the inner retina before irreversible structural damage occurs provides useful information for developing new diagnostic tools and treatments for glaucoma in human patients. PMID:25675503

  13. Grafted c-kit(+)/SSEA1(-) eye-wall progenitor cells delay retinal degeneration in mice by regulating neural plasticity and forming new graft-to-host synapses.

    PubMed

    Chen, Xi; Chen, Zehua; Li, Zhengya; Zhao, Chen; Zeng, Yuxiao; Zou, Ting; Fu, Caiyun; Liu, Xiaoli; Xu, Haiwei; Yin, Zheng Qin

    2016-12-30

    Despite diverse pathogenesis, the common pathological change observed in age-related macular degeneration and in most hereditary retinal degeneration (RD) diseases is photoreceptor loss. Photoreceptor replacement by cell transplantation may be a feasible treatment for RD. The major obstacles to clinical translation of stem cell-based cell therapy in RD remain the difficulty of obtaining sufficient quantities of appropriate and safe donor cells and the poor integration of grafted stem cell-derived photoreceptors into the remaining retinal circuitry. Eye-wall c-kit(+)/stage-specific embryonic antigen 1 (SSEA1)(-) cells were isolated via fluorescence-activated cell sorting, and their self-renewal and differentiation potential were detected by immunochemistry and flow cytometry in vitro. After labeling with quantum nanocrystal dots and transplantation into the subretinal space of rd1 RD mice, differentiation and synapse formation by daughter cells of the eye-wall c-kit(+)/SSEA1(-) cells were evaluated by immunochemistry and western blotting. Morphological changes of the inner retina of rd1 mice after cell transplantation were demonstrated by immunochemistry. Retinal function of rd1 mice that received cell grafts was tested via flash electroretinograms and the light/dark transition test. Eye-wall c-kit(+)/SSEA1(-) cells were self-renewing and clonogenic, and they retained their proliferative potential through more than 20 passages. Additionally, eye-wall c-kit(+)/SSEA1(-) cells were capable of differentiating into multiple retinal cell types including photoreceptors, bipolar cells, horizontal cells, amacrine cells, Müller cells, and retinal pigment epithelium cells and of transdifferentiating into smooth muscle cells and endothelial cells in vitro. The levels of synaptophysin and postsynaptic density-95 in the retinas of eye-wall c-kit(+)/SSEA1(-) cell-transplanted rd1 mice were significantly increased at 4 weeks post transplantation. The c-kit(+)/SSEA1(-) cells were

  14. Characterization of dsRed2-positive cells in the doublecortin-dsRed2 transgenic adult rat retina.

    PubMed

    Trost, A; Schroedl, F; Marschallinger, J; Rivera, F J; Bogner, B; Runge, C; Couillard-Despres, S; Aigner, L; Reitsamer, H A

    2014-12-01

    Doublecortin (DCX) is predominantly expressed in neuronal precursor cells and young immature neurons of the developing and adult brain, where it is involved in neuronal differentiation, migration and plasticity. Moreover, its expression pattern reflects neurogenesis, and transgenic DCX promoter-driven reporter models have been previously used to investigate adult neurogenesis. In this study, we characterize dsRed2 reporter protein-expressing cells in the adult retina of the transgenic DCX promoter-dsRed2 rat model, with the aim to identify cells with putative neurogenic activity. Additionally, we confirmed the expression of the dsRed2 protein in DCX-expressing cells in the adult hippocampal dentate gyrus. Adult DCX-dsRed2 rat retinas were analyzed by immunohistochemistry for expression of DCX, NF200, Brn3a, Sox2, NeuN, calbindin, calretinin, PKC-a, Otx2, ChAT, PSA-NCAM and the glial markers GFAP and CRALBP, followed by confocal laser-scanning microscopy. In addition, brain sections of transgenic rats were analyzed for dsRed2 expression and co-localization with DCX, NeuN, GFAP and Sox2 in the cortex and dentate gyrus. Endogenous DCX expression in the adult retina was confined to horizontal cells, and these cells co-expressed the DCX promoter-driven dsRed2 reporter protein. In addition, we encountered dsRed2 expression in various other cell types in the retina: retinal ganglion cells (RGCs), a subpopulation of amacrine cells, a minority of bipolar cells and in perivascular cells. Since also RGCs expressed dsRed2, the DCX-dsRed2 rat model might offer a useful tool to study RGCs in vivo under various conditions. Müller glial cells, which have previously been identified as cells with stem cell features and with neurogenic potential, did express neither endogenous DCX nor the dsRed2 reporter. However, and surprisingly, we identified a perivascular glial cell type expressing the dsRed2 reporter, enmeshed with the glia/stem cell marker GFAP and colocalizing with the

  15. Passive membrane properties and electrotonic signal processing in retinal rod bipolar cells.

    PubMed

    Oltedal, Leif; Veruki, Margaret Lin; Hartveit, Espen

    2009-02-15

    Rod bipolar cells transmit visual signals from their dendrites, where they receive input from rod photoreceptors, to their axon terminals, where they synapse onto amacrine cells. Little is known, however, about the transmission and possible transformation of these signals. We have combined axon terminal recording in retinal slices, quantitative, light-microscopic morphological reconstruction and computer modelling to obtain detailed compartmental models of rat rod bipolar cells. Passive cable properties were estimated by directly fitting the current responses of the models evoked by voltage pulses to the physiologically recorded responses. At a holding potential of -60 mV, the average best-fit parameters were 1.1 microF cm(-2) for specific membrane capacitance (C(m)), 130 Omega cm for cytoplasmic resistivity (R(i)), and 24 kOmega cm(2) for specific membrane resistance (R(m)). The passive integration of excitatory and inhibitory synaptic inputs was examined by computer modelling with physiologically realistic synaptic conductance waveforms. For both transient and steady-state synaptic inhibition, the inhibitory effect was relatively insensitive to the location of the inhibition. For transient synaptic inhibition, the time window of effective inhibition depended critically on the relative timing of inhibition and excitation. The passive signal transmission between soma and axon terminal was examined by the electrotonic transform and quantified as the frequency-dependent voltage attenuation of sinusoidal voltage waveforms. For the range of parameters explored (axon diameter and length, R(i)), the lowest cutoff frequency observed was approximately 300 Hz, suggesting that realistic scotopic visual signals will be faithfully transmitted from soma to axon terminal, with minimal passive attenuation along the axon.

  16. Angiotensin II receptor heterogeneity

    SciTech Connect

    Herblin, W.F.; Chiu, A.T.; McCall, D.E.; Ardecky, R.J.; Carini, D.J.; Duncia, J.V.; Pease, L.J.; Wong, P.C.; Wexler, R.R.; Johnson, A.L. )

    1991-04-01

    The possibility of receptor heterogeneity in the angiotensin II (AII) system has been suggested previously, based on differences in Kd values or sensitivity to thiol reagents. One of the authors earliest indications was the frequent observation of incomplete inhibition of the binding of AII to adrenal cortical membranes. Autoradiographic studies demonstrated that all of the labeling of the rat adrenal was blocked by unlabeled AII or saralasin, but not by DuP 753. The predominant receptor in the rat adrenal cortex (80%) is sensitive to dithiothreitol (DTT) and DuP 753, and is designated AII-1. The residual sites in the adrenal cortex and almost all of the sites in the rat adrenal medulla are insensitive to both DTT and DuP 753, but were blocked by EXP655. These sites have been confirmed by ligand binding studies and are designated AII-2. The rabbit adrenal cortex is unique in yielding a nonuniform distribution of AII-2 sites around the outer layer of glomerulosa cells. In the rabbit kidney, the sites on the glomeruli are AII-1, but the sites on the kidney capsule are AII-2. Angiotensin III appears to have a higher affinity for AII-2 sites since it inhibits the binding to the rabbit kidney capsule but not the glomeruli. Elucidation of the distribution and function of these diverse sites should permit the development of more selective and specific therapeutic strategies.

  17. Cholinergic neurotransmission in the mammalian retina. Annual report (Summary), 30 September 1983-29 September 1984

    SciTech Connect

    Pourcho, R.G.

    1984-11-30

    This study is directed toward the cytochemical localization of cholinergic markers in a mammalian (cat) retina and biochemical characterization of the interactions of cholinergic neurons with other neurotransmitters in the retina. Particular attention is paid to localization of acetylcholinesterase and the effects of anticholinesterase organophosphates on normal retinal function. Studies to date have shown the presence of newly synthesized acetylcholine in amacrine and displaced amacrine cells. Acetylcholinesterase was localized in both amacrine and ganglion cells. The presumed cholinotoxin, AF64A, causes severe destruction in the cat retina, involving both amacrine and ganglion cells. Although the evidence to date indicates that only amacrine cells are cholinergic, ganglion cells appear to play a major role in cholinergic or related pathways and may be particularly susceptible to organophosphate poisoning. The biochemical component of the study has centered on the development of a superfusion system in which to monitor the release of various amino acid transmitters in response to application of acetylcholine. Preliminary experiments suggest that cholinergic amacrine cells are presynaptic to glycinergic cells in the cat retina. After the normal pattern has been established, it should be possible to investigate the effects of changes in the level of acetylcholinesterase on these responses.

  18. Mechanisms creating transient and sustained photoresponses in mammalian retinal ganglion cells.

    PubMed

    Zhao, Xiwu; Reifler, Aaron N; Schroeder, Melanie M; Jaeckel, Elizabeth R; Chervenak, Andrew P; Wong, Kwoon Y

    2017-03-06

    Retinal neurons use sustained and transient light responses to encode visual stimuli of different frequency ranges, but the underlying mechanisms remain poorly understood. In particular, although earlier studies in retinal ganglion cells (RGCs) proposed seven potential mechanisms, all seven have since been disputed, and it remains unknown whether different RGC types use different mechanisms or how many mechanisms are used by each type. Here, we conduct a comprehensive survey in mice and rats of 12 candidate mechanisms that could conceivably produce tonic rod/cone-driven ON responses in intrinsically photosensitive RGCs (ipRGCs) and transient ON responses in three types of direction-selective RGCs (TRHR+, Hoxd10+ ON, and Hoxd10+ ON-OFF cells). We find that the tonic kinetics of ipRGCs arises from their substantially above-threshold resting potentials, input from sustained ON bipolar cells, absence of amacrine cell inhibition of presynaptic ON bipolar cells, and mGluR7-mediated maintenance of light-evoked glutamatergic input. All three types of direction-selective RGCs receive input from transient ON bipolar cells, and each type uses additional strategies to promote photoresponse transience: presynaptic inhibition and dopaminergic modulation for TRHR+ cells, center/surround antagonism and relatively negative resting potentials for Hoxd10+ ON cells, and presynaptic inhibition for Hoxd10+ ON-OFF cells. We find that the sustained nature of ipRGCs' rod/cone-driven responses depends neither on melanopsin nor on N-methyl-d-aspartate (NMDA) receptors, whereas the transience of the direction-selective cells' responses is influenced neither by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor desensitization nor by glutamate uptake. For all cells, we further rule out spike frequency adaptation and intracellular Ca(2+) as determinants of photoresponse kinetics. In conclusion, different RGC types use diverse mechanisms to produce sustained or

  19. Study of retinal alterations in a high fat diet-induced type ii diabetes rodent: Meriones shawi.

    PubMed

    Hammoum, Imane; Mbarek, Sihem; Dellaa, Ahmed; Dubus, Elisabeth; Baccouche, Basma; Azaiz, Rached; Charfeddine, Ridha; Picaud, Serge; Ben Chaouacha-Chekir, Rafika

    2017-01-01

    Diabetic retinopathy is a common complication of type 2 diabetes and the leading cause of blindness in adults of working age. The aim of this work was to study the repercussions of high fat diet (HFD) induced diabetes on the retina of Meriones shawi (M.sh). Two groups of six M.sh each was studied. Group I was a normal control, fed with standard laboratory granules. In Group II, rodents received a HFD of enriched laboratory granules, for a period of 3 months. Body weight and plasma glucose were determined in the two groups. Retinal sections of the two groups were stained with the Hematoxylin-Eosin. Photoreceptors were identified by immunolabeling for rhodopsin (rods) and PNA (cones). Gliosis and microglial activation were identified by immunolabeling for GFAP and Iba-1. Labeling of calretinin and parvalbumin were also carried out to study the AII amacrine cells. Retinal layers thicknesses, gliosis, and specific neural cell populations were quantified by microscopy. The body weight (+77%) and plasma glucose (+108%) were significantly greater in the HFD rodents. Three months of HFD induced a significant loss of 38.77% of cone photoreceptors, as well as gliosis and an increase of 70.67% of microglial cells. Calcium homeostatic enzymes were depleted. This work shows that HFD in Meriones shawi induces a type II diabetes-like condition that causes loss of retinal neurons and photoreceptors, as well as gliosis. Meriones shawi could be a useful experimental animal model for this physiopathology particularly in the study of retinal neuro-glial alterations in Type II diabetes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Melatonin potentiates glycine currents through a PLC/PKC signalling pathway in rat retinal ganglion cells.

