1. Temporally precise in vivo control of intracellular signaling pathways through optogene expression 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 transmembrane receptor domains responsible for key signal transduction pathways.

4. Signal Transduction Background

5. Goals Make a GPCR-Opsin Chimaera 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, cGTP and Ca 2+

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-a 1 AR (expected to recruit [Ca 2+ ] via G q – Image for presence of [Ca 2+ ]up-regulation to confirm Transfect HEK cells with opto-β 2 AR (expected to recruit cAMP via G s – Image for presence of cAMP up-regulation to confirm

9. Specificity Test Transfected HEK cells illuminated with green light – 3 mW mm -1 504 nm for 60 sec – Lysed and analyzed for cAMP, cGMP and IP 1 via immuno assays Canonical results: – opto-β 2 AR transfected cells showed significant cAMP levels as compared to β 2 AR wild-type cells without IP 3 recruitment – opto-a 1 AR transfected cells showed significant up-regulation of IP 3 when compared to a 1 AR 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 G s /G q 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 [Ca 2+ ]

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 Optrode to measure multiunit in vivo neuronal firing – No differences of firing rates between dark and photo-stimulated cells – opto-β 2 AR transfected cells showed decreased network firing when stimulated (supports current research) – opto-a 1 AR 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-a 1 AR show higher preference for conditioned chamber based on reward Results reproducible across 2 cohorts of opto-a 1 AR expressing mice.

15. Preference after conditioning

16. Questions