Light Up Your Research: Use and Selection of Optogenetic Mouse Models 1 Presented by: Brian W. Soper, PhD Technical Information Services Hosted by Charles River, exclusive distributor of JAX ® Mice in 27 European Countries
Light Up Your
Research: Use and Selection of
Optogenetic Mouse
Models
1
Presented by:
Brian W. Soper, PhD
Technical Information Services
Hosted by Charles River, exclusive
distributor of JAX® Mice in 27 European
Countries
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Online Resources to Expedite
Research
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Online Resources to Expedite
Research
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Online Resources to Expedite
Research
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www.informatics.jax.org
Mouse Phenome Database
www.jax.org/phenome
And many more unique
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Optogenetics Mouse Models: Presentation Overview
7
Historical relationship between light tools & neuroscience
Optogenetics defined
Benefits of optogenetic tools
Origins of optogenetics and opsin subtypes
Opsins in the mammalian system
JAX selection of optogenetic transgenic & cre-conditional strains
o In vitro and in vivo data
Additional optogenetic strains
Finding JAX optogenetic strains
Optogenetics Resource |
Using Light to Understand the Brain
8
Wilt et al. 2009. Annu Rev Neurosci 32:435-506. PMID:19555292
De Carlo and Borrel. 2007. Brain Res Rev 55: 8-16 PMID:17490748
http://www.cell.com/cell_picture_show-weaving
Light Microscope
Confocal & 2-photon Microscopy
http://mcb.berkeley.edu/labs2/robey/content/2-photon-imaging
http://web.mit.edu/newsoffice/2011/illuminating-brain-0128.html
Glutamate Uncaging
http://www.rsc.org/ejga/CC/2005/b504922a-ga.gif
Fluorescence Labeling
Optogenetics Resource |
Optogenetics
Combining Light & Genetics
Optogenetics
Light-induced control of neuron activity via
genetically introduced microbial opsins
http://www.psychologicalscience.org/redesign/
wp-content/uploads/2012/02/neuron.jpg
9 Optogenetics Resource |
Traditional Methods for Understanding Brain
Function Unsatisfactory
10
Deisseroth 2011. Nature Methods 8(1):26-29. PMID:21191368
Electrical Stimulation
Marin
a C
orr
al
Chemical Stimulation
http://www.chem.duke.edu/~wwarren/neuroimaging.php
Optogenetics Resource |
o Poor spatial resolution o Poor temporal resolution
Optogenetics: Improved Spatial and Temporal Resolution
11
Deisseroth 2011. Nature Methods 8(1):26-29. PMID:21191368
Improved spatial resolution
o Only specific neural populations stimulated
Improved temporal resolution
o Activation by light faster than by chemicals
Optogenetics Resource |
Original Function of Opsins
12
Berthold et al. 2008. The Plant Cell 20(6):1665-77. PMID:18552201
Phototaxis response to light by unicellular green algae
(Chlamydomonas reinhardtii)
http://remf.dartmouth.edu/images/algae
SEM/source/2.html
Optogenetics Resource |
Categories of Opsins
13
Adapted from Mei & Zhang 2012. Biol Psychiatry 71(12):1033-8. PMID:22480664
Channelrhodopsin
Halorhodopsin
Bacteriorhodopsin
(Archaerhodopsin)
OptoXR
Optogenetics Resource |
Categories of Opsins
14
Adapted from Mei & Zhang 2012. Biol Psychiatry 71(12):1033-8. PMID:22480664
Channelrhodopsin
Halorhodopsin
OptoXR
all-trans-Retinal
Optogenetics Resource |
Bacteriorhodopsin
(Archaerhodopsin)
Channelrhodopsin
15
Mei & Zhang 2012. Biol Psychiatry 71(12):1033-8. PMID:22480664
Yizhar et al. 2011. Cold Spring Harb Protoc (3). PMID21363959
Cation-conducting channel
(Na+, K+, H+, Ca2+)
o Chr2 == Chlamyopsin4; COP4
(C. reinhardtii)
o VChr1 == Chlamyospin 3; COP3
(Volvox carterii)
Activates neuron activity
