Quercetin protects ARPE-19 cells against photic stress mediated by the products of rhodopsin photobleaching.

Although the primary biological function of retina photoreceptors is to absorb light and provide visual information, exposure to intense light could increase the risk of phototoxic reactions mediated by rhodopsin photobleaching products (RPBP) that might accumulate in photoreceptor outer segments (POS). Here we investigated whether quercetin can modify the phototoxic potential of RPBP under in vitro photic stress conditions. ARPE-19 cells or quercetin enriched cultures pre-loaded with rhodopsin-rich POS isolated from bovine retinas were irradiated with green light to photobleach rhodopsin, and subsequently with blue light. Survival of cells was determined by MTT assay and propidium iodide staining. Changes in mitochondrial membrane potential (MMP) were assessed by JC-1 staining. Protein hydroperoxides, formed by photosensitized oxidation, mediated by RPBP, were analyzed in cells and in a model system with bovine serum albumin (BSA), using the coumarin boronic acid fluorogenic probe. The effect of photic stress on specific phagocytosis of RPE cells was determined by flow cytometry. Photoreactivity of POS with and without quercetin was analyzed by EPR oximetry and EPR spin trapping. Cytotoxicity measurements and MMP analyses confirmed that supplementation with quercetin protected ARPE-19 cells against photic stress mediated by rhodopsin-rich POS. Quercetin significantly reduced the inhibitory effect of RPBP-mediated stress on POS phagocytosis and the RPBP ability to photooxidize cellular proteins or BSA. The data support the hypothesis that quercetin may efficiently diminish the phototoxic action of retinoids, necessary for restoring the phagocytic function of ARPE-19 cells.

Neuromodulatory Regulation of Behavioral Individuality in Zebrafish.

Inter-individual behavioral variation is thought to increase fitness and aid adaptation to environmental change, but the underlying mechanisms are poorly understood. We find that variation between individuals in neuromodulatory input contributes to individuality in short-term habituation of the zebrafish (Danio Rerio) acoustic startle response (ASR). ASR habituation varies greatly between individuals, but differences are stable over days and are heritable. Acoustic stimuli that activate ASR-command Mauthner cells also activate dorsal raphe nucleus (DRN) serotonergic neurons, which project to the vicinity of the Mauthner cells and their inputs. DRN neuron activity decreases during habituation in proportion to habituation and a genetic manipulation that reduces serotonin content in DRN neurons increases habituation, whereas serotonergic agonism or DRN activation with ChR2 reduces habituation. Finally, level of rundown of DRN activity co-segregates with extent of behavioral habituation across generations. Thus, variation between individuals in neuromodulatory input contributes to individuality in a core adaptive behavior. VIDEO ABSTRACT.

Activation of GABAergic neurons in the interpeduncular nucleus triggers physical nicotine withdrawal symptoms.

BACKGROUND: Chronic exposure to nicotine elicits physical dependence in smokers, yet the mechanism and neuroanatomical bases for withdrawal symptoms are unclear. As in humans, rodents undergo physical withdrawal symptoms after cessation from chronic nicotine characterized by increased scratching, head nods, and body shakes. RESULTS: Here we show that induction of physical nicotine withdrawal symptoms activates GABAergic neurons within the interpeduncular nucleus (IPN). Optical activation of IPN GABAergic neurons via light stimulation of channelrhodopsin elicited physical withdrawal symptoms in both nicotine-naive and chronic-nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blockade of IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the ß4 subunit were upregulated in somatostatin interneurons clustered in the dorsal region of the IPN. Blockade of these receptors induced withdrawal signs more dramatically in nicotine-dependent compared to nicotine-naive mice and activated nonsomatostatin neurons in the IPN. CONCLUSIONS: Together, our data indicate that therapeutic strategies to reduce IPN GABAergic neuron excitability during nicotine withdrawal, for example, by activating nicotinic receptors on somatostatin interneurons, may be beneficial for alleviating withdrawal symptoms and facilitating smoking cessation.

Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging.

The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.

Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo.