    PubMed

    Zhao, Wen-Jie; Zhang, Min; Miao, Yanying; Yang, Xiong-Li; Wang, Zhongfeng

    2010-07-15

    In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin-induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT(2) receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT(2) receptor antagonist 4-P-PDOT. The melatonin effect was blocked by intracellular dialysis of GDP-beta-S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)-PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin-induced potentiation. The melatonin effect persisted when [Ca(2+)](i) was chelated by BAPTA, and melatonin induced no increase in [Ca(2+)](i). Neither cAMP-PKA nor cGMP-PKG signalling pathways seemed to be involved because 8-Br-cAMP or 8-Br-cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H-89 and the PKG inhibitor KT5823 did not block the melatonin-induced potentiation. In consequence, a distinct PC-PLC/PKC signalling pathway, following the activation of G(i/o)-coupled MT(2) receptors, is most likely responsible for the melatonin-induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light-evoked glycine receptor-mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner

  1. Mechanisms creating transient and sustained photoresponses in mammalian retinal ganglion cells

    PubMed Central

    Zhao, Xiwu; Jaeckel, Elizabeth R.; Chervenak, Andrew P.

    2017-01-01

    Retinal neurons use sustained and transient light responses to encode visual stimuli of different frequency ranges, but the underlying mechanisms remain poorly understood. In particular, although earlier studies in retinal ganglion cells (RGCs) proposed seven potential mechanisms, all seven have since been disputed, and it remains unknown whether different RGC types use different mechanisms or how many mechanisms are used by each type. Here, we conduct a comprehensive survey in mice and rats of 12 candidate mechanisms that could conceivably produce tonic rod/cone-driven ON responses in intrinsically photosensitive RGCs (ipRGCs) and transient ON responses in three types of direction-selective RGCs (TRHR+, Hoxd10+ ON, and Hoxd10+ ON-OFF cells). We find that the tonic kinetics of ipRGCs arises from their substantially above-threshold resting potentials, input from sustained ON bipolar cells, absence of amacrine cell inhibition of presynaptic ON bipolar cells, and mGluR7-mediated maintenance of light-evoked glutamatergic input. All three types of direction-selective RGCs receive input from transient ON bipolar cells, and each type uses additional strategies to promote photoresponse transience: presynaptic inhibition and dopaminergic modulation for TRHR+ cells, center/surround antagonism and relatively negative resting potentials for Hoxd10+ ON cells, and presynaptic inhibition for Hoxd10+ ON-OFF cells. We find that the sustained nature of ipRGCs’ rod/cone-driven responses depends neither on melanopsin nor on N-methyl-d-aspartate (NMDA) receptors, whereas the transience of the direction-selective cells’ responses is influenced neither by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor desensitization nor by glutamate uptake. For all cells, we further rule out spike frequency adaptation and intracellular Ca2+ as determinants of photoresponse kinetics. In conclusion, different RGC types use diverse mechanisms to produce sustained or

  2. Angiotensin II receptors in testes

    SciTech Connect

    Millan, M.A.; Aguilera, G.

    1988-05-01

    Receptors for angiotensin II (AII) were identified and characterized in testes of rats and several primate species. Autoradiographic analysis of the binding of 125I-labeled (Sar1,Ile8)AII to rat, rhesus monkey, cebus monkey, and human testicular slide-mounted frozen sections indicated specific binding to Leydig cells in the interstitium. In rat collagenase-dispersed interstitial cells fractionated by Percoll gradient, AII receptor content was parallel to that of hCG receptors, confirming that the AII receptors are in the Leydig cells. In rat dispersed Leydig cells, binding was specific for AII and its analogs and of high affinity (Kd, 4.8 nM), with a receptor concentration of 15 fmol/10(6) cells. Studies of AII receptors in rat testes during development reveals the presence of high receptor density in newborn rats which decreases toward the adult age (4934 +/- 309, 1460 +/- 228, 772 +/- 169, and 82 +/- 12 fmol/mg protein at 5, 15, 20, and 30 days of age, respectively) with no change in affinity. At all ages receptors were located in the interstitium, and the decrease in binding was parallel to the decrease in the interstitial to tubular ratio observed with age. AII receptor properties in membrane-rich fractions from prepuberal testes were similar in the rat and rhesus monkey. Binding was time and temperature dependent, reaching a plateau at 60 min at 37 C, and was increased by divalent cations, EGTA, and dithiothreitol up to 0.5 mM. In membranes from prepuberal monkey testes, AII receptors were specific for AII analogs and of high affinity (Kd, 4.2 nM) with a receptor concentration of 7599 +/- 1342 fmol/mg protein. The presence of AII receptors in Leydig cells in rat and primate testes in conjunction with reports of the presence of other components of the renin-angiotensin system in the testes suggests that the peptide has a physiological role in testicular function.

  3. In Vitro Cytotoxic Effects of Gold Nanoparticles Coated with Functional Acyl Homoserine Lactone Lactonase Protein from Bacillus licheniformis and Their Antibiofilm Activity against Proteus Species

    PubMed Central

    Vinoj, Gopalakrishnan; Pati, Rashmirekha; Sonawane, Avinash

    2014-01-01

    N-acylated homoserine lactonases are known to inhibit the signaling molecules of the biofilm-forming pathogens. In this study, gold nanoparticles were coated with N-acylated homoserine lactonase proteins (AiiA AuNPs) purified from Bacillus licheniformis. The AiiA AuNPs were characterized by UV-visible spectra, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and X-ray diffraction (XRD). The synthesized AiiA AuNPs were found to be spherical in shape and 10 to 30 nm in size. Treatment with AiiA protein-coated AuNPs showed maximum reduction in exopolysaccharide production, metabolic activities, and cell surface hydrophobicity and potent antibiofilm activity against multidrug-resistant Proteus species compared to treatment with AiiA protein alone. AiiA AuNPs exhibited potent antibiofilm activity at 2 to 8 μM concentrations without being harmful to the macrophages. We conclude that at a specific dose, AuNPs coated with AiiA can kill bacteria without harming the host cells, thus representing a potential template for the design of novel antibiofilm and antibacterial protein drugs to decrease bacterial colonization and to overcome the problem of drug resistance. In summary, our data suggest that the combined effect of the lactonase and the gold nanoparticles of the AiiA AuNPs has promising antibiofilm activity against biofilm-forming and multidrug-resistant Proteus species. PMID:25403677

  4. In vitro cytotoxic effects of gold nanoparticles coated with functional acyl homoserine lactone lactonase protein from Bacillus licheniformis and their antibiofilm activity against Proteus species.

    PubMed

    Vinoj, Gopalakrishnan; Pati, Rashmirekha; Sonawane, Avinash; Vaseeharan, Baskaralingam

    2015-02-01

    N-acylated homoserine lactonases are known to inhibit the signaling molecules of the biofilm-forming pathogens. In this study, gold nanoparticles were coated with N-acylated homoserine lactonase proteins (AiiA AuNPs) purified from Bacillus licheniformis. The AiiA AuNPs were characterized by UV-visible spectra, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and X-ray diffraction (XRD). The synthesized AiiA AuNPs were found to be spherical in shape and 10 to 30 nm in size. Treatment with AiiA protein-coated AuNPs showed maximum reduction in exopolysaccharide production, metabolic activities, and cell surface hydrophobicity and potent antibiofilm activity against multidrug-resistant Proteus species compared to treatment with AiiA protein alone. AiiA AuNPs exhibited potent antibiofilm activity at 2 to 8 μM concentrations without being harmful to the macrophages. We conclude that at a specific dose, AuNPs coated with AiiA can kill bacteria without harming the host cells, thus representing a potential template for the design of novel antibiofilm and antibacterial protein drugs to decrease bacterial colonization and to overcome the problem of drug resistance. In summary, our data suggest that the combined effect of the lactonase and the gold nanoparticles of the AiiA AuNPs has promising antibiofilm activity against biofilm-forming and multidrug-resistant Proteus species.

  5. Surface-active state of semiconductor materials based on CdTe-AIIS systems

    NASA Astrophysics Data System (ADS)

    Kirovskaya, I. A.; Nor, P. E.; Ushakov, O. V.; Pogodin, S. N.

    2017-08-01

    Surface properties (surface chemical and acid-base state) of binary and multicomponent semiconductors, i.e. CdTe-CdS and CdTe-ZnS systems components, were studied. The influence of mutual binary component CdTe on the surface acid-base state is demonstrated. Similar regularities in acid-base properties changes due to composition were determined, as well as their correlations with the regularities in bulk properties changes in the row of initial binary compounds. The conclusion was made that semiconductors - the components of the systems with acid surfaces (pHiso<7) - can be used as the materials for sensors on basic gases micro-impurities. The possibility of applying the stated regularities and correlations to facilitate the search for such materials is also noted.

  6. Apolipoprotein A-II polymorphism: relationships to behavioural and hormonal mediators of obesity

    USDA-ARS?s Scientific Manuscript database

    New obesity loci continue to be identified through genomewide association studies in populations of increasing size and ethnic diversity but understanding of the mechanisms by which known genetic variants contribute to obesity remains limited. Several well-established obesity candidates encode prote...

  7. Metabolism of Apolipoprotein A-II Containing Triglyceride Rich ApoB Lipoproteins in Humans

    PubMed Central

    Desai, Nirav K.; Ooi, Esther M.; Mitchell, Paul D.; Furtado, Jeremy; Sacks, Frank M.

