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Optogenetics
Ying Xu
GHM Institute of CNS Regeneration
2016 Dec
What’s optogenetics?
• Optogenetics is the combination of genetic and optical methods
to control specific events in targeted cells of living tissue, even
within freely moving mammals and other animals, with the
temporal precision (millisecond-timescale) needed to keep pace
with functioning intact biological systems.
Publication timeline for microbial opsins and
optogenetics over 45 years
Karl Deisseroth， Nature Neurosci, 2015
Light sensitive proteins
Light sensitive proteins in animal retina
rhodopsin
melanopsin
Light sensitive proteins in microbio
Channelrhodopsin(ChR)
found in green algae
Halorhodopsin (NpHR)
found in halobacteria
嗜盐菌
Channelrhodopsin (ChR)
first reported by Nagel et al., Science，2002
小鼠海马神经元
Tohoku Neuroscience Global COE
Structure of ChR
Kato et al., 2012 Nature
Native rhodopsin
channelrhodopsin
Halorhodopsin (NpHR)
Han and Boyden, 2007
Many others…
Optogenetic tools
light-activated
membrane-bound G
protein-coupled (OptoXR)
or soluble (bacterial
cyclase) receptors that
mimic various signalling
cascades
bacteriorhodopsin
or proteorhodopsin
(BR/PR)
wavelength activation spectra and decay kinetics
Tye and Deisseroth, Nature Reviews, 2012
Application of optogenetics in
Neuroscience
Principle of optogenetics in neuroscience
Targeted excitation or inhibition , conferring cellular specificity and even projection specificity
not feasible with electrodes while maintaining high temporal (action-potential scale) precision.
Karl Deisseroth, 2010
Millisecond-timescale, genetically
targeted optical control of neural activity.
Boyden ES.. Deisseroth , 2005, Nature Neurosci
Hippocampal neurons
expressing ChR2-YFP
Bi-directional optical control of voltage with blue
and yellow light pulses
In cultured hippocampal neurons
Han and Boyden, 2007
Zhang et al. Deisseroth lab, 2007
In Vivo Light-Induced Activation of Neural Circuitry in
Transgenic Mice Expressing Channelrhodopsin-2
The Thy1 Promoter Drives Transgenic Expression of ChR2-YFP
in Subsets of Neurons in the Central Nervous System
Arenkiel et al., 2007, Neuron
Optical deconstruction of parkinsonian
neural circuitry.
Gradinaru .. Deisseroth , 2009 Science
Direct optical inhibition of local STN neurons
subthalamic nucleus
…to systematically drive or inhibit an array of distinct
circuit elements in freely moving parkinsonian rodents
…demonstrate an optical approach for dissection of
disease circuitry…
Selective optical control of
afferent fibers in the STN
Prosthetic systems built up
In monkey
cortex
(Bernstein et al., 2008)
Control Animal’s Behavior
Boyden’s lab in MIT
Deisseroth’s lab in Stanford
Designing possible optogenetic
neuromodulation therapies
Parkinson's disease. Deep brain stimulation devices have been efficacious in correcting movement
disorders in patients with advanced stage Parkinson's disease. High frequency stimulation is thought to
suppress firing of neurons in the subthalamic nucleus (STN). Optical neuromodulation could be used to shut
down excitatory glutamatergic neurons in the STN that express halorhodopsin (NpHR) under the control of
an excitatory neuron-specific promoter; this cell type specificity will result in a more direct inhibition, and thus
less side-effects, compared with electrode-based DBS.
Depression. DBS to the subgenual cingulate area 25 (Cg25) appears to alleviate depression symptoms.
NpHR could be introduced into Cg25 to specifically and potently reduce neuronal activity in this area.
Alternatively, channelrhodopsin-2 (ChR2) could be expressed in the nucleus accumbens to model proposed
DBS-mediated excitation.
Chronic pain. Electrical stimulation methods include local peripheral nerve stimulation, local cranial
nerve stimulation and 'subthreshold' motor cortex stimulation. Reasonable optogenic approaches might
include NpHR-mediated inhibition of specific pain fibres or foci, sparing other fibre types. ChR2 could also
be used to provide effective pain relief by driving inhibitory or analgesic neurons. Development of tolerance
and dependence might be less of an issue with this method of selectively driving analgesic neurons
compared with pharmacological treatments.
Epilepsy. Quenching or blocking epileptogenic activity is an exciting prospect. Many epilepsy patients
have a stereotyped pattern of activity spread resulting from an epileptogenic focus. Brief activation of NpHR
could be used to suppress the abnormal activity before it spreads, or to truncate the abnormal activity early
in its course. Activation of ChR2 in -aminobutyric acid (GABA)-releasing interneurons could provide a
similar result.
