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Steven Barnes

Publications and source records attributed to Steven Barnes.

At least 19 recordsLinked to original sources
PubMed2006

Cannabinoid receptor-mediated inhibition of calcium signaling in rat retinal ganglion cells.

PURPOSE: The physiological actions of CB(1) cannabinoid receptors (CB(1)Rs) in mammalian retina have yet to be fully described in all cell types. Here we investigate the actions of CB(1)R activation on high-voltage-activated (HVA) Ca(2+) channel currents in purified cultures of rat retinal ganglion cells (RGCs). METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry were used to determine the presence of CB(1)R mRNA and protein in a purified RGC culture generated from neonatal rats using a two-step panning procedure. Ruptured-patch whole-cell voltage clamp was used to test the effect of CB(1)R agonists (WIN 55,212-2) and antagonists (SR141716A, AM281) on HVA Ca(2+) channel currents. RESULTS: RT-PCR analysis confirmed CB(1)R mRNA in cultured RGCs and immunocytochemistry for CB(1)R protein revealed labeling in both the cell body and neurites of isolated RGCs. Patch-clamp recording from cultured rat RGCs showed that the CB(1)R agonist WIN 55,212-2 inhibited HVA Ca(2+) channel currents up to 50% in a concentration-dependent manner (0.5, 1, and 5 muM). The Ca(2+) channel current inhibition by WIN 55,212-2 was blocked by CB(1)R antagonists AM281 and SR141716. CONCLUSIONS: Activation of CB(1)Rs in cultured RGCs inhibits HVA Ca(2+) channel currents. These data show that cannabinoids can modify the excitability of RGCs and could affect retinal output. This finding has implications for retinal signal processing as it suggests that endogenous cannabinoids have inhibitory effects on RGCs and that exogenous cannabinoids could modulate retinal function by this pathway as well.

Animals↗
PubMed2006

Calcium-activated chloride channels in müller cells acutely isolated from tiger salamander retina.

Ca(2+)-activated chloride channels were identified with whole-cell patch-clamp recording techniques in salamander retinal Müller cells. Cl(Ca) channels were activated by membrane depolarizations that elicited Ca2+ influx or the application of the Ca2+ ionophore, ionomycin. The Ca channel blocker, Cd2+, abolished the Cl(Ca) channel tail currents. Increasing the duration of the depolarizing pulse resulted in enhancement of the Cl(Ca) channel tail current. Repetitive depolarizations with rapid pulses to +20 mV produced a buildup of I(Cl(Ca)), which reversed at 0 mV in symmetrical [Cl-] and at -40 mV when intracellular [Cl-] was reduced to 10% of the external concentration. I(Cl(Ca)) was blocked by the Cl channel blocker niflumic acid, while niflumic acid had no effect on voltage-gated Ca channels. These results offer the first demonstration of Cl(Ca) channels in a nonastrocytic glial cell and expand our understanding of the functional capacities of retinal glial cells.

Ambystoma↗
PubMed2005

Mutation of the calcium channel gene Cacna1f disrupts calcium signaling, synaptic transmission and cellular organization in mouse retina.