    2015-01-01

    Objective To characterize human triglyceride-rich lipoproteins (TRL) with and without apoA-II and to study their metabolism in vivo. Methods Plasma from 11 participants on a controlled diet given a bolus infusion of [D5]L-phenylalanine to label apoB was combined into four pools and applied to anti-apoA-II immunoaffinity columns. Fractions with and without apoA-II were separated into VLDL and IDL by ultracentrifugation; lipids and apolipoproteins were measured. For kinetic measurements, apoB was isolated and hydrolyzed to the constituent amino acids. Tracer enrichment was measured by GCMS. Metabolic rates were determined by SAAM-II. Results VLDL and IDL with apoA-II comprised 7% and 9% of total VLDL and IDL apoB respectively. VLDL with apoA-II was enriched in apoC-III, apoE, and cholesterol compared to VLDL without apoA-II. Mean apoB FCR of VLDL with apoA-II was significantly lower than for VLDL without apoA-II (2.80±0.96 pools/day v.s. 5.09±1.69 pools/day, P=0.009). A higher percentage of VLDL with apoA-II was converted to IDL than was cleared from circulation, compared to VLDL without apoA-II (96±8% vs. 45±22%; P=0.007). The rate constants for conversion of VLDL to IDL were similar for VLDL with and without apoA-II. Thus, a very low rate constant for clearance accounted for the lower FCR of VLDL with apoA-II. Conclusion VLDL with apoA-II represents a small pool of VLDL particles that has a slow FCR and is predominantly converted to IDL rather than cleared from the circulation. PMID:26071654

  8. Hot spots in apolipoprotein A-II misfolding and amyloidosis in mice and men

    PubMed Central

    Gursky, Olga

    2014-01-01

    ApoA-II is the second-major protein of high-density lipoproteins. C-terminal extension in human apoA-II or point substitutions in murine apoA-II cause amyloidosis. The molecular mechanism of apolipoprotein misfolding, from the native predominantly α-helical conformation to cross-β-sheet in amyloid, is unknown. We used 12 sequence-based prediction algorithms to identify two ten-residue segments in apoA-II that probably initiate β-aggregation. Previous studies of apoA-II fragments experimentally verify this prediction. Together, experimental and bioinformatics studies explain why the C-terminal extension in human apoA-II causes amyloidosis and why, unlike murine apoA-II, human apoA-II normally does not cause amyloidosis despite its unusually high sequence propensity for β-aggregation. PMID:24561203

  9. Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas.

    PubMed

    Deng, P; Cuenca, N; Doerr, T; Pow, D V; Miller, R; Kolb, H

    2001-06-01

    We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells.

  10. Design of angiotensin II derivatives suitable for indirect affinity techniques: potential applications to receptor studies.

    PubMed

    Bonnafous, J C; Seyer, R; Tence, M; Marie, J; Kabbaj, M; Aumelas, A

    1988-01-01

    The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-AII (Bio-Ahx-AII) and dinitrophenyl- aminohexanoyl-AII (Dnp-Ahx-AII). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (Kd values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala1,Phe(4N3)8)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido AII-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.

  11. Effect of angiotensin II on the apical K+ channel in the thick ascending limb of the rat kidney

    PubMed Central

    1996-01-01

    We have used the patch-clamp technique to study the effect of angiotensin II (AII) on the activity of the apical 70 pS K+ channel and used Na(+)-sensitive fluorescent dye (SBFI) to investigate the effect of AII on intracellular Na+ concentration (Na+i) in the thick ascending limb (TAL) of the rat kidney. Addition of 50 pM AII reversibly reduced NPo, a product of channel open probability (Po) and channel number (N), to 40% of the control value and reduced the Na+i by 26%. The AII (50 pM)-induced decrease in channel activity defined by NPo was partially reversed by addition of 5 microM 17-octadecynoic acid (17-ODYA), an agent which blocks the cytochrome P450 monooxygenase. The notion that P450 metabolites of arachidonic acid (AA) may mediate the inhibitory effect of AII was further suggested by experiments in which addition of 10 nM of 20-hydroxyeicosatetraenoic acid (20-HETE) blocked the channel activity in cell-attached patches in the presence of 17-ODYA. We have used gas chromatography mass spectrometry (GC/MS) to measure the production of 20-HETE, a major AA metabolite of the P450-dependent pathway in the TAL of the rat. Addition of 50 pM AII increased the production of 20-HETE to 260% of the control value, indicating that 20- HETE may be involved in mediating the effect of AII (50 pM). In contrast to the inhibitory effect of 50 pM AII, addition of 50-100 nM AII increased the channel activity to 270% of the control value and elevated the Na+i by 45%. The effect of AII on the activity of the 70 pS K+ channel was also observed in the presence of 5 microM 17-ODYA and 5 microM calphostin C, an inhibitor of protein kinase C. However, addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50- 100 nM)-induced increase in channel activity and addition of an exogenous nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine (SNAP), increased channel activity in the presence of L-NAME. These

  12. Comparative analysis of three purification protocols for retinal ganglion cells from rat.

    PubMed

    Gao, Fengjuan; Li, Tingting; Hu, Jianyan; Zhou, Xujiao; Wu, Jihong; Wu, Qiang

    2016-01-01

    To make comparative analyses of the common three purification protocols for retinal ganglion cells (RGCs), providing a solid practical basis for selecting the method for purifying RGCs for use in subsequent experiments. Rat RGCs were isolated and purified using three methods, including two-step immunopanning (TIP) separation, two-step immunopanning-magnetic (TIPM) separation, and flow cytometric (FC) separation. Immunocytochemical staining, quantitative real-time PCR, flow cytometry, electrophysiology, and Cell Counting Kit-8 (CCK-8) analyses were performed to compare the purity, yield, and viability of the RGCs. The RGC yields from the TIP, TIPM, and FC methods were 24.60±15.98 × 10(4), 5.28±4.42 × 10(4), and 5.4±2.7 × 10(3) per retina, respectively. We easily controlled the relative purity of the RGCs with the FC method and even reached 100% of the maximum expected purity. However, the RGC purity was only 80.97±5.45% and 95.41±3.23% using the TIP and TIPM methods, respectively. The contaminant cells were mainly large, star-shaped, glial fibrillary acidic protein (GFAP)-positive astrocytes and small, round, syntaxin 1-positive amacrine cells with multiple short neurites. The RGCs purified with FC could not be cultured successively in our study; however, the TIP-RGCs survived more than 20 days with good viability, while the TIPM-RGCs survived less than 9 days. The three protocols for purifying the RGCs each had its own pros and cons. The RGCs isolated by the TIP method exhibited the highest viability and yield but had low purity. The purity of the RGCs isolated with the FC method could reach approximately 100% but had a low yield and cell viability. The TIPM method was reliable and produced RGCs with considerable purity, yield, and viability. This study provides a solid practical basis for selecting the method for purifying RGCs for use in subsequent experiments.

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

    SciTech Connect

    O'Malley, D.M.; Masland, R.H.

    1989-05-01

    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.

  14. Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells

    PubMed Central

    1991-01-01

    Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [3H]palmitoyl-PC. At 37 degrees C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4 degrees C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and AII by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organelles labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38th h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium

  15. Angiotensin II receptor blocker inhibits tumour necrosis factor-alpha-induced cell damage in human renal proximal tubular epithelial cells.

    PubMed

    Kagawa, Toru; Takao, Toshihiro; Horino, Taro; Matsumoto, Reiko; Inoue, Kousuke; Morita, Tatsuhito; Hashimoto, Kozo

    2008-06-01

    We investigated the effect of angiotensin II (AII) type 1 (AT1) and angiotensin II type 2 (AT2) receptor blockers on tumour necrosis factor alpha (TNF-alpha)-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). The lactate dehydrogenase (LDH) and N-acetyl-beta-glucosaminidase (NAG) release into the medium after TNF-alpha treatment in RPTEC were determined using modified commercial procedures. In addition, the levels of caspase 3/7 activity in RPTEC were measured after TNF-alpha treatment with AlphaTau1 or AT2 receptor blockers. Finally we investigated the change of p22phox protein levels after TNF-alpha with AlphaTau1 or AT2 receptor blockers in RPTEC. Tumour necrosis factor alpha (10(-8) mol/L) significantly increased LDH and NAG release into the medium from RPTEC. AlphaTau1 receptor blockers, olmesartan and valsartan (10(-9)-10(-6) mol/L) showed a significant reduction on TNF-alpha-induced LDH and NAG release in RPTEC. AT2 receptor blocker, PD123319 (10(-7)-10(-5) mol/L) also decreased TNF-alpha-induced LDH and NAG release in RPTEC. Blockade of both AlphaTau1 and AT2 receptor indicated additional reduction on TNF-alpha-induced LDH and NAG release. TNF-alpha (10(-8) mol/L) treatment showed small but significant increases of caspase 3/7 activity in RPTEC, and AT1 and AT2 receptor blockers (10(-8) mol/L) comparably decreased TNF-alpha-induced caspase 3/7 activity. Significant increases of p22phox protein levels were observed in TNF-alpha-treated group in RPTEC. However, only AlphaTau1 (10(-8) mol/L) but not AT2 (10(-5) mol/L) receptor blocker significantly decreased TNF-alpha-induced p22phox protein levels. The present study demonstrates that TNF-alpha induces renal tubular cell damage in RPTEC and AT1/AT2 receptor blockers showed cytoprotective effects probably via at least partly different mechanism.

  16. Na(+)-H+ exchanger kinetics in adrenal glomerulosa cells and its activation by angiotensin II

    SciTech Connect

    Conlin, P.R.; Kim, S.Y.; Williams, G.H.; Canessa, M.L. )

    1990-07-01

    We have studied the kinetic properties of basal and angiotensin II (ANG II) stimulated Na(+)-H+ exchange in adrenal glomerulosa cells by measuring changes in cytosolic pH (pHi) and initial rates of 22Na uptake in the presence or absence of dimethylamiloride (DMA). The cells were studied under basal conditions, at constant pHi with varied external sodium (Na+o), and at varied pHi with constant Na+o (50 mM). In 2,7-biscarboxyethyl-5(6)-carboxyfluorescein loaded cells under basal conditions, pHi rose from 7.09 +/- 0.02 to 7.19 +/- 0.02. Similarly, DMA-sensitive Na influx was enhanced from 9.2 +/- 1.3 to 14.8 +/- 2.1 nmol Na+/mg protein x min (P less than 0.01) by ANG II. In cells acid-loaded by preincubation in Na(+)-free media (pHi 6.8), addition of varying Na+o resulted in a rapid H+ efflux that was markedly inhibited by DMA. DMA-sensitive Na+ influx into these acidified cells with varied Na+o exhibited a Michaelis-Menten constant (Km) of 23 mM and a maximum velocity (Vmax) of 43 nmol Na+/mg protein x min. By varying pHi (from pHi 7.1 to 6.2), DMA-sensitive Na+ influx likewise showed activation with cellular acidification with a pK at pHi 7.09. At pHi 6.8, ANG II decreased the Km for Na+o from 23 to 17 mM and increased the Vmax from 43 to 53 nmol Na+/mg protein x min. The pHi dependence of DMA-sensitive Na+ influx was not affected by ANG II (pK at pHi 7.03). DMA also inhibited AII-stimulated aldosterone secretion and Na+ influx similarly. These results indicate that Na(+)-H+ exchange in adrenal glomerulosa cells is functioning under basal conditions, and is modulated by ANG II with enhanced Na+o affinity and Vmax but without a shift in pHi dependence (similar to ANG II effects on vascular smooth muscle cells). These effects suggest an important role for Na(+)-H+ exchange during ANG II stimulation of aldosterone production by glomerulosa cells.