Optogenetic control of seizure
Tung et al., 2016 Brain Stimulation
Restore Vision
（2015）
Restore visual responses in mice with
photoreceptor degeneration by ChR2
Expression of Chop2GFP in Retinal Neurons
Visual Evoked Potential in rd1/rd1 Mice
Multielectrode Array Recordings of the
ChR2-Expressing Retinas of rd1/rd1 Mice
Bi et al., Pan ZH, 2006, Neuron
Light-activated channels targeted to ON bipolar cells
restore visual function in retinal degeneration
(Lagali et al., Roska, 2008,
Nature Neurosci)
Restoration of visual function by expression of a
light-gated mammalian ion channel in retinal ganglion cells or ONbipolar cells
(Gaub et al., PNAS, 2014)
light-gated ionotropic glutamate receptor (LiGluR)
Optopharmacology
rd1
mice
(mutated kainate receptor+MAG)
LiGluR expression restores innate and learned light-guided behavior in rd1 mice in vivo
LiGluR expression in RGCs restores light responses in the rcd1 canine retina in vitro.
Engineering the Blind to See
Aug 2010
Method of
the Year
How?
Four steps:
Choose right light-sensitive proteins
Deliver the genes
Control the illumination
Read the outcome
First step: light-activated proteins—the
toolbox
Tools for modulating the
membrane potential
Tools for modulating cell
signaling
Second step: delivering the genes
• Transfection
• Viral transducion
• Creation of transgenic animal
Targeting strategies with optogenetic tools in
vivo (1)
Targeting strategies with optogenetic tools in
vivo (2)
Third step: controlled illumination
( Packer et al., 2013, Nature Neurosci)
Patterned illumination strategies
( Packer et al., 2013, Nature Neurosci)
Optical dissection of brain circuits with patterned illumination
through the phase modulation of light
(Bovetti and Felli, 2015, J Neurosci Methods)
Fourth step: reading the outcome
Optical imaging:
to measure the fluorescence changes
Electrophysiological recording:
to monitor the effect of changes in membrane voltage.
Behavioral test:
to assess the effect of modulating cellular activity in whole animals.
Designs of fiber-optics and multimodal probes
Wang F et al. Probing pain pathways with light. Neuroscience (2016)
(Illuminating the brain, Nature, 2011)
Ongoing research and future
direction
Dissect neuronal circuits
Targeting specific neuron
Control neuronal signaling
Optogenetic dissection of medial prefrontal
cortex circuitry
Optogenetic evidence for the involvement of the mPFC in depressive-like behavior and anxiety.
(Riga et al., 2014, Frontiers in SYSTEMS NEUROSCIENCE )
Targeting Specific Neuron by Optogenetics
Liu and Tonegawa, 2010, Cell
Optogenetic pharmacology for control of
native neuronal signaling proteins
Kramer et al., Nature Neurosci, 2013
photosensitive reagents that act on channels and receptors
Caged glutamate
Chemical photoswitches
Optogenetic pharmacology combines optics and chemistry and adds genetics to the mix,
simultaneously solving both the subtype-specificity and cell-targeting problems. The idea is to
attach a synthetic photosensitive ligand onto a genetically engineered protein to allow activation
or inhibition of only that specific protein with light
(Kramer et al., Nature Neurosci, 2013)
Optogenetic control of intracellular
signaling pathways
(light, oxygen, and voltage (LOV) domains)
Zhang and Cui, 2015, Trends in Biotechnology
Modes of signaling control by photoactivatable proteins
Light-induced conformational change
Optogenetic modulation of postsynaptic neuronal
function at multiple levels of cellular activity
(Stuber and Mason, 2013, Pharmacological Reviews)
Beyond Neuroscience
Optogenetic Control of Cardiac Function
Arrenberg et al.,Science. 2010
Movie1
Movie2
Altered Heart Cells Controlled with Light
BONN, Germany, Oct. 13,
2010 — Using a method called
photostimulation, scientists at
the University of Bonn have
altered cardiac muscle cells to
make them controllable with light.
They were able to use directed
blue light to cause conditions
such as arrhythmia in genetically
modified mice.
 Using light to create a safer, more reliable pacemaker
Pacing lightly: optogenetics gets to the heart. Knollmann BC. Nat Methods. 2010
Optogenetic control of cardiac function. Arrenberg et al.,Science. 2010
(Boyle et al., 2015, Trends in Cardiovascualar Medicine )
Using light to reinstate respiratory plasticity.
C2 section
Light-induced rescue of breathing after spinal cord injury.
Alilain et al., J Neurosci. 2008
Restore motion function
… We generated murine embryonic stem cell–derived motor neurons that express the light-sensitive ion channel
channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult
mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their
function to be restored in a controllable manner using optogenetic stimulation.
( Optical control of muscle function by transplantation of stem cell-derived motor
neurons in mice. Science, 2014)
Optogenetic Immunomodulation
Cancer-Immunity Cycle
Tan et al., 2016 Trends in Biotechnology
The Cancer-Immunity Cycle and Opportunities for Optogenetic
Interventions to Improve Cancer Immunotherapies
Strategies for In Vivo Optogenetic Immunomodulation
Lots to explore
Promising future…
Noble Prize?