The present study describes a robust 50-fold increase in rhodopsin gene transcription by cAMP in cultured retinal precursor cells of chicken embryo. Retinal cells isolated at embryonic day 8 (E8) and cultured for 3 days in serum-supplemented medium differentiated mostly into red-sensitive cones and to a lesser degree into green-sensitive cones, as indicated by real-time RT-PCR quantification of each specific opsin mRNA. In contrast, both rhodopsin mRNA concentration and rhodopsin gene promoter activity required the presence of cAMP-increasing agents [forskolin and 3-isobutyl-1-methylxanthine (IBMX)] to reach significant levels. This response was rod-specific and was sufficient to activate rhodopsin gene transcription in serum-free medium. The increase in rhodopsin mRNA levels evoked by a series of cAMP analogs suggested the response was mediated by protein kinase A, not by EPAC. Membrane depolarization by high KCl concentration also increased rhodopsin mRNA levels and this response was strongly potentiated by IBMX. The rhodopsin gene response to cAMP-increasing agents was developmentally gated between E6 and E7. Rod-specific transducin alpha subunit mRNA levels also increased up to 50-fold in response to forskolin and IBMX, while rod-specific phosphodiesterase-VI and rod arrestin transcripts increased 3- to 10-fold. These results suggest a cAMP-mediated signaling pathway may play a role in rod differentiation.

Inhibition of the visual cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night blindness in isotretinoin therapy.

Isotretinoin (13-cis retinoic acid) is frequently prescribed for severe acne [Peck, G. L., Olsen, T. G., Yoder, F. W., Strauss, J. S., Downing, D. T., Pandya, M., Butkus, D. & Arnaud-Battandier, J. (1979) N. Engl. J. Med. 300, 329-333] but can impair night vision [Fraunfelder, F. T., LaBraico, J. M. & Meyer, S. M. (1985) Am. J. Ophthalmol. 100, 534-537] shortly after the beginning of therapy [Shulman, S. R. (1989) Am. J. Public Health 79, 1565-1568]. As rod photoreceptors are responsible for night vision, we administered isotretinoin to rats to learn whether night blindness resulted from rod cell death or from rod functional impairment. High-dose isotretinoin was given daily for 2 months and produced systemic toxicity, but this caused no histological loss of rod photoreceptors, and rod-driven electroretinogram amplitudes were normal after prolonged dark adaptation. Additional studies showed, however, that even a single dose of isotretinoin slowed the recovery of rod signaling after exposure to an intense bleaching light, and that rhodopsin regeneration was markedly slowed. When only a single dose was given, rod function recovered to normal within several days. Rods and cones both showed slow recovery from bleach after isotretinoin in rats and in mice. HPLC analysis of ocular retinoids after isotretinoin and an intense bleach showed decreased levels of rhodopsin chromophore, 11-cis retinal, and the accumulation of the biosynthetic intermediates, 11-cis and all-trans retinyl esters. Isotretinoin was also found to protect rat photoreceptors from light-induced damage, suggesting that strategies of altering retinoid cycling may have therapeutic implications for some forms of retinal and macular degeneration.

High-level inducible expression of visual pigments in transfected cells.

A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.

Characterization of the Xenopus rhodopsin gene.

The abundant Xenopus rhodopsin gene and cDNA have been cloned and characterized. The gene is composed of five exons spanning 3.5 kilobase pairs of genomic DNA and codes for a protein 82% identical to the bovine rhodopsin. The cDNA was expressed in COS1 cells and regenerated with 11-cis-retinal, forming a light-sensitive pigment with maximal absorbance at 500 nm. Both Southern blots and polymerase chain reaction amplification of intron 1 revealed multiple products, indicating more than one allele for the rhodopsin gene. Comparisons with other vertebrate rhodopsin 5 upstream sequences showed significant nucleotide homologies in the 200 nucleotides proximal to the transcription initiation site. This homology included the TATA box region, Ret 1/PCE1 core sequence (CCAATTA), and surrounding nucleotides. To functionally characterize the rhodopsin promoter, transient embryo transfections were used to assay transcriptional control elements in the 5 upstream region using a luciferase reporter. DNA sequences encompassing -5500 to +41 were able to direct luciferase expression in embryo heads. Reporter gene expression was also observed in embryos microinjected with reporter plasmids during early blastomere stages. These results locate transcriptional control elements upstream of the Xenopus rhodopsin gene and show the feasibility of embryo transfections for promoter analysis of rod-specific genes.

Maturation of major Drosophila rhodopsin, ninaE, requires chromophore 3-hydroxyretinal.

Opsin expression is extremely suppressed by carotenoid deprivation in Drosophila. Carotenoid replacement in deprived flies promotes the recovery of visual pigment with an increase in opsin, as well as the chromophore 11-cis-3-hydroxyretinal. Here, we show that opsin mRNA and opsin peptide in an intermediate step of posttranslational processing were present in carotenoid-deprived flies. By supplementing chromophore to photoreceptor cells, intermediate opsin was made mature. During this process, opsin peptide underwent multiple modifications involving glycosylation. Based on these results, we present a novel mechanism of protein regulatory expression; that is, chromophore posttranslationally controls the expression of apoprotein by promoting its maturation.