Blue/green light activation
(470/540 nm)
Optogenetics Resource |
Halorhodopsin
16
Mei & Zhang 2012. Biol Psychiatry 71(12):1033-8. PMID:22480664
Yizhar et al. 2011. Cold Spring Harb Protoc (3). PMID21363959
Anion-conducting (Cl-) pump
- isolated from Natronobacterium pharaonis
Inhibits neuron activity
o Hyperpolarizes the cell membrane
Yellow light activation (580 nm)
Optogenetics Resource |
Bacteriorhodopsin
(Archaerhodopsin)
17
Mei & Zhang 2012. Biol Psychiatry 71(12):1033-8. PMID:22480664
Yizhar et al. 2011. Cold Spring Harb Protoc (3). PMID21363959
Proton pump (H+)
Inhibits neuron activity
o Hyperpolarizes the cell membrane
Green light activation (568 nm)
Optogenetics Resource |
OptoXR
18
Mei & Zhang 2012. Biol Psychiatry 71(12):1033-8. PMID:22480664
Yizhar et al. 2011. Cold Spring Harb Protoc (3). PMID21363959
Derived from vertebrate rhopodsin
Engineered to include G-protein
coupled intracellular domain
Activate or inhibit neuron
depending on GPCR domain
choice
Green light activation (487-587 nm)
Optogenetics Resource |
Channelrhodopsin in Mammalian Neurons
19
Boyden et al. 2005. Nature Neurosci 8(9):1263-8. PMID:16116447
Two different neurons:
same pulse series
Cultured rat hippocampal neurons transduced with ChR2-YFP lentivirus
Light stimulation generates action potentials & synaptic transmission
Light
stimulation
Action
potentials
Optogenetics Resource |
(glutamate inhibitor)
(GABA inhibitor)
Further Development of Optogenetic Tools
Fenno et al. 2011. Annu Rev Neurosci 34:389-412. PMID:21692661
Mattis J et al. 2012. Nature Meth. 9(2):159-72 PMID:22179551
Channels with different “speeds” or activation wavelengths
o Amino acid alterations (ChR2*H134R; ChETA (E123T); ChETAA (E123A))
o Chimeras of different channelrhodopsins (ChIEF; C1V1)
Multiple approaches to introduce opsins
o Lenti or adeno-associated viral vectors
o Transgenic mice
o Cre system Cre-dependent viruses, Cre-driver mice, optogenetic “reporters”
20 Optogenetics Resource |
Viral Versus Transgenic Delivery
Viral Delivery
Advantage:
o High levels of optogenetic protein expression
deliver many viral copies of construct per neuron
Complications:
o Incomplete coverage; not all target neurons take up virus
o Variable opsin expression; gradient of expression from
injection site out
o Laborious validation of each animal
o Virus may not be able to infect all target populations
Zeng and Madisen. 2012. Prog Brain Res. 196: 193–213. PMID:22341327
Madisen et al. 2012. Nat Neurosci 15(5): 793-802. PMID:22446880
http://openwetware.org/wiki/Image:Lentivirus2.jpg#file
Lentivirus
particles
21 Optogenetics Resource |
Viral Versus Transgenic Delivery
Transgenic Delivery
Advantages:
o Stable integration & inheritance of optogenetic transgenes
o Consistent distribution of optogenetic proteins
o Can take advantage of established cre-lox conditional expression systems
Many neuron-specific cre mice available & well characterized
Complications:
o Mosaicism
o “Ectopic” expression in non-target cells
o Genetic and epigenetic silencing
Zeng and Madisen. 2012. Prog Brain Res. 196: 193–213. PMID:22341327
Madisen et al. 2012. Nat Neurosci 15(5): 793-802. PMID:22446880
22 Optogenetics Resource |
Fiberoptic Neural Interface
Aravanis et al. 2007. J Neural Eng 4(3):S143-56. PMID:17873414
Airan et al. 2009. Nature 458(7241):1025-9. PMID:19295515
23 Optogenetics Resource |
Fiberoptic Neural Interface
24
Aravanis et al. 2007. J Neural Eng 4(3):S143-56. PMID:17873414
Optogenetics Resource |
Optogenetics: Method of the Year 2010
25 Optogenetics Resource |
Research Areas Using Optogenetics
Depression
26 Optogenetics Resource |
Research Areas Using Optogenetics
Schizophrenia
