in vivo control of intracellular signaling pathways through optogene expression By Michael DeSalvio Bio 570 September 24 2010 Agenda Definition and Clarification Optogenetics Opsin ID: 238088
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Slide1
Temporally precise in vivo control of intracellular signaling pathways through optogene expression
By: Michael DeSalvio
Bio 570
September 24, 2010Slide2
AgendaDefinition and ClarificationOptogeneticsOpsinGPCROverview
Goals
Validation Test
Specificity Test
Test in Neural Tissue
ConclusionsSlide3
DefinitionsWhat 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. Slide4
Signal Transduction BackgroundSlide5
GoalsMake a GPCR-Opsin Chimaera and specifically target cells in vivoDifferentiate 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 cascadesSer 133-phosphorylated CREB, cAMP, cGTP and Ca2+Slide6
Making a ChimaeraAligned Human, Hamster and Bovine adrenergic receptorsReplaced 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 chimaeraSlide7
Rhodopsin StructureSlide8
Validation TestTransfect HEK cells with opto-a1AR (expected to recruit [Ca2+] via Gq
Image for presence of [Ca
2+
]up-regulation to confirm
Transfect HEK cells with
opto
-
β
2
AR (expected to recruit cAMP via GsImage for presence of cAMP up-regulation to confirmSlide9
Specificity TestTransfected HEK cells illuminated with green light3 mW mm-1
504 nm for 60 sec
Lysed
and analyzed for
cAMP
,
cGMP
and IP
1
via immuno assaysCanonical results:opto-β2AR 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 pathwaysSlide10
Test in Neural TissueUsed 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 cellsStereotactically injected into nucleus accumbens of adult miceTargeting 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+
]Slide11
Neural Tissue Tests cont…No supplementation of retinoids were given but significant amounts of pCREB were observedIndicates 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 stimulatedSlide12
ConclusionsOptoXR’s can be functionally expressed in vivoPermits differential photo-activatable control of intracellular cascadesModulate network physiologySlide13
Behavioral StudiesGoals: to show a correlation between photo-stimulation of OptoXR’s and physical behaviors in mice through operant conditioningWhat is operant conditioning?Altering behavioral decisions using a rewards/punishment system
Parenting
101
Drug Addiction
Study design:
3 trials
2 blind scorers
Conditioned chamberSlide14
Behavioral StudiesDay 1Mouse free moving, no conditioningDay 2Mouse receives photo-stimulation upon entering conditioned chamberSignal 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.Slide15
Preference after conditioningSlide16
Questions