Optogenetics
Genevieve Bell
Daniel Blackman
Brandon Chelette
Membrane Biophysics - Fall 2014
Optogenetics
• Integration of optics and genetics that allows for experimental
control of events within a specific cell
Before Optogenetics
• The spatial and temporal resolution of modulation in the brain
left much to be desired
• Spatial resolution: stimulation with electrodes does not
distinguish between different cell types (Francis Crick, 1979)
• Temporal resolution: multi-component systems require a long
time to occur
• Insufficient for millisecond time scales needed to analyze cellular
events such as action potentials
Before Optogenetics
• It was known that there were single component, light activated
ion pumps in bacteria
• Vast array of microbial opsin genes
Optogenetics – the basic concept
• Express a light-activated ion channel (isolated
from bacteria) in a specific sub-population of
neurons
• Illuminate neurons to modulate activity
• Modulation depends on type of channel
• Record output (e-phys, fMRI, behavioral, etc.)
Initial Problems
• It was presumed that:
• Microbial proteins would not be expressed very well in mammalian
cells
• Photocurrents not strong enough to control neurons
• Retinal (co-factor) would have to be added to the cell type of interest
Initial Problems
• It was presumed that:
• Microbial proteins would not be expressed very well in mammalian
cells (bacterial opsin expressed reliably in mammalian neurons.
Boyden 2005)
• Photocurrents not strong enough to control neurons
• Retinal, a requisite cofactor for opsins, would have to be added to the
cell type of interest
Initial Problems
• It was presumed that:
• Microbial proteins would not be expressed very well in mammalian
cells (bacterial opsin expressed reliably in mammalian neurons.
Boyden 2005)
• Photocurrents not strong enough to control neurons (bacterial opsin
provided sustained control of action potential. Boyden 2005)
• Retinal, a requisite cofactor for opsins, would have to be added to the
cell type of interest
Initial Problems
• It was presumed that:
• Microbial proteins would not be expressed very well in mammalian
cells (bacterial opsin expressed reliably in mammalian neurons.
Boyden 2005)
• Photocurrents not strong enough to