27 Optogenetics Resource |
Research Areas Using Optogenetics
Parkinson’s
Disease
28 Optogenetics Resource |
Research Areas Using Optogenetics
Addiction
29 Optogenetics Resource |
Channelrhodopsin Strains from JAX
**cryopreserved
Brain
(Thy1-COP4/EYFP)18Gfng (007612)
(Thy1-COP4/EYFP)9Gfng (007615)
(Thy1-COP4*H134R/EYFP)20Gfng (012350)
(Thy1-COP3/EYFP)1Gfng (012341)**
(Thy1-COP3/EYFP)4Gfng (012344)**
(Thy1-COP3/EYFP)8Gfng (012348)**
Cholinergic neurons
(Chat-COP4*H134R/EYFP)5Gfng (014545)
(Chat-COP4*H134R/EYFP)6Gfng (014546)
GABAergic Neurons
(Slc32a1-COP4*H134R/EYFP)8Gfng (014548)
Parvalbumin Neurons
(Pvalb-COP4*H134R/EYFP)15Gfng
(012355)
Serotonergic Neurons
(Tph2-COP4*H134R/EYFP)5Gfng
(014555)
Spinal Cord & Hindbrain
(Slc17a6-COP4*H134R/EYFP)2Oki
(017978)
Olfactory Neurons
Omptm1.1(COP4*/EYFP)Tboz (014173)
Olfr160tm1.1(COP4*/EYFP)Tboz (021206)
30 Optogenetics Resource |
Photostimulation Induces Action Potentials B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J (007612)
Kim et al. 2012. Proc Natl Acad Sci 109(50): 20673-8. PMID:23169668
Thalamic Reticular Nucleus o Sleep spindles
o Burst of 7-15Hz oscillations
o Sleep stabilization
Light
stimulation
Light
stimulation
Light
stimulation
Light
stimulation
Light
stimulation
Light
stimulation
Enlargement of
Action Potentials
31 Optogenetics Resource |
62ms, 8Hz
Inducing Sleep Spindles Via Photostimulation
Increases NREM Sleep B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J (007612)
Kim et al. 2012. Proc Natl Acad Sci 109(50): 20673-8.PMID:23169668
Increased NREM Transitions Between
NREM and REM
Increased
Periods Spent
Awake Shorter
32 Optogenetics Resource |
Halorhodopsin Strains from JAX
33
B6;SJL-Tg(Thy1-hop/EYFP)2Gfng/J (012332)
B6;SJL-Tg(Thy1-hop/EYFP)4Gfng/J (012334)
Unpublished lines
Expression reported in: dentate gyrus, thalamus, superior colliculus, inferior colliculus, brainstem, amygdala and cerebellum
Pyramidal neurons in cortex
o Layer 5 (line 2)
o Layer 2/3 & 5 (line 4)
Express improved Halorhodopsin/EYFP fusion protein (eNpHR::YFP)
o Less retention of halorhodopsin in ER/Golgi; reduced toxicity
Optogenetics Resource |
JAX Optogenetic Cre-conditional Mice
34
Channelrhodopsin
B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4*H134R/tdTomato)Hze/J (012567)
B6;129-Gt(ROSA)26Sortm1(CAG-COP4*E123T*H134R,-tdTomato)Gfng/J (017455)
B6;129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J (012569)
Halorhodopsin
B6;129S-Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/J (014539)
Archaerhopsin
B6;129S-Gt(ROSA)26Sortm35.1(CAG-aop3/GFP)Hze/J (012735)
B6.Cg-Gt(ROSA)26Sortm40.1(CAG-aop3/EGFP)Hze/J (021188) ‡
‡ under development
Madisen et al. 2012. Nat Neurosci 15(5): 793-802. PMID:22446880
Optogenetics Resource |
Promoter loxP loxP Ai39: Halorhodopsin/EYFP STOP
Promoter loxP
hop/EYFP
+
X
B6;129S-Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/J (014539)
Cre
Halorhodopsin/EYFP
Halorhodopsin Cre-conditional Mice
35 Optogenetics Resource |
Generating Forebrain-specific Halorhodopsin
Strain with Cre-lox System
36 Madisen et al. 2012. Nat Neurosci 15(5): 793-802. PMID:22446880
x Emx1 cre
loxP loxP
loxP loxP
Forebrain-specific cre transgenic mouse B6.129S2-Emx1tm1(cre)Krj/J (005628)
homozygous loxP (“floxed”) mouse
Emx1 cre
Heterozygous for Halorhodopsin/EYFP after 1 generation
STOP
STOP
hop
hop
hop
B6;129S-Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/J
(014539)
loxP
Light Activation of Halorhodopsin Induces
Voltage Response in Cortical Neurons B6;129S-Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/J (014539)
B6.129S2-Emx1tm1(cre)Krj/J (005628)
Madisen et al. 2012. Nat Neurosci
15(5): 793-802. PMID:22446880 100 μM
Cortical Pyramidal Neuron