  17. Amino acid neurotransmitters in the retina: a functional overview.

    PubMed

    Wu, S M; Maple, B R

    1998-05-01

    Physiological and pharmacological mechanisms of glutamatergic, GABAergic and glycinergic synapses in the tiger salamander retina were studied. We used immunocytochemical and autoradiographic methods to study localizations of these neurotransmitters and their uptake transporters; and electrophysiological methods (intracellular, extracellular and whole cell patch electrode recordings) to study the light responses, miniature postsynaptic currents and neurotransmitter-induced postsynaptic currents in various retinal neurons. Our results are consistent with the following scheme: Glutamate is used by the photoreceptor and bipolar cell output synapses and the release of glutamate is largely mediated by calcium-dependent vesicular processes. The postsynaptic glutamate receptors in DBCs are L-AP4 receptors, in HBCs, HCs and ganglion cells are the kainate/AMPA and NMDA receptors. Subpopulations of HCs make GABAergic synapses on cones and gate chloride condunctance through GABAA receptors. GABAergic HCs do not make feedforward synapses on bipolar cell dendrites and the neurotransmitter identity of the HCs making feedforward synapses is unknown. Subpopulations of amacrine cells make GABAergic synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and GABA gates chloride conductances in theses cells. Glycinergic amacrine cells make synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and glycine opens postsynaptic chloride channels. Glycinergic interplexiform cells make synapses on bipolar cells in the outer retina and glycine released from these cells open chloride channels in bipolar cell dendrites.

  18. Immunocytochemical identification of serotonin-synthesizing neurons in the vertebrate retina: a comparative study.

    PubMed

    Wilhelm, M; Zhu, B; Gábriel, R; Straznicky, C

    1993-02-01

    Serotonin-synthesizing neurons in the retinas of goldfish, axolotl, turtle, chick, rabbit and cat were identified using double labelling with anti-serotonin and anti-phenylalanine hydroxylase antibodies. The latter antibody recognizes tryptophan 5-hydroxylase, one of the synthesizing enzymes for serotonin. Neurons labelled by both markers were considered to be serotonin-synthesizing neurons, while those only with serotonin-immunoreactivity were assumed to be serotonin-accumulating neurons. In the goldfish and chick retinas, all serotonin-immunoreactive amacrine cells (S1) were positive for phenylalanine hydroxylase. In the axolotl and turtle retinas, all the S1 amacrine cells, and only 52.8% and 40.5% of S2 amacrine cells were double-labelled. Although serotonin-immunoreactive bipolar cells were observed in the turtle and chick retinas, the synthesizing enzyme for serotonin could not be detected in these cells. In the rabbit and cat retinas, tryptophan hydroxylase could not be revealed in any cell type with immunocytochemistry. In control experiments SLI neurons in the raphe nuclei of the brain stem always exhibited PH-LI in all species examined, including mammals, indicating that our anti-PH antibody is able to recognize tryptophan hydroxylase across vertebrate classes. These results indicate that the majority of serotonin-immunoreactive amacrine cells are able to synthesize serotonin and are the source of endogenous serotonin in the non-mammalian retina, while some serotonin-immunoreactive amacrine and bipolar cells possibly only accumulate serotonin. We also suggest that serotonin may not be a primary neurotransmitter in the serotonin-accumulating bipolar and amacrine cells of the non-mammalian retina.

  19. Multiple Independent Oscillatory Networks in the Degenerating Retina.

    PubMed

    Euler, Thomas; Schubert, Timm

    2015-01-01

    During neuronal degenerative diseases, microcircuits undergo severe structural alterations, leading to remodeling of synaptic connectivity. This can be particularly well observed in the retina, where photoreceptor degeneration triggers rewiring of connections in the retina's first synaptic layer (e.g., Strettoi et al., 2003; Haq et al., 2014), while the synaptic organization of inner retinal circuits appears to be little affected (O'Brien et al., 2014; Figures 1A,B). Remodeling of (outer) retinal circuits and diminishing light-driven activity due to the loss of functional photoreceptors lead to spontaneous activity that can be observed at different retinal levels (Figure 1C), including the retinal ganglion cells, which display rhythmic spiking activity in the degenerative retina (Margolis et al., 2008; Stasheff, 2008; Menzler and Zeck, 2011; Stasheff et al., 2011). Two networks have been suggested to drive the oscillatory activity in the degenerating retina: a network of remnant cone photoreceptors, rod bipolar cells (RBCs) and horizontal cells in the outer retina (Haq et al., 2014), and the AII amacrine cell-cone bipolar cell network in the inner retina (Borowska et al., 2011). Notably, spontaneous rhythmic activity in the inner retinal network can be triggered in the absence of synaptic remodeling in the outer retina, for example, in the healthy retina after photo-bleaching (Menzler et al., 2014). In addition, the two networks show remarkable differences in their dominant oscillation frequency range as well as in the types and numbers of involved cells (Menzler and Zeck, 2011; Haq et al., 2014). Taken together this suggests that the two networks are self-sustained and can be active independently from each other. However, it is not known if and how they modulate each other. In this mini review, we will discuss: (i) commonalities and differences between these two oscillatory networks as well as possible interaction pathways; (ii) how multiple self

  20. A Role for Synaptic Input Distribution in a Dendritic Computation of Motion Direction in the Retina.

    PubMed

    Vlasits, Anna L; Morrie, Ryan D; Tran-Van-Minh, Alexandra; Bleckert, Adam; Gainer, Christian F; DiGregorio, David A; Feller, Marla B

    2016-03-16

    The starburst amacrine cell in the mouse retina presents an opportunity to examine the precise role of sensory input location on neuronal computations. Using visual receptive field mapping, glutamate uncaging, two-photon Ca(2+) imaging, and genetic labeling of putative synapses, we identify a unique arrangement of excitatory inputs and neurotransmitter release sites on starburst amacrine cell dendrites: the excitatory input distribution is skewed away from the release sites. By comparing computational simulations with Ca(2+) transients recorded near release sites, we show that this anatomical arrangement of inputs and outputs supports a dendritic mechanism for computing motion direction. Direction-selective Ca(2+) transients persist in the presence of a GABA-A receptor antagonist, though the directional tuning is reduced. These results indicate a synergistic interaction between dendritic and circuit mechanisms for generating direction selectivity in the starburst amacrine cell.

  1. Plasma Lipoproteins as Mediators of the Oxidative Stress Induced by UV Light in Human Skin: A Review of Biochemical and Biophysical Studies on Mechanisms of Apolipoprotein Alteration, Lipid Peroxidation, and Associated Skin Cell Responses

    PubMed Central

    Filipe, Paulo; Morlière, Patrice; Silva, João N.; Mazière, Jean-Claude; Patterson, Larry K.; Freitas, João P.; Santus, R.

    2013-01-01

    There are numerous studies concerning the effect of UVB light on skin cells but fewer on other skin components such as the interstitial fluid. This review highlights high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as important targets of UVB in interstitial fluid. Tryptophan residues are the sole apolipoprotein residues absorbing solar UVB. The UVB-induced one-electron oxidation of Trp produces •Trp and •O2 − radicals which trigger lipid peroxidation. Immunoblots from buffered solutions or suction blister fluid reveal that propagation of photooxidative damage to other residues such as Tyr or disulfide bonds produces intra- and intermolecular bonds in apolipoproteins A-I, A-II, and B100. Partial repair of phenoxyl tyrosyl radicals (TyrO•) by α-tocopherol is observed with LDL and HDL on millisecond or second time scales, whereas limited repair of α-tocopherol by carotenoids occurs in only HDL. More effective repair of Tyr and α-tocopherol is observed with the flavonoid, quercetin, bound to serum albumin, but quercetin is less potent than new synthetic polyphenols in inhibiting LDL lipid peroxidation or restoring α-tocopherol. The systemic consequences of HDL and LDL oxidation and the activation and/or inhibition of signalling pathways by oxidized LDL and their ability to enhance transcription factor DNA binding activity are also reviewed. PMID:23738035

  2. Administration of angiotensin II and a bradykinin B2 receptor blocker in midpregnancy impairs gestational outcome in guinea pigs

    PubMed Central

    2014-01-01

    Background The opposing renin-angiotensin system (RAS) and kallikrein-kinin system (KKS) are upregulated in pregnancy and localize in the utero-placental unit. To test their participation as counter-regulators, circulating angiotensin II (AII) was exogenously elevated and the bradykinin B2 receptor (B2R) was antagonized in pregnant guinea-pigs. We hypothesized that disrupting the RAS/KKS balance during the period of maximal trophoblast invasion and placental development would provoke increased blood pressure, defective trophoblast invasion and a preeclampsia-like syndrome. Methods Pregnant guinea-pigs received subcutaneous infusions of AII (200 μg/kg/day), the B2R antagonist Bradyzide (BDZ; 62.5 microg/kg/day), or both (AII + BDZ) from gestational day 20 to 34. Non-pregnant cycling animals were included in a control group (C NP) or received AII + BDZ (AII + BDZ NP) during 14 days. Systolic blood pressure was determined during cycle in C NP, and on the last day of infusion, and 6 and 26 days thereafter in the remaining groups. Twenty six days after the infusions blood and urine were extracted, fetuses, placentas and kidneys were weighed, and trophoblast invasion of spiral arteries was defined in the utero-placental units by immunocytochemistry. Results Systolic blood pressure transiently rose in a subgroup of the pregnant females while receiving AII + BDZ infusion, but not in AII + BDZ NP. Plasma creatinine was higher in AII- and BDZ-treated dams, but no proteinuria or hyperuricemia were observed. Kidney weight increased in AII + BDZ-treated pregnant and non-pregnant females. Aborted and dead fetuses were increased in dams that received AII and AII + BDZ. The fetal/placental weight ratio was reduced in litters of AII + BDZ-treated mothers. All groups that received interventions during pregnancy showed reduced replacement of endothelial cells by extravillous trophoblasts in lateral and myometrial spiral arteries. Conclusions The acute effects on fetal viability, and

  3. Stem Cells

    MedlinePlus

    Stem cells are cells with the potential to develop into many different types of cells in the body. ... the body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  4. Cells, cells, and more cells.

    PubMed

    Bhatti, M Tariq; Gres, Katherine E; Petitto, Virginia B; Cross, Shelley Ann

    2007-01-01

    A 64-year-old woman presented with bilateral optic nerve swelling, vitreous cells, and cerebrospinal fluid monocytic pleocytosis. A chest radiograph and computed tomography demonstrated a lesion in the left lung, which histologically was confirmed to be a small-cell lung carcinoma. The serum was positive for the anti-CV2 (anti-CRMP-5) antibody. Following treatment with chemoradiation the optic nerve swelling and vitritis resolved. The differential diagnosis of uveal-meningeal diseases is discussed and the pathophysiology and clinical manifestations of paraneoplastic syndromes reviewed.

  5. Crossover inhibition in the retina: circuitry that compensates for nonlinear rectifying synaptic transmission.

    PubMed

    Molnar, Alyosha; Hsueh, Hain-Ann; Roska, Botond; Werblin, Frank S

    2009-12-01

    In the mammalian retina, complementary ON and OFF visual streams are formed at the bipolar cell dendrites, then carried to amacrine and ganglion cells via nonlinear excitatory synapses from bipolar cells. Bipolar, amacrine and ganglion cells also receive a nonlinear inhibitory input from amacrine cells. The most common form of such inhibition crosses over from the opposite visual stream: Amacrine cells carry ON inhibition to the OFF cells and carry OFF inhibition to the ON cells ("crossover inhibition"). Although these synapses are predominantly nonlinear, linear signal processing is required for computing many properties of the visual world such as average intensity across a receptive field. Linear signaling is also necessary for maintaining the distinction between brightness and contrast. It has long been known that a subset of retinal outputs provide exactly this sort of linear representation of the world; we show here that rectifying (nonlinear) synaptic currents, when combined thorough crossover inhibition can generate this linear signaling. Using simple mathematical models we show that for a large set of cases, repeated rounds of synaptic rectification without crossover inhibition can destroy information carried by those synapses. A similar circuit motif is employed in the electronics industry to compensate for transistor nonlinearities in analog circuits.