Retinal neural transmission represents a key function of the eye. Identifying the molecular components of this vital process is helped by studies of selected human genetic eye disorders. For example, mutations in the calcium channel subunit gene CACNA1F cause incomplete X-linked congenital stationary night blindness (CSNB2 or iCSNB), a human retinal disorder with abnormal electrophysiological response and visual impairments consistent with a retinal neurotransmission defect. To understand the subcellular basis of this retinal disorder, we generated a mouse with a loss-of-function mutation by inserting a self-excising Cre-lox-neo cassette into exon 7 of the murine orthologue, Cacna1f. Electroretinography of the mutant mouse revealed a scotopic a-wave of marginally reduced amplitude compared with the wild-type mouse and absence of the post-receptoral b-wave and oscillatory potentials. Cone ERG responses together with visual evoked potentials and multi-unit activity in the superior colliculus were also absent. Calcium imaging in Fluo-4 loaded retinal slices depolarized with KCl showed 90% less peak signal in the photoreceptor synapses of the Cacna1f mutant than in wild-type mice. The absence of post-receptoral ERG responses and the diminished photoreceptor calcium signals are consistent with a loss of Ca((2+)) channel function in photoreceptors. Immunocytochemistry showed no detectable Ca(v)1.4 protein in the outer plexiform layer of Cacna1f-mutant mice, profound loss of photoreceptor synapses, and abnormal dendritic sprouting of second-order neurons in the photoreceptor layer. Together, these findings in the Cacna1f-mutant mouse reveal that the Ca(v)1.4 calcium channel is vital for the functional assembly and/or maintenance and synaptic functions of photoreceptor ribbon synapses. Moreover, the outcome of this study provides critical clues to the pathophysiology of the human retinal channelopathy of X-linked incomplete CSNB.

Animals↗
PubMed2005

Proton-mediated feedback inhibition of presynaptic calcium channels at the cone photoreceptor synapse.

Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K(+) increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K(+)-induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2005

High-voltage-activated calcium channels in Muller cells acutely isolated from tiger salamander retina.

Muller cells mediate retinal function by stabilizing the ionic environment and signal glial network activity via calcium waves. Using whole-cell patch clamp recording, we describe a high-voltage-activated, slowly inactivating Ca channel current in isolated salamander Muller cells that has unusual pharmacological properties. The Ca channel current has an activation midpoint of approximately -8 mV and an inactivation midpoint of approximately -26 mV in 10 mM Ba2+. The time constant for inactivation is approximately 380 ms at potentials positive to zero. The current is blocked by Cd2+ with an EC50 of <100 nM. nisoldipine (10 microM) blocks approximately 50%, while nifedipine (1 microM), diltiazem (20 microM), and verapamil (50 microM) each block one-third of the current. In contrast to its typical actions, BayK 8644 blocks the current by approximately 25%. Blockers of other Ca channel subtypes were also tested: omega-agatoxin IVA (200 nM) blocked only 13% of the Ca channel current, while omega-conotoxin GVIA (1 microM) blocked 84% of the current. Immnohistochemistry supported the presence of alpha1A, alpha1B, alpha1C, and alpha1D Ca channel subunits. Mapping of dihydropyridine-binding sites with DM-BODIPY revealed a distribution of channels over the entire membrane of the Muller cell with a higher density at the apical region. Overall, these observations suggest either the presence of a mix of L- and N-type Ca channels or a single, unconventional HVA Ca channel subtype sharing L- and N-type Ca channel characteristics.

Ambystoma↗
PubMed2004

Carbenoxolone inhibition of voltage-gated Ca channels and synaptic transmission in the retina.

We show that carbenoxolone, a drug used to block hemichannels in the retina to test the ephaptic model of horizontal cell inhibitory feedback, has strong inhibitory effects on voltage-gated Ca channels. Carbenoxolone (100 microM) reduced photoreceptor-to-horizontal cell synaptic transmission by 92%. Applied to patch-clamped, isolated cone photoreceptors, carbenoxolone inhibited Ca channels with an EC(50) of 48 microM. At 100 microM, it reduced cone Ca channel current by 37%, reduced depolarization-evoked [Ca(2+)] signals in fluo-4 loaded retinal slices by 57% and inhibited Ca channels in Müller cells by 52%. A synaptic transfer model suggests that the degree of block of Ca channels accounts for the reduction in synaptic transmission. These results suggest broad inhibitory actions for carbenoxolone in the retina that must be considered when interpreting its effects on inhibitory feedback.

Ambystoma↗
PubMed2004

Reciprocal modulation of calcium dynamics at rod and cone photoreceptor synapses by nitric oxide.