Negative
Current
Injection
Light
Pulse
Similar voltage
responses produced
by current injection or
light pulse
37 Optogenetics Resource |
In Vivo Neural Activity Is Inhibited By Light
Activation of Halorhodopsin
B6;129S-Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/J (014539)
B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/J (012362)
Madisen et al. 2012. Nat Neurosci 15(5): 793-802. PMID:22446880
100 μM
Green light off Green light off Green light on
Neural firing inhibited
Cortical
pyramidal
neurons
38 Optogenetics Resource |
In Vivo Neural Activity Is Inhibited By Light
Activation of Archaerhodopsin
B6;129S-Gt(ROSA)26Sortm35.1(CAG-aop3/GFP)Hze/J (012735)
B6.129S2-Emx1tm1(cre)Krj/J (005628)
Madisen et al. 2012. Nat Neurosci
15(5): 793-802. PMID:22446880
Green light off Green light off Green light on
Neural firing inhibited
39 Optogenetics Resource |
Activation of Archaerhodopsin by Different
Light Sources Inhibits Cortical Cell Firing
B6;129S-Gt(ROSA)26Sortm35.1(CAG-aop3/GFP)Hze/J (012735)
B6.129S2-Emx1tm1(cre)Krj/J (005628)
Madisen et al. 2012. Nat Neurosci 15(5): 793-802. PMID:22446880
100 μM
Yellow Light White Light Red Light
Whole cell recording in cortical pyramidal cells
from acute slice preparations
40 Optogenetics Resource |
Additional Optogenetic JAX Strains
41
Calcium Sensors
B6;CBA-Tg(Thy1-GCaMP2.2c)8Gfng/J (017892)
B6;CBA-Tg(Thy1-GCaMP3)6Gfng/J (017893)
B6;129S-Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (014538)
STOCK Tg(tetO-GCAMP2)12iRyu/J (017755)
STOCK Tg(Myh6*/tetO-GCaMP2)1Mik/J (012477)**
Chloride Sensors
B6.Cg-Tg(Thy1-Clomeleon)1Gjau/J (013161)**
STOCK Tg(Thy1-Clomeleon)12Gjau/J (013162)**
STOCK Tg(Thy1-Clomeleon)13Gjau/J (013163)**
**cryopreserved
Optogenetics Resource |
In Vivo Observation of Ca++ Transients in
GCaMP Transgenic Mouse Neurons
42
B6;CBA-Tg(Thy1-GCaMP2.2c)8Gfng/J (017892)
Chen et al. 2007. Neuron
76: 297-308. PMID:23083733
Layer 5 pyramidal
neurons in vivo
(8 weeks old)
Optogenetics Resource |
Locating JAX Optogenetic Strains
www.jax.org
43 Optogenetics Resource |
Locating JAX Optogenetic Strains
www.jaxmice.jax.org/query
44 Optogenetics Resource |
Locating JAX Optogenetic Strains
www.jaxmice.jax.org/research
45 Optogenetics Resource |
Locating JAX Optogenetic Strains
www.jaxmice.jax.org/neurobiology
46 Optogenetics Resource |
Optogenetics Resources at JAX
www.jax.org/reserch/grs/optogenetics
Optogenetic
strain
offerings
&
more
47 Optogenetics Resource |
Optogenetics Resources at JAX
www.jax.org/reserch/grs/optogenetics
48 Optogenetics Resource |
Additional Optogenetics Resources
www.jax.org/reserch/grs/optogenetics
49 Optogenetics Resource |
Collaboration Between JAX &
Allen Institute for Brain Science
50
Allen Institute for Brain Science
o Developing new cre & cre-conditonal tools
Optogenetic conditional strains
o Characterizing new strains & publishing results
The Jackson Laboratory
o Archiving and distributing newly developed optogenetic strains
o Characterizing cre expression patterns
Optogenetics Resource |
Allen Institute for Brain Science
www.alleninstitute.org
51 Optogenetics Resource |
Allen Institute for Brain Science
www.alleninstitute.org
52 Optogenetics Resource |
Allen Institute for Brain Science
www.alleninstitute.org
53
Allen Institute for Brain Science
www.alleninstitute.org
54
Allen Institute for Brain Science
www.alleninstitute.org
55
Allen Institute for Brain Science
www.alleninstitute.org
56
Accessing Optogenetics Data from
the Allen Institute for Brain Science
www.alleninstitute.org
57
JAX Strain Datasheet Links to Data from the
Allen Institute for Brain Science
58
59
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