Light Up Your Research - Trinity College Dublin · Optogenetics Mouse Models: Presentation Overview...

Light Up Your

Research: Use and Selection of

Optogenetic Mouse

Models

1

Presented by:

Brian W. Soper, PhD

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Optogenetics Mouse Models: Presentation Overview

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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 |

http://research.jax.org/grs/optogenetics.html


Using Light to Understand the Brain

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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

http://www.ncbi.nlm.nih.gov/pubmed/19555292

http://www.ncbi.nlm.nih.gov/pubmed/?term=17490748




















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 |

http://www.psychologicalscience.org/redesign/wp-content/uploads/2012/02/neuron.jpg











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


o Poor spatial resolution o Poor temporal resolution

http://www.ncbi.nlm.nih.gov/pubmed/?term=Karl+Deisseroth+Optogenetics+Nature+Methods+2010

http://www.chem.duke.edu/~wwarren/neuroimaging.php



Optogenetics: Improved Spatial and Temporal Resolution

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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


http://www.ncbi.nlm.nih.gov/pubmed/?term=Karl+Deisseroth+Optogenetics+Nature+Methods+2010



Original Function of Opsins

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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



http://remf.dartmouth.edu/images/algaeSEM/source/2.html

http://remf.dartmouth.edu/images/algaeSEM/source/2.html



Categories of Opsins

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Adapted from Mei & Zhang 2012. Biol Psychiatry 71(12):1033-8. PMID:22480664

Channelrhodopsin

Halorhodopsin

Bacteriorhodopsin

(Archaerhodopsin)

OptoXR





Categories of Opsins

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Adapted from Mei & Zhang 2012. Biol Psychiatry 71(12):1033-8. PMID:22480664

Channelrhodopsin

Halorhodopsin

OptoXR

all-trans-Retinal


Bacteriorhodopsin

(Archaerhodopsin)




Channelrhodopsin

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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)






Halorhodopsin

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Anion-conducting (Cl-) pump

- isolated from Natronobacterium pharaonis

Inhibits neuron activity

o Hyperpolarizes the cell membrane

Yellow light activation (580 nm)






Bacteriorhodopsin

(Archaerhodopsin)

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Proton pump (H+)

Inhibits neuron activity

o Hyperpolarizes the cell membrane

Green light activation (568 nm)






OptoXR

18



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)






Channelrhodopsin in Mammalian Neurons

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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


(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”






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



http://www.ncbi.nlm.nih.gov/pubmed/22446880?dopt=Abstract

http://openwetware.org/wiki/Image:Lentivirus2.jpg



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







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






Fiberoptic Neural Interface

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Aravanis et al. 2007. J Neural Eng 4(3):S143-56. PMID:17873414





Optogenetics: Method of the Year 2010




Research Areas Using Optogenetics

Depression





Schizophrenia





Parkinson’s

Disease





Addiction




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)**



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)


http://jaxmice.jax.org/strain/007612.html
















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


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






Halorhodopsin Strains from JAX

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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






JAX Optogenetic Cre-conditional Mice

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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












Promoter loxP loxP Ai39: Halorhodopsin/EYFP STOP

Promoter loxP

hop/EYFP

+

X


Cre

Halorhodopsin/EYFP

Halorhodopsin Cre-conditional Mice





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







In Vivo Neural Activity Is Inhibited By Light

Activation of Halorhodopsin


B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/J (012362)


100 μM

Green light off Green light off Green light on

Neural firing inhibited

Cortical

pyramidal

neurons







In Vivo Neural Activity Is Inhibited By Light

Activation of Archaerhodopsin



Madisen et al. 2012. Nat Neurosci

15(5): 793-802. PMID:22446880

Green light off Green light off Green light on

Neural firing inhibited







Activation of Archaerhodopsin by Different

Light Sources Inhibits Cortical Cell Firing




100 μM

Yellow Light White Light Red Light

Whole cell recording in cortical pyramidal cells

from acute slice preparations







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












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)






Locating JAX Optogenetic Strains

www.jax.org


http://www.jax.org/




www.jaxmice.jax.org/query


http://jaxmice.jax.org/query

http://jaxmice.jax.org/query




www.jaxmice.jax.org/research


http://www.jaxmice.jax.org/research

http://www.jaxmice.jax.org/research




www.jaxmice.jax.org/neurobiology


http://www.jaxmice.jax.org/neurobiology

http://www.jaxmice.jax.org/neurobiology



Optogenetics Resources at JAX

www.jax.org/reserch/grs/optogenetics

Optogenetic

strain

offerings

&

more






Optogenetics Resources at JAX







Additional Optogenetics Resources







Collaboration Between JAX &

Allen Institute for Brain Science

50


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





www.alleninstitute.org


http://www.alleninstitute.org/





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Accessing Optogenetics Data from

the Allen Institute for Brain Science


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JAX Strain Datasheet Links to Data from the


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Light Up Your Research - Trinity College Dublin · Optogenetics Mouse Models: Presentation Overview...

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