  6. Neurokinin 1 Receptor Expression in the Rat Retina

    PubMed Central

    Casini, Giovanni; Rickman, Dennis W.; Sternini, Catia; Brecha, Nicholas C.

    2010-01-01

    Tachykinin (TK) peptides influence neuronal activity in the inner retina of mammals. The aim of this investigation was to determine the cellular localization of the neurokinin 1 receptor (NK1), whose preferred ligand is the TK peptide substance P (SP), in the rat retina. These studies used a polyclonal antiserum directed to the C-terminus of rat NK1. The majority of NK1-immunoreactive (IR) cells were located in the proximal inner nuclear layer (INL), and very rarely they were found in the distal INL. Some small and large NK1-IR somata were present in the ganglion cell layer. NK1-IR processes were densely distributed across the inner plexiform layer (IPL) with a maximum density over lamina 2 of the IPL. Immunoreactive processes also crossed the INL and ramified in the outer plexiform layer where they formed a sparse meshwork. NK1-IR processes were rarely observed in the optic nerve fiber layer. Double-label immunofluorescence studies with different histochemical markers for bipolar cells indicated that NK1 immunoreactivity was not present in bipolar cells. Together, these observations indicate that NK1 immunoreactivity is predominantly expressed by amacrine, displaced amacrine, interplexiform, and some ganglion cells. Double-label immunofluorescence experiments were also performed to characterize NK1-containing amacrine cells. Sixty-one percent of the γ-aminobutyric acid (GABA)-IR cells, 71% of the large tyrosine hydroxylase (TH)-IR cells, and 100% of the small TH-IR cells contained NK1 immunoreactivity. In addition, most (91%) of the NK1-IR cells had GABA immunoreactivity. In contrast, vasoactive intestinal polypeptide-, TK-, choline acetyltransferase-, and parvalbumin-IR amacrine cells did not express NK1 immunoreactivity. Overall, the present findings suggest that SP acts directly upon several cell populations, including GABA-containing amacrine cells and ganglion cells, to influence visual information processing in the inner retina. J. Comp. Neurol. 389:496

  7. Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate Modeling and Active Site Mutations

    SciTech Connect

    Momb, Jessica; Wang, Canhui; Liu, Dali; Thomas, Pei W.; Petsko, Gregory A.; Guo, Hua; Ringe, Dagmar; Fast, Walter

    2008-12-02

    The N-acyl-l-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo-{beta}-lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme-product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily.

  8. 75 FR 20645 - Order Instituting Administrative Proceedings Pursuant to Section 15E(a)(2)(A)(ii) of the...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-20

    ... Credit Rating Co., Ltd. April 14, 2010. Dagong Global Credit Rating Co., Ltd. (``Dagong''), a credit rating agency based in Beijing, China, submitted an application with the Securities and Exchange Commission (``Commission'') for registration as a nationally recognized statistical rating organization...

  9. Instructional Challenges: Understanding the Needs of Novice Special Education Teachers. Induction Insights. Supporting Special Education Teachers - Administrators [AII-04

    ERIC Educational Resources Information Center

    National Center to Inform Policy and Practice in Special Education Professional Development, 2010

    2010-01-01

    Novice special education teachers struggle with many of the same pedagogical challenges as their general education counterparts. They often need help learning the curriculum, acquiring and adapting necessary materials, and addressing challenging student behavior. A complicating factor is that novice special education teachers typically have…

  10. 78 FR 64430 - Mixed Straddles; Straddle-by-Straddle Identification Under Section 1092(b)(2)(A)(i)(I); Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-29

    ... Internal Revenue Service 26 CFR Part 1 RIN 1545-BK99 Mixed Straddles; Straddle-by-Straddle Identification...: Notice of proposed rulemaking by cross-reference to temporary regulations and notice of public hearing... taxpayers electing to establish a mixed straddle using straddle-by-straddle identification. These amendments...

  11. 78 FR 46854 - Mixed Straddles; Straddle-by-Straddle Identification Under Section 1092(b)(2)(A)(i)(I)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-02

    ...In the Rules and Regulations section of this issue of the Federal Register the Treasury Department and the IRS are issuing temporary regulations that explain how to account for unrealized gain or loss on a position held by a taxpayer prior to the time the taxpayer establishes a mixed straddle using straddle-by-straddle identification. The text of the temporary regulations also serves as the text of these proposed regulations. This document also provides notice of a public hearing on these proposed regulations.

  12. 78 FR 46807 - Mixed Straddles; Straddle-by-Straddle Identification Under Section 1092(b)(2)(A)(i)(I)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-02

    ...This document contains guidance for those taxpayers electing to establish a mixed straddle using straddle-by-straddle identification. These temporary regulations explain how to account for unrealized gain or loss on a position held by a taxpayer prior to the time the taxpayer establishes a mixed straddle using straddle-by- straddle identification. The text of these temporary regulations also serves as the text of the proposed regulations (REG-112815-12) set forth in the Proposed Rules section in this issue of the Federal Register.

  13. Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 1. Product-Bound Structures

    SciTech Connect

    Liu, Dali; Momb, Jessica; Thomas, Pei W.; Moulin, Aaron; Petsko, Gregory A.; Fast, Walter; Ringe, Dagmar

    2008-08-06

    Enzymes capable of hydrolyzing N-acyl-l-homoserine lactones (AHLs) used in some bacterial quorum-sensing pathways are of considerable interest for their ability to block undesirable phenotypes. Most known AHL hydrolases that catalyze ring opening (AHL lactonases) are members of the metallo-{beta}-lactamase enzyme superfamily and rely on a dinuclear zinc site for catalysis and stability. Here we report the three-dimensional structures of three product complexes formed with the AHL lactonase from Bacillus thuringiensis. Structures of the lactonase bound with two different concentrations of the ring-opened product of N-hexanoyl-l-homoserine lactone are determined at 0.95 and 1.4 {angstrom} resolution and exhibit different product configurations. A structure of the ring-opened product of the non-natural N-hexanoyl-l-homocysteine thiolactone at 1.3 {angstrom} resolution is also determined. On the basis of these product-bound structures, a substrate-binding model is presented that differs from previous proposals. Additionally, the proximity of the product to active-site residues and observed changes in protein conformation and metal coordination provide insight into the catalytic mechanism of this quorum-quenching metalloenzyme.

  14. 76 FR 53312 - Airworthiness Directives; Agusta S.p.A. Model A109A and A109AII Helicopters

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-26

    ..., P/N 109-0133-16-103 and 109-0130-89-1, and the static stop, P/ N 109-0130-27-5, for damage or...,884 9,309 7 (assume 10% of 65,163 Spacers, Static Stop. rate of $85. fleet). Total cost impact for.... Using the search function of the docket Web site, you can find and read the comments to any of our...

  15. Neurokinin 1 receptor expression in the rat retina.

    PubMed

    Casini, G; Rickman, D W; Sternini, C; Brecha, N C

    1997-12-22

    Tachykinin (TK) peptides influence neuronal activity in the inner retina of mammals. The aim of this investigation was to determine the cellular localization of the neurokinin 1 receptor (NK1), whose preferred ligand is the TK peptide substance P (SP), in the rat retina. These studies used a polyclonal antiserum directed to the C-terminus of rat NK1. The majority of NK1-immunoreactive (IR) cells were located in the proximal inner nuclear layer (INL), and very rarely they were found in the distal INL. Some small and large NK1-IR somata were present in the ganglion cell layer. NK1-IR processes were densely distributed across the inner plexiform layer (IPL) with a maximum density over lamina 2 of the IPL. Immunoreactive processes also crossed the INL and ramified in the outer plexiform layer where they formed a sparse meshwork. NK1-IR processes were rarely observed in the optic nerve fiber layer. Double-label immunofluorescence studies with different histochemical markers for bipolar cells indicated that NK1 immunoreactivity was not present in bipolar cells. Together, these observations indicate that NK1 immunoreactivity is predominantly expressed by amacrine, displaced amacrine, interplexiform, and some ganglion cells. Double-label immunofluorescence experiments were also performed to characterize NK1-containing amacrine cells. Sixty-one percent of the gamma-aminobutyric acid (GABA)-IR cells, 71% of the large tyrosine hydroxylase (TH)-IR cells, and 100% of the small TH-IR cells contained NK1 immunoreactivity. In addition, most (91%) of the NK1-IR cells had GABA immunoreactivity. In contrast, vasoactive intestinal polypeptide-, TK-, choline acetyltransferase-, and parvalbumin-IR amacrine tells did not express NK1 immunoreactivity. Overall, the present findings suggest that SP acts directly upon several cell populations, including GABA-containing amacrine cells and ganglion cells, to influence visual information processing in the inner retina.

  16. Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons

    PubMed Central

    Chintala, Shravan; Cheng, Mei; Zhang, Xiao

    2015-01-01

    The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and

  17. Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons.

    PubMed

    Chintala, Shravan; Cheng, Mei; Zhang, Xiao

    2015-01-01

    The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and

  18. High affinity binding of 125I-angiotensin II to rat glomerular basement membranes.

    PubMed Central

    Sraer, J; Baud, L; Cosyns, J P; Verroust, P; Nivez, M P; Ardaillou, R

    1977-01-01

    125I-angiotensin II (AII) specifically bound to rat glomerular basement membrane (GBM). The kinetics of binding were similar to those obtained with the total glomeruli. The apparent dissociation constant was close to 50 pM with both preparations. The number of sites related to the amount of protein was two times greater with GBM than with total glomeruli. Since the amount of GBM protein extracted from a given amount of glomerular protein was about 10%, it was possible to estimate the share of the GBM binding sites for AII as representing 20% of the total number present in the entire glomerulus. Binding studies at equilibrium as a function of 125I-AII concentration and competitive binding experiments suggested either multiplicity of the binding sites or cooperativity in the binding reaction. Degradation of 125I-AII in the presence of GBM was slight and did not increase with time. The difference in the degrees of degradation of 125I-AII was too small to account for the observed difference in binding when the results obtained with GBM and isolated glomeruli preparations were compared. 125I-AII binding to GBM was increased after treatment of these membranes with collagenase, slightly diminished with neuraminidase, and almost completely abolished with trypsin suggesting the proteic nature of the receptor. 125I-AII binding to GBM was diminished after incubation of GBM with anti-GBM antibodies as a result of a decrease in the number of binding sites. 125I-AII binding was even more diminished in preparations of glomeruli isolated from rats passively immunized with anti-GBM antibodies when compared with glomeruli from control animals. This resulted from both smaller affinity for AII and decrease in the number of the binding sites. The present data provides evidence for specific binding sites for AII localized on GBM. This is noteworthy since receptors for polypeptide hormones are currently observed on the surface of cell membranes. These findings also suggest a new

  19. T Cells

    MedlinePlus

    ... Cells Share this page Facebook Twitter Email T Cells Definition of MS Myelin Immune-Mediated Disease T ... other immune cells. Three broad categories of T cells Helper T cells augment the immune response by ...

