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– Conclusions

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.

Signal Transduction Background

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 Ca2+

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

Rhodopsin Structure

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

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 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

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+]

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-β2AR transfected cells showed decreased network firing when

stimulated (supports current research)– opto-a1AR transfected cells showed increased firing when stimulated

Conclusions

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

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

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.

Preference after conditioning

Questions