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Temporally precise in vivo control of intracellular signaling pathways through optogene expression

Temporally precise in vivo control of intracellular signaling pathways through optogene expression. By: Michael DeSalvio Bio 570 September 24, 2010. Agenda. Definition and Clarification Optogenetics Opsin GPCR Overview Goals Validation Test Specificity Test Test in Neural Tissue

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Temporally precise in vivo control of intracellular signaling pathways through optogene expression

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  1. Temporally precise in vivo control of intracellular signaling pathways through optogeneexpression By: Michael DeSalvio Bio 570 September 24, 2010

  2. Agenda • Definition and Clarification • Optogenetics • Opsin • GPCR • Overview • Goals • Validation Test • Specificity Test • Test in Neural Tissue • Conclusions

  3. Definitions • What is optogenetics? • Optical and genetic techniques used in conjunction to target and probe mammalian neural circuits in millisecond timescale to better understand spatiotemporal relationships within the brain? • What is Opsin? • A group of light sensitive 35-55 kDA membrane-bound GPCR’s in the retinylidene protein family of photoreceptor cells located in the retina. • What is GPCR? • A transmembrane protein consisting of 7 transmembranereceptor domains responsible for key signal transduction pathways.

  4. Signal Transduction Background

  5. Goals • Make a GPCR-OpsinChimaera and specifically target cells in vivo • Differentiate between pulsatile and tonic modulation • Determine syncrony between different modulatory systems at varying timeframes • Determine if retinal cofactors must be used in mammalian cells • Verify successful expression of OptoXRs by checking for products of biochemical signaling cascades • Ser 133-phosphorylated CREB, cAMP, cGTPand Ca2+

  6. Making a Chimaera • Aligned Human, Hamster and Bovine adrenergic receptors • Replaced intracellular loops of rhodopsin with intracellular loops of specific adrenergic receptors resulting from alignment • Exchanges made to transform GPCR of Gt (Bovine Rhodopsin “rhod”) and combine with opsin to form chimaera

  7. Rhodopsin Structure

  8. Validation Test • Transfect HEK cells with opto-a1AR (expected to recruit [Ca2+] via Gq • Image for presence of [Ca2+]up-regulation to confirm • Transfect HEK cells with opto-β2AR (expected to recruit cAMP via Gs • Image for presence of cAMP up-regulation to confirm

  9. Specificity Test • TransfectedHEK cells illuminated with green light • 3 mW mm-1 504 nm for 60 sec • Lysed and analyzed for cAMP, cGMP and IP1 via immuno assays • Canonical results: • opto-β2AR transfected cells showed significant cAMP levels as compared to β2AR wild-type cells without IP3 recruitment • opto-a1AR transfected cells showed significant up-regulation of IP3 when compared to a1AR wild-type cells • Non-canonical results: • Optical stimulation was not sufficient to modulate cGMP levels • Specificity indicates an ability to integrate over range of biologically suitable light fluxes to activate non-canonical pathways

  10. Test in Neural Tissue • Used a Lentiviral vector with Synapsin-I promotor to deliver optoXR gene • Targets biochemical modulation to local neurons only • Excludes Gs/Gq responsive cells such as glia and endothelial cells • Stereotactically injected into nucleus accumbens of adult mice • Targeting biochemical modulation to neurons with somatodendritic compartments • ~95% GABAergic neurons • Pre-synaptic terminals • After 2 weeks, accute coronal slices of accumbens were prepared in artificial CSF • Optically stimulated for 10min, immediately fixed and stained for Ser 133-phosphorylated CREB • Biochemical indicator of cAMP and [Ca2+]

  11. Neural Tissue Tests cont… • No supplementation of retinoids were given but significant amounts of pCREB were observed • Indicates that adequate amounts of retinoids exist in mammalian cells • Next, measured electrical activity in neuronal tissue • Used an Optrodeto measure multiunit in vivo neuronal firing • No differences of firing rates between dark and photo-stimulated cells • opto-β2AR transfected cells showed decreased network firing when stimulated (supports current research) • opto-a1AR transfected cells showed increased firing when stimulated

  12. Conclusions • OptoXR’s can be functionally expressed in vivo • Permits differential photo-activatable control of intracellular cascades • Modulate network physiology

  13. Behavioral Studies • Goals: to show a correlation between photo-stimulation of OptoXR’s and physical behaviors in mice through operant conditioning • What is operant conditioning? • Altering behavioral decisions using a rewards/punishment system • Parenting 101 • Drug Addiction • Study design: • 3 trials • 2 blind scorers • Conditioned chamber

  14. Behavioral Studies • Day 1 • Mouse free moving, no conditioning • Day 2 • Mouse receives photo-stimulation upon entering conditioned chamber • Signal approximates intensity of monoaminergic input during strong reward • Day 3 • Mouse free moving, no conditioning • opto-a1AR show higher preference for conditioned chamber based on reward • Results reproducible across 2 cohorts of opto-a1AR expressing mice.

  15. Preference after conditioning

  16. Questions

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