  20. Function and Circuitry of VIP+ Interneurons in the Mouse Retina

    PubMed Central

    Park, Silvia J.H.; Borghuis, Bart G.; Rahmani, Pouyan; Zeng, Qiang

    2015-01-01

    Visual processing in the retina depends on coordinated signaling by interneurons. Photoreceptor signals are relayed to ∼20 ganglion cell types through a dozen excitatory bipolar interneurons, each responsive to light increments (ON) or decrements (OFF). ON and OFF bipolar cell pathways become tuned through specific connections with inhibitory interneurons: horizontal and amacrine cells. A major obstacle for understanding retinal circuitry is the unknown function of most of the ∼30–40 amacrine cell types, each of which synapses onto a subset of bipolar cell terminals, ganglion cell dendrites, and other amacrine cells. Here, we used a transgenic mouse line in which vasoactive intestinal polypeptide-expressing (VIP+) GABAergic interneurons express Cre recombinase. Targeted whole-cell recordings of fluorescently labeled VIP+ cells revealed three predominant types: wide-field bistratified and narrow-field monostratified cells with somas in the inner nuclear layer (INL) and medium-field monostratified cells with somas in the ganglion cell layer (GCL). Bistratified INL cells integrated excitation and inhibition driven by both ON and OFF pathways with little spatial tuning. Narrow-field INL cells integrated excitation driven by the ON pathway and inhibition driven by both pathways, with pronounced hyperpolarizations at light offset. Monostratified GCL cells integrated excitation and inhibition driven by the ON pathway and showed center-surround spatial tuning. Optogenetic experiments showed that, collectively, VIP+ cells made strong connections with OFF δ, ON-OFF direction-selective, and W3 ganglion cells but weak, inconsistent connections with ON and OFF α cells. Revealing VIP+ cell morphologies, receptive fields and synaptic connections advances our understanding of their role in visual processing. SIGNIFICANCE STATEMENT The retina is a model system for understanding nervous system function. At the first stage, rod and cone photoreceptors encode light and

  1. The erythropoietin receptor is not required for the development, function, and aging of rods and cells in the retinal periphery

    PubMed Central

    Caprara, Christian; Britschgi, Corinne; Samardzija, Marijana

    2014-01-01

    . Analysis of the retinal morphology in the two knockdown lines did not reveal any developmental defects or signs of accelerated degeneration in the senescent tissue. Similarly, retinal function was not altered under scotopic and photopic conditions. In addition, EpoR knockdown had no influence on cell viability under acute hypoxic conditions. Retinal angiogenesis and vasculature were normal in the absence of EPOR. However, expression of some EPOR-signaling target genes was significantly altered in the retinas of the EpoRflox/flox;α-Cre mice. Conclusions Our data suggest that expression of EPOR in rod photoreceptors, Müller cells, and amacrine, horizontal, and ganglion cells of the peripheral retina is not required for the maturation, function, and survival of these cells in aging tissue. Based on the expression of the EPOR-signaling target genes, we postulate that expression of soluble EPOR in the retina may modulate endogenous EPO-EPOR signaling. PMID:24644405

  2. A hierarchical artificial retina architecture

    NASA Astrophysics Data System (ADS)

    Parker, Alice C.; Azar, Adi N.

    2009-05-01

    Connectivity in the human retina is complex. Over one hundred million photoreceptors transduce light into electrical signals. These electrical signals are sent to the ganglion cells through amacrine and bipolar cells. Lateral connections involving horizontal and amacrine cells span throughout the outer plexiform layer and inner plexiform layer respectively. Horizontal cells are important for photoreceptor regulation by depolarizing them after an illumination occurs. Horizontal cells themselves form an electrical network that communicates by gap junctions, and these cells exhibit plasticity (change in behavior and structure) with respect to glycine receptors. The bipolar and amacrine cells transfer electrical signals from photoreceptors to the ganglion cells. Furthermore, amacrine cells are responsible for further processing the retinal image. Finally, the ganglion cells receive electrical signals from the bipolar and amacrine cells and will spike at a faster rate if there is a change in the overall intensity for a group of photoreceptors, sending a signal to the brain. Dramatic progress is being made with respect to retinal prostheses, raising hope for an entire synthetic retina in the future. We propose a bio-inspired 3D hierarchical pyramidal architecture for a synthetic retina that mimics the overall structure of the human retina. We chose to use a 3D architecture to facilitate connectivity among retinal cells, maintaining a hierarchical structure similar to that of the biological retina. The first layer of the architecture contains electronic circuits that model photoreceptors and horizontal cells. The second layer contains amacrine and bipolar electronic cells, and the third layer contains ganglion cells. Layer I has the highest number of cells, and layer III has the lowest number of cells, resulting in a pyramidal architecture. In our proposed architecture we intend to use photodetectors to transduce light into electrical signals. We propose to employ

  3. Cell division

    MedlinePlus Videos and Cool Tools

    ... the first 12 hours after conception, the fertilized egg cell remains a single cell. After approximately 30 ... at the end of 3 days, the fertilized egg cell has become a berry-like structure made ...

  4. The organization of the turtle inner retina. I. ON- and OFF-center pathways.

    PubMed

    Ammermüller, J; Kolb, H

    1995-07-17

    Intracellular recordings and dye injections of Lucifer yellow, horseradish peroxidase, or Neurobiotin were made in bipolar, amacrine, and ganglion cells of the Pseudemys turtle retina. By using a standard light-stimulation protocol in a sample of 375 labeled neurons, we were able to identify morphological and physiological characteristics of 11 types of bipolar cell, 37 types of amacrine cell, and 24 types of ganglion cell. To make sense of these data, we have chosen to group the 72 essentially different neuron types into traditional, functionally significant pathways. In this paper we look at the neuronal types in the inner plexiform layer (IPL) in terms of their contribution to generalized luminosity responses such as sustained ON- or OFF-center and transient ON-OFF ganglion cells; in the companion paper (J. Ammermüller, J.F. Muller, and H. Kolb, 1995, J. Comp. Neurol. 358:35-62) we look at them in terms of their involvement in color opponency and directional selectivity. A functional organization of the turtle IPL into OFF sublaminae (strata 1 and 2) and ON sublaminae (strata 3, 4, and 5), as has been described for other vertebrate retinas, was quite clear for two varieties of OFF-center bipolar cells (B4 and B5) and for all four types of sustained ON-center bipolar cell (B1, B2, B6, and B7). Thus, we found no sustained ON-center bipolar cell terminating in strata 1 and 2. We did, however, see three varieties of sustained OFF-center bipolar cells (B3, B9, and B10) having axon terminals in strata 3-5 (the ON sublamina) in addition to their terminations in stratum 1 or 2 (the OFF sublamina). Monostratified sustained ON- and OFF-center amacrine and ganglion cells rigidly obeyed the border of ON and OFF sublaminae. However, multistratified and diffuse sustained amacrine and ganglion cells could be either ON-center or OFF-center, and they did not strictly obey the border: such ON-center cells always had processes in one of the ON sublaminae (strata 3-5), and the

  5. Galvanic Cells

    ERIC Educational Resources Information Center

    Young, I. G.

    1973-01-01

    Many standard physical chemistry textbooks contain ambiguities which lead to confusion about standard electrode potentials, calculating cell voltages, and writing reactions for galvanic cells. This article shows how standard electrode potentials can be used to calculate cell voltages and deduce cell reactions. (Author/RH)

  6. Convergence of Protein Kinase C and JAK-STAT Signaling on Transcription Factor GATA-4

    PubMed Central

    Wang, Jun; Paradis, Pierre; Aries, Anne; Komati, Hiba; Lefebvre, Chantal; Wang, Hao; Nemer, Mona

    2005-01-01

    Angiotensin II (AII), a potent vasoactive hormone, acts on numerous organs via G-protein-coupled receptors and elicits cell-specific responses. At the level of the heart, AII stimulation alters gene transcription and leads to cardiomyocyte hypertrophy. Numerous intracellular signaling pathways are activated in this process; however, which of these directly link receptor activation to transcriptional regulation remains undefined. We used the atrial natriuretic factor (ANF) gene (NPPA) as a marker to elucidate the signaling cascades involved in AII transcriptional responses. We show that ANF transcription is activated directly by the AII type 1 receptor and precedes the development of myocyte hypertrophy. This response maps to STAT and GATA binding sites, and the two elements transcriptionally cooperate to mediate signaling through the JAK-STAT and protein kinase C (PKC)-GATA-4 pathways. PKC phosphorylation enhances GATA-4 DNA binding activity, and STAT-1 functionally and physically interacts with GATA-4 to synergistically activate AII and other growth factor-inducible promoters. Moreover, GATA factors are able to recruit STAT proteins to target promoters via GATA binding sites, which are sufficient to support synergy. Thus, STAT proteins can act as growth factor-inducible coactivators of tissue-specific transcription factors. Interactions between STAT and GATA proteins may provide a general paradigm for understanding cell specificity of cytokine and growth factor signaling. PMID:16260600

  7. Stem cells.

    PubMed

    Behr, Björn; Ko, Sae Hee; Wong, Victor W; Gurtner, Geoffrey C; Longaker, Michael T

    2010-10-01

    Stem cells are self-renewing cells capable of differentiating into multiple cell lines and are classified according to their origin and their ability to differentiate. Enormous potential exists in use of stem cells for regenerative medicine. To produce effective stem cell-based treatments for a range of diseases, an improved understanding of stem cell biology and better control over stem cell fate are necessary. In addition, the barriers to clinical translation, such as potential oncologic properties of stem cells, need to be addressed. With renewed government support and continued refinement of current stem cell methodologies, the future of stem cell research is exciting and promises to provide novel reconstructive options for patients and surgeons limited by traditional paradigms.

  8. Developmental expression of neurokinin-1 and neurokinin-3 receptors in the rat retina.

    PubMed

    Casini, G; Brecha, N C; Bosco, L; Rickman, D W

    2000-05-29

    Tachykinin (TK) peptides act on retinal neurons through neurokinin (NK) receptors. We examined the expression of neurokinin-1 (NK1; the substance P receptor), NK3 [the neurokinin B (NKB) receptor], and TK peptides in developing rat retinas. NK1 immunolabeling was found in newborn retinas in rare amacrine cells and in putative ganglion cells. At postnatal day 2 (PND 2), NK1 immunostaining was reduced greatly among ganglion cells, and it appeared in many amacrine cells and in fibers in the inner plexiform layer (IPL), with the highest density in laminae 1, 3, and 5. A similar pattern was found at PND 7. At PND 12, interplexiform NK1-immunoreactive (-IR) cells were detected, and NK1-IR fibers in the IPL were concentrated in lamina 2, similar to what was seen in adults. NK3 was expressed mainly by OFF-cone bipolar cells, and the developmental pattern of NK3 was compared with that of cone bipolar cells that were labeled with antibodies to recoverin. Immature recoverin-IR cone bipolar cells were seen at PND 2. NK3 immunolabeling was detected first in the outer plexiform layer and in sparse bipolar cell somata at PND 10, when recoverin-IR cone bipolar cells are nearly mature. By PND 15, both the NK3 immunostaining pattern and the recoverin immunostaining pattern were similar to the patterns seen in adults. TK immunoreactivity was present at PND 0 in amacrine cells and displaced amacrine cells. By PND 10, the morphologic maturation of TK-IR cells was complete. These findings indicate that, in early postnatal retinas, substance P may act on NK1 receptors, whereas NKB/NK3 interactions are unlikely, suggesting that there are different levels of importance for different TK peptides in the developing retina. Copyright 2000 Wiley-Liss, Inc.

  9. Engineering Cell-Cell Signaling

    PubMed Central

    Milano, Daniel F.; Natividad, Robert J.; Asthagiri, Anand R.

    2014-01-01

    Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling based on quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilizing synthetic cells, advanced ‘chassis’ and predictive modeling to engineer the form and function of living tissues. PMID:23856592

  10. Engineering cell-cell signaling.

    PubMed

    Blagovic, Katarina; Gong, Emily S; Milano, Daniel F; Natividad, Robert J; Asthagiri, Anand R

    2013-10-01

    Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling on the basis of quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilize synthetic cells, advanced 'chassis' and predictive modeling to engineer the form and function of living tissues.

  11. Fuel cells

    NASA Astrophysics Data System (ADS)

    1984-12-01

    The US Department of Energy (DOE), Office of Fossil Energy, has supported and managed a fuel cell research and development (R and D) program since 1976. Responsibility for implementing DOE's fuel cell program, which includes activities related to both fuel cells and fuel cell systems, has been assigned to the Morgantown Energy Technology Center (METC) in Morgantown, West Virginia. The total United States effort of the private and public sectors in developing fuel cell technology is referred to as the National Fuel Cell Program (NFCP). The goal of the NFCP is to develop fuel cell power plants for base-load and dispersed electric utility systems, industrial cogeneration, and on-site applications. To achieve this goal, the fuel cell developers, electric and gas utilities, research institutes, and Government agencies are working together. Four organized groups are coordinating the diversified activities of the NFCP. The status of the overall program is reviewed in detail.

  12. Cell Phones

    MedlinePlus

    ... These base stations operate at higher power than cell phones. The RF exposures people experience from base stations are typically much lower than from cell phones because base station antennas are mounted on ...

  13. Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

    PubMed Central

    Sargoy, Allison; Barnes, Steven; Brecha, Nicholas C.; Pérez De Sevilla Müller, Luis

    2014-01-01

    In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer. PMID:25349920

  14. Immunohistochemical and calcium imaging methods in wholemount rat retina.

    PubMed

    Sargoy, Allison; Barnes, Steven; Brecha, Nicholas C; Pérez De Sevilla Müller, Luis

    2014-10-13

    In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

  15. The neuronal organization of the rat subfornical organ in vitro and a test of the osmo- and morphine-receptor hypotheses.

    PubMed Central

    Buranarugsa, P; Hubbard, J I

    1979-01-01

    1. Extracellular action potentials (units) were recorded from rat subfornical organ explants in vitro in response to addition of angiotensin II (AII) or carbamyl-choline (carbachol) or serotonin (5-HT) to the superfusion solution. The frequency recorded was dose dependent over a wide range (AII, 0.05--5 nM; carbachol, 2.7--2700 nM; 5-HT, 1--100 nM). Appropriate antagonists, sarc1-ala2 angiotensin (saralasin) for AII, atropine sulphate for carbachol and methysergide maleate for 5-HT, blocked these excitations. The effects were reversible except for that of atropine. 2. Two populations of AII-excited units were found. A superficial population lying between 15 and 45 microns from the ependymal surface was blocked only by saralasin and another population lying more than 55 microns below the ependymal surface could be blocked by atropine as well as saralasin. Carbachol-evoked units generally lay below 45 microns, and 5-HT-evoked units were scattered evenly over the subfornical organ. It is suggested that superficial AII-excited neurones have a cholinergic excitatory synapse with the deeper carbachol-excited neurones. 3. No evidence was found for the hypothesis that neurones of the subfornical organ are excited by morphine or by changes in extracellular osmotic pressure. 4. All types of drug-excited unit, both superficial (15--55 microns) and deep (below 55 microns), could be driven polysynaptically from the body or columns of the fornix. Units driven antidromically or antidromically and synaptically were almost all more than 55 microns from the surface. 5-HT-evoked units were driven antidromically only by stimulation of the columns of the fornix. AII- and carbachol-evoked units could be driven antidromically or antidromically and synaptically by stimulation of the body or the columns of the fornix. It is suggested that AII units driven antidromically are actually carbachol-sensitive neurones driven by the more superficial AII-sensitive cells. 5. A model of the neuronal

  16. Photoelectrochemical cells

    NASA Astrophysics Data System (ADS)

    Nozik, A. J.

    1980-02-01

    The application of photoelectrochemical systems based on photoactive semiconducting electrodes to the problem of solar energy conversion and chemical synthesis is discussed. Three types of cells are described: electrochemical photovoltaic cells (wherein optical energy is converted into electrical energy); photoelectrolysis cells (wherein optical energy is converted into chemical free energy); and photocatalytic cells (wherein optical energy provides the activation energy for exoergic chemical reactions). The critical semiconductor electrode properties for these cells are the band gap, the flat-band potential, and the photoelectrochemical stability. No semiconductor electrode material is yet known for which all three parameters are simultaneously optimized. An interesting configurational variation of photoelectrolysis cells, labelled 'photochemical diodes', is described. These diodes comprise cells that have been collapsed into monolithic particles containing no external wires. Recent advances in several areas of photoelectrochemical systems are also described.

  17. Characterization of glucagon-expressing neurons in the chicken retina

    PubMed Central

    Fischer, Andy J.; Skorupa, Dana; Schonberg, David L.; Walton, Nathaniel A.

    2008-01-01

    We have recently identified large glucagon-expressing neurons that densely ramify neurites in the peripheral edge of the retina and regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken eye (Fischer et al., 2005). However, nothing is known about the transmitters and proteins that are expressed by the glucagon-expressing neurons in the avian retina. We used antibodies to cell-distinguishing markers to better characterize the different types of glucagon-expressing neurons. We found that the large glucagon-expressing neurons were immunoreactive for substance P, neurofilament, Pax6, AP2α, HuD, calretinin, trkB and trkC. Colocalization of glucagon and substance P in the large glucagon-expressing neurons indicates that these cells are the “bullwhip cells” that have been briefly described by Ehrlich, Keyser and Karten (1987). Similar to the bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for calretinin, HuD, Pax6, and AP2α. Unlike bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for GABA. While glucagon-immunoreactive amacrine cells were negative for substance P in central regions of the retina, a subset of this type of amacrine cell was immunoreactive for substance P in far peripheral regions of the retina. An additional type of glucagon/substance P-expressing neuron, resembling the bullwhip cells, was found in far peripheral and dorsal regions of the retina. Based on morphology, distribution within the retina, and histological markers, we conclude that there may be 4 different types of glucagon-expressing neurons in the avian retina. PMID:16572462

  18. Types of Stem Cells

    MedlinePlus

    ... Stem Cell Glossary Search Toggle Nav Types of Stem Cells Stem cells are the foundation from which all ... Learn About Stem Cells > Types of Stem Cells Stem cells Stem cells are the foundation for every organ ...

  19. Electrolytic cell

    NASA Astrophysics Data System (ADS)

    Bullock, J. S.; Hale, B. D.

    1984-09-01

    An apparatus is described for the separation of the anolyte and the catholyte during electrolysis. The electrolyte flows through an electrolytic cell between the oppositely charged electrodes. The cell is equipped with a wedge-shaped device, the tapered end is located between the electrodes on the effluent side of the cell. The wedge diverts the flow of the electrolyte to either side of the wedge, substantially separating the anolyte and the catholyte.

  20. Cell Chauvinism

    ERIC Educational Resources Information Center

    Keller, Dolores Elaine

    1972-01-01

    Indicates that biological terminology, such as mother cell'' and labels of sex factors in bacteria, reflect discrimination against females by reinforcing perpetuation of stereotyped gender roles. (AL)

  1. Cell Migration

    PubMed Central

    Trepat, Xavier; Chen, Zaozao; Jacobson, Ken

    2015-01-01

    Cell migration is fundamental to establishing and maintaining the proper organization of multicellular organisms. Morphogenesis can be viewed as a consequence, in part, of cell locomotion, from large-scale migrations of epithelial sheets during gastrulation, to the movement of individual cells during development of the nervous system. In an adult organism, cell migration is essential for proper immune response, wound repair, and tissue homeostasis, while aberrant cell migration is found in various pathologies. Indeed, as our knowledge of migration increases, we can look forward to, for example, abating the spread of highly malignant cancer cells, retarding the invasion of white cells in the inflammatory process, or enhancing the healing of wounds. This article is organized in two main sections. The first section is devoted to the single-cell migrating in isolation such as occurs when leukocytes migrate during the immune response or when fibroblasts squeeze through connective tissue. The second section is devoted to cells collectively migrating as part of multicellular clusters or sheets. This second type of migration is prevalent in development, wound healing, and in some forms of cancer metastasis. PMID:23720251

  2. Cell Chauvinism

    ERIC Educational Resources Information Center

    Keller, Dolores Elaine

    1972-01-01

    Indicates that biological terminology, such as mother cell'' and labels of sex factors in bacteria, reflect discrimination against females by reinforcing perpetuation of stereotyped gender roles. (AL)

  3. Plasmalemmal and Vesicular γ-Aminobutyric Acid Transporter Expression in the Developing Mouse Retina

    PubMed Central

    GUO, CHENYING; STELLA, SALVATORE L.; HIRANO, ARLENE A.; BRECHA, NICHOLAS C.

    2009-01-01

    Plasmalemmal and vesicular γ-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week. PMID:18975268

  4. Multiple γ-Aminobutyric Acid Plasma Membrane Transporters (GAT-1, GAT-2, GAT-3) in the Rat Retina

    PubMed Central

    JOHNSON, JULIETTE; CHEN, TY K.; RICKMAN, DENNIS W.; EVANS, CHRIS; BRECHA, NICHOLAS C.

    2010-01-01

    γ-Aminobutyric acid (GABA) plasma membrane transporters (GATS) influence synaptic neurotransmission by high-affinity uptake and release of GABA. The distribution and cellular localization of GAT-1, GAT-2, and GAT-3 in the rat retina have been evaluated by using affinity-purified polyclonal antibodies directed to the C terminus of each of these GAT subtypes. Small GAT-1–immunoreactive cell bodies were located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL), and processes were distributed to all laminae of the interplexiform layer (IPL). Varicose processes were in the optic fiber layer (OFL) and the outer plexiform layer (OPL). Weak GAT-1 immunostaining surrounded cells in the INL and GCL, and it was found in the OFL and OPL and in numerous processes in the outer nuclear layer (ONL) that ended at the outer limiting membrane. GAT-1 is therefore strongly expressed by amacrine, displaced amacrine, and interplexiform cells and weakly expressed by Müller cells. GAT-2 immunostaining was observed in the retina pigment epithelium and the nonpigmented ciliary epithelium. GAT-3 immunoreactivity was distributed to the OFL, to all laminae of the IPL, GCL and INL, and to processes in the ONL that ended at the outer limiting membrane. Small GAT-3–immunoreactive cell bodies were in the proximal INL and GCL. GAT-3 is therefore strongly expressed by Müller cells, and by some amacrine and displaced amacrine cells. Together, these observations demonstrate a heterologous distribution of GATs in the retina. These transporters are likely to take up GABA from, and perhaps release GABA into, the synaptic cleft and extracellular space. This suggests that GATs regulate GABA levels in these areas and thus influence synaptic neurotransmission. PMID:8915826

  5. Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina.

    PubMed

    Matsuoka, Ryota L; Nguyen-Ba-Charvet, Kim T; Parray, Aijaz; Badea, Tudor C; Chédotal, Alain; Kolodkin, Alex L

    2011-02-10

    In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.

