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

Light Up Your

Research: Use and Selection of

Optogenetic Mouse

Models

Presented by:

Brian W. Soper, PhD

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Optogenetics Mouse Models: Presentation Overview

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

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

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

Deisseroth 2011. Nature Methods 8(1):26-29. PMID:21191368

Electrical Stimulation

Chemical Stimulation

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

o Poor spatial resolution o Poor temporal resolution

Optogenetics: Improved Spatial and Temporal Resolution

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

Original Function of Opsins

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

Categories of Opsins

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

Channelrhodopsin

Halorhodopsin

Bacteriorhodopsin

(Archaerhodopsin)

OptoXR

Categories of Opsins

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

Channelrhodopsin

Halorhodopsin

OptoXR

all-trans-Retinal

Bacteriorhodopsin

(Archaerhodopsin)

Channelrhodopsin

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

Anion-conducting (Cl-) pump

- isolated from Natronobacterium pharaonis

Inhibits neuron activity

o Hyperpolarizes the cell membrane

Yellow light activation (580 nm)

Bacteriorhodopsin

(Archaerhodopsin)

Proton pump (H+)

Inhibits neuron activity

o Hyperpolarizes the cell membrane

Green light activation (568 nm)

OptoXR

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

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

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

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

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

(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)

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

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

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

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

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

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

B6;129S-Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/J

(014539)

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

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

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

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

www.jaxmice.jax.org/query

www.jaxmice.jax.org/research

www.jaxmice.jax.org/neurobiology

Optogenetics Resources at JAX

www.jax.org/reserch/grs/optogenetics

Optogenetic

strain

offerings

Optogenetics Resources at JAX

Additional Optogenetics Resources

Collaboration Between JAX &

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

www.alleninstitute.org

Accessing Optogenetics Data from

the Allen Institute for Brain Science

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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