Download OptoXR control - Mike DeSalvio

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 133phosphorylated 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