  6. Transmembrane semaphorin signaling controls laminar stratification in the mammalian retina

    PubMed Central

    Matsuoka, Ryota L.; Nguyen-Ba-Charvet, Kim T.; Parray, Aijaz; Badea, Tudor C.; Chédotal, Alain; Kolodkin, Alex L.

    2010-01-01

    In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells, and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL)1–3: a laminar region that is conventionally divided into five major parallel sublaminae1,2. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here, we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs), and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo with respect to the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes. PMID:21270798

  7. Unit Cells

    ERIC Educational Resources Information Center

    Olsen, Robert C.; Tobiason, Fred L.

    1975-01-01

    Describes the construction of unit cells using clear plastic cubes which can be disassembled, and one inch cork balls of various colors, which can be cut in halves, quarters, or eighths, and glued on the inside face of the cube, thus simulating a unit cell. (MLH)

  8. Unit Cells

    ERIC Educational Resources Information Center

    Olsen, Robert C.; Tobiason, Fred L.

    1975-01-01

    Describes the construction of unit cells using clear plastic cubes which can be disassembled, and one inch cork balls of various colors, which can be cut in halves, quarters, or eighths, and glued on the inside face of the cube, thus simulating a unit cell. (MLH)

  9. Fuel Cells

    ERIC Educational Resources Information Center

    Hawkins, M. D.

    1973-01-01

    Discusses the theories, construction, operation, types, and advantages of fuel cells developed by the American space programs. Indicates that the cell is an ideal small-scale power source characterized by its compactness, high efficiency, reliability, and freedom from polluting fumes. (CC)

  10. Fuel Cells

    ERIC Educational Resources Information Center

    Hawkins, M. D.

    1973-01-01

    Discusses the theories, construction, operation, types, and advantages of fuel cells developed by the American space programs. Indicates that the cell is an ideal small-scale power source characterized by its compactness, high efficiency, reliability, and freedom from polluting fumes. (CC)

  11. Host cells and cell banking.

    PubMed

    Stacey, Glyn N; Merten, Otto-Wilhelm

    2011-01-01

    Gene therapy based on the use of viral vectors is entirely dependent on the use of animal cell lines, mainly of mammalian origin, but also of insect origin. As for any biotechnology product for clinical use, viral -vectors have to be produced with cells derived from an extensively characterized cell bank to maintain the appropriate standard for assuring the lowest risk for the patients to be treated. Although many different cell types and lines have been used for the production of viral vectors, HEK293 cells or their derivatives have been extensively used for production of different vector types: adenovirus, oncorectrovirus, lentivirus, and AAV vectors, because of their easy handling and the possibility to grow them adherently in serum-containing medium as well as in suspension in serum-free culture medium. Despite this, these cells are not necessarily the best for the production of a given viral vector, and there are many other cell lines with significant advantages including superior growth and/or production characteristics, which have been tested and also used for the production of clinical vector batches. This chapter presents basic -considerations concerning the characterization of cell banks, in the first part, and, in the second part, practically all cell lines (at least when public information was available) established and developed for the production of the most important viral vectors (adenoviral, oncoretroviral, lentiviral, AAV, baculovirus).

  12. BK channels modulate pre- and postsynaptic signaling at reciprocal synapses in retina

    PubMed Central

    Grimes, William N.; Li, Wei; Chávez, Andrés E.; Diamond, Jeffrey S.

    2009-01-01

    In the mammalian retina, A17 amacrine cells provide reciprocal inhibitory feedback to rod bipolar cells, thereby shaping the time course of visual signaling in vivo. Previous results indicate that A17 feedback can be triggered by Ca2+ influx through Ca2+ permeable AMPARs and can occur independently of voltage-gated Ca2+ (Cav) channels, whose presence and functional role in A17 dendrites have not been explored. Here, we combine electrophysiology, calcium imaging and immunohistochemistry to show that L-type Cav channels in rat A17 amacrine cells are located at the sites of reciprocal synaptic feedback, but their contribution to GABA release is diminished by large-conductance Ca2+-activated potassium (BK) channels, which suppress postsynaptic depolarization in A17s and limit Cav channel activation. We also show that BK channels, by limiting GABA release from A17s, regulate the flow of excitatory synaptic transmission through the rod pathway. PMID:19363492

  13. Cell polarity

    PubMed Central

    Romereim, Sarah M

    2011-01-01

    Despite extensive genetic analysis of the dynamic multi-phase process that transforms a small population of lateral plate mesoderm into the mature limb skeleton, the mechanisms by which signaling pathways regulate cellular behaviors to generate morphogenetic forces are not known. Recently, a series of papers have offered the intriguing possibility that regulated cell polarity fine-tunes the morphogenetic process via orienting cell axes, division planes and cell movements. Wnt5a-mediated non-canonical signaling, which may include planar cell polarity, has emerged as a common thread in the otherwise distinct signaling networks that regulate morphogenesis in each phase of limb development. These findings position the limb as a key model to elucidate how global tissue patterning pathways direct local differences in cell behavior that, in turn, generate growth and form. PMID:22064549

  14. Fatty acids are potential endogenous regulators of aldosterone secretion.

    PubMed

    Goodfriend, T L; Ball, D L; Elliott, M E; Morrison, A R; Evenson, M A

    1991-05-01

    Adrenal glomerulosa cells washed with delipidated albumin produced increased amounts of aldosterone in response to angiotensin-II (AII) or (Bu)2cAMP. Albumin treatment also increased binding of 125I-labeled AII to high affinity binding sites on adrenal cells. Lipid extracts of albumin solutions that were used to wash cells inhibited AII binding and aldosterone responses by washed glomerulosa cells. Chromatographic fractionation and mass spectroscopic analysis indicated that the inhibitors removed from cells by albumin were long chain fatty acids. Exogenous fatty acids not only inhibited AII binding, but they inhibited basal aldosterone production and increments in aldosterone caused by AII or dbcAMP, suggesting an effect on postreceptor steps in aldosteronogenesis. The most potent and most abundant fatty acids removed from adrenal cells were oleic, linoleic, and arachidonic. These fatty acids inhibited at micromolar concentrations in the absence of albumin and at somewhat higher concentrations in its presence. Cells that had been washed, then inhibited by exogenous oleic acid in vitro, were restored to their enhanced responsiveness by a second albumin wash, making it unlikely that cell damage is the mechanism of inhibition by fatty acids. Responses of fasciculata cells were not potentiated by albumin washes, and cortisol production was less sensitive than aldosterone production to exogenous fatty acids. Binding of ANP to glomerulosa cells was not affected by albumin or fatty acids. These results combined with clinical correlations make it plausible that unesterified fatty acids are naturally occurring regulators of the adrenal glomerulosa. Insulin's ability to lower plasma levels of fatty acids may be one way that it causes sodium retention.

  15. Fuel cells 101

    SciTech Connect

    Hirschenhofer, J.H.

    1999-07-01

    This paper discusses the various types of fuel cells, the importance of cell voltage, fuel processing for natural gas, cell stacking, fuel cell plant description, advantages and disadvantages of the types of fuel cells, and applications. The types covered include: polymer electrolyte fuel cell, alkaline fuel cell, phosphoric acid fuel cell; molten carbonate fuel cell, and solid oxide fuel cell.

  16. In vivo effects of glycine on retinal ultrastructure and averaged electroretinogram.

    PubMed

    Korol, S; Leuenberger, P M; Englert, U; Babel, J

    1975-10-31

    Glycine, when injected intravitreally, has an inhibitory action on the electroretinogram (ERG) of the rabbit resulting in a transient loss of the oscillatory potentials of the b-wave. This inhibitory action is reversible within 24 h after glycine injection. In autoradiographs, after [3H] glycine administration, the radioactive label is mainly found over the inner nuclear layer (INL) and inner plexiform layer (IPL) without predilection of specific cell types and/or synapses. Electron microscopy reveals cytopathological changes in amacrine cells, in particular their cell membranes. These changes are conspicuous especially 1-2 h after the injection and the cells become normal again within 24 h. It is concluded that glycine has an inhibitory action upon the rabbit ERG in vivo. This action, on the basis of our morphological observations under our experimental conditions, may be due to an overall somatic membrane action rather than to an action as inhibitory neurotransmitter at the synaptic level. The reversible cellular lesions of amacrine cells after glycine administration with a concomitant, transient loss of oscillatory potentials (OP) supports the hypothesis that the cellular origin of OP is situated in amacrine cells.

  17. Functional Networks of Parvalbumin-immunoreactive Neurons in Cat Auditory Cortex

    PubMed Central

    Yuan, Kexin; Shih, Jonathan Y.; Winer, Jeffery A.; Schreiner, Christoph E.

    2011-01-01

    Inhibitory interneurons constitute ~20% of auditory cortical cells and are essential for shaping sensory processing. Connectivity patterns of interneurons in relation to functional organization principles are not well understood. We contrasted the connection patterns of parvalbumin-immunoreactive cells in two functionally distinct cortical regions, the tonotopic, narrowly frequency-tuned module (cNB) of cat central primary auditory cortex (AI), and the non-tonotopic, broadly tuned second auditory field (AII). Interneuronal connectivity patterns and laminar distribution were identified by combining a retrograde tracer (WAHG) with labeling of the Ca2+ binding protein, parvalbumin (Pv), a marker for the GABAergic interneurons usually described physiologically as fast-spiking neurons. In AI, PV+ cells constituted 13% of the retrograde labeled cells in the immediate vicinity of the injection site, compared to 10% in AII. The retrograde labeling of Pv+ cells along isofrequency countours was confined to cNB. The spatial spread of labeled excitatory neurons in AI was more than twice that found for Pv+ cells. By contrast, in AII, the spread of Pv+ cells was nearly equal to that of excitatory neurons. The retrograde labeling of Pv+ cells was anisotropic in AI and isotropic in AII. This demonstration of inhibitory networks in auditory cortex reveals that the connections of cat GABAergic AI and AII cells follow different anatomical plans and, thus, contribute differently to the shaping of neural response properties. The finding that local connectivity of parvalbumin-immunoreactive neurons in AI is closely aligned with spectral integration properties demonstrates the critical role of inhibition in creating distinct processing modules in AI. PMID:21917816

  18. 9. ENGINE TEST CELL BUILDING INTERIOR. CELL ACCESS ELEVATOR, CELLS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. ENGINE TEST CELL BUILDING INTERIOR. CELL ACCESS ELEVATOR, CELLS 2 AND 4, BASEMENT LEVEL. LOOKING SOUTHEAST. - Fairchild Air Force Base, Engine Test Cell Building, Near intersection of Arnold Street & George Avenue, Spokane, Spokane County, WA

  19. Bi-Cell Unit for Fuel Cell.

    DTIC Science & Technology

    The patent concerns a bi-cell unit for a fuel cell . The bi-cell unit is comprised of two electrode packs. Each of the electrode packs includes an...invention relates in general to a bi-cell unit for a fuel cell and in particular, to a bi-cell unit for a hydrazine-air fuel cell .

  20. Tryptophan hydroxylase and serotonin receptor 1A expression in the retina of the sea lamprey.

    PubMed

    Cornide-Petronio, María Eugenia; Anadón, Ramón; Barreiro-Iglesias, Antón; Rodicio, María Celina

    2015-06-01

    The dual development of the retina of lampreys is exceptional among vertebrates and offers an interesting EvoDevo (evolutionary developmental biology) model for understanding the origin and evolution of the vertebrate retina. Only a single type of photoreceptor, gangl