The abundance of nitric oxide (NO) synthesizing enzymes identified in the vertebrate retina highlight the importance of NO as a signaling molecule in this tissue. Here we describe opposing actions of NO on the rod and cone photoreceptor synapse. Depolarization-induced increases of calcium concentration in rods and cones were enhanced and inhibited, respectively, by the NO donor S-nitrosocysteine. NO suppressed calcium current in cones by decreasing the maximum conductance, whereas NO facilitated rod Ca channel activation. NO also activated a nonselective voltage-independent conductance in both rods and cones. Suppression of NO production in the intact retina with N(G)-nitro-l-arginine favored cone over rod driven postsynaptic signals, as would be expected if NO enhanced rod and suppressed cone synaptic activity. These findings may imply involvement of NO in regulating the strength of rod and cone pathways in the retina during different states of adaptation.

Ambystoma↗
PubMed2004

Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane.

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl(-) conductance (I(Cl,swell)), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I(Cl,swell) following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl(-) current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I(Cl,swell). Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I(Cl,swell). Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I(Cl,swell) following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.

Animals↗
PubMed2004

Adenosine A1-receptor modulation of glutamate-induced calcium influx in rat retinal ganglion cells.

PURPOSE: Although adenosine receptors (A(1)-Rs and A(2)-Rs) have been identified in the mammalian retina, the role of adenosine in this tissue is not fully understood. The purpose of this work was to investigate the action of adenosine on glutamate-induced calcium influx in rat retinal ganglion cells (RGCs) and to determine whether adenosine modulates RGC voltage-gated calcium channels. METHODS: Purified RGC cultures were generated from neonatal rats with a two-step panning procedure. Isolated RGCs were loaded with the ratiometric calcium-indicator dye fura-2, and the effect of adenosine (and related agonists and antagonists) on intracellular calcium levels ([Ca(2+)](i)) during exposure to glutamate (10 microM with 10 microM glycine) was assessed. The effect of adenosine on calcium channel currents was also studied in isolated RGCs with whole-cell patch-clamp techniques. In addition, the effect of adenosine on [Ca(2+)](i) was investigated in fura dextran-loaded RGCs in an intact adult rat retina preparation. RESULTS: In isolated RGCs, adenosine (10 and 100 microM) significantly reduced the glutamate-induced increase in [Ca(2+)](i) ( approximately 30%). The effect of adenosine was blocked by the A(1)-R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), but not by the A(2)-R antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Adenosine (10 microM) inhibited calcium channel currents by 43%, and again this effect was blocked by DPCPX, but not DMPX. Adenosine (100 microM) also significantly reduced the elevation of [Ca(2+)](i) in RGCs in the intact retina during exposure to N-methyl-d-aspartate (NMDA; 100 microM). CONCLUSIONS: Adenosine can inhibit glutamate-induced calcium influx and voltage-gated calcium currents in rat RGCs through A(1)-R activation. This work supports a role for adenosine as a neuromodulator of mammalian RGCs.

Adenosine↗
PubMed2003

Protons and calcium alter gating of the hyperpolarization-activated cation current (I(h)) in rod photoreceptors.

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I(h), in rod photoreceptors. I(h) is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I(h) activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca(2+)] from 3 to 20 mM resulted in a large (approximately 17 mV) positive shift in I(h) activation. External [Ca(2+)] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (-9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I(h) activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I(h) activation.

Ambystoma↗
PubMed2003

Inhibitory action of diltiazem on voltage-gated calcium channels in cone photoreceptors.

The benzothiazepine, diltiazem, is commonly used as an inhibitor of vascular L-type Ca channels, and is a clinically important anti-anginal and antihypertensive medication. In the retina, diltiazem also inhibits cyclic-nucleotide gated (CNG) channels, including the cGMP-gated channels in photoreceptors, and has been suggested to be a neuroprotectant in an animal model of retinitis pigmentosa, a degenerative disease of photoreceptors. In contrast to CNG channels, the actions of diltiazem on photoreceptor Ca channels have not been studied. We show that D-cis-diltiazem can block Ca channels in cone photoreceptors and that the potency and efficacy of cone photoreceptor Ca channel inhibition by this drug is unconventional. Over the concentration range of 5-500 microM diltiazem, the dose response curve was biphasic with a high affinity saturation level of approximately 30% block in the 20-50 microM range (IC(50)=4.9 microM) and a low affinity saturation block (near 100%) with concentrations up to 500 microM (IC(50)=100.4 microM). The degree of block was found to be equivalent when Bay K 8644 was used to increase Ca channel current, indicating that the levels of block do not result from multiple Ca channel subtypes having differing sensitivities to diltiazem. Calcium imaging showed that the relatively low efficacy of the high-affinity Ca channel block was not due to the species of charge-carrying divalent cation nor that it was associated with dialysis of cellular contents. These data contribute to an emerging perspective that the photoreceptor Ca channel has properties unique from other L-type channels, an important consideration should these channels become a target for testing putative neuroprotective therapies.

Animals↗
PubMed2003

Regulation of alpha1G T-type calcium channel gene (CACNA1G) expression during neuronal differentiation.

Down-regulation of T-type Ca channel current and mRNA occurs following differentiation of Y79 retinoblastoma cells. To understand how the decrease in expression is linked to cell differentiation, we examined transcriptional regulation of the Cav3.1 Ca channel gene, CACNA1G. We identified two putative promoters (A and B) in 1.3 kb of cloned genomic DNA. Reverse transcriptase-polymerase chain reaction and 5' rapid amplification of cDNA ends-polymerase chain reaction analyses demonstrated that two transcripts with different 5' untranslated regions are generated by different transcription start sites, with promoter A favoured in undifferentiated cells and promoter B favoured in differentiated cells. Functional analyses of the promoter sequence revealed that both promoters are active. Enhancer and repressor sequences were identified upstream of promoter A and B, respectively. These results suggest that the down-regulation of alpha1G mRNA in differentiated Y79 cells is mediated primarily by decreased activity of promoter A, which could occur in conjunction with repression of the activity of promoter B. The decrease in T-type Ca channel expression in Y79 cells may be an essential signal affecting phenotypic maturation and expression of other ion channel subtypes in the differentiated cells.

Amino Acid Sequence↗
PubMed2002

Plasticity-driven gene expression in the rat retina.

Animals exposed to an enriched environment display features of neural plasticity such as an increased brain volume, enhanced number of dendritic spines, as well as enlarged synapses. Here we report the first description of molecular plasticity in the mammalian retina, as revealed by gene expression. A marked upregulation of both NGFI-A and Arc, two candidate-plasticity genes, was observed in adult rats that had been exposed to an enriched environment for 3 weeks. This increase was paralleled by an increase in the expression of the late genes GAP-43 and Synapsin I, which also indicated changes in retinal connectivity. Our results suggest that both NGFI-A and Arc may regulate mechanisms of plasticity that had been invoked by heightened complexity of the visual environment.

Animals↗
PubMed2002

Calcium channels at the photoreceptor synapse.

Presynaptic Ca2+ channels mediate early stages of visual information processing in photoreceptors by facilitating the release of neurotransmitter and by receiving modulatory input that alters transmission. Two types of L-type Ca2+ channels, composed of alpha1F and alpha1D subunits and having similar biophysical andpharmacological properties, appear to form the principle voltage-dependent Ca2+ influx pathways in rods and cones, respectively. The role played by these channels in neurotransmitter release at these graded potential, non-spiking synapses, has been well described. The channels mediate sustained glutamate release in darkness where the cells rest at potentials near -40 mV, and signal increases in light intensity as the cells hyperpolarize negative to this value. Synaptic modulation and integration mediated by these channels has not yet been as fully described but appears to involve GABA, nitric oxide (NO), glutamate, and dopamine. Ca2+ permeable cyclic nucleotide gated (CNG) channels appear to have supporting roles at the photoreceptor output synapse and may transduce NO signals from other cells by either directly permitting Ca2+ influx or by providing depolarizing influences that gate voltage dependent Ca2+ channels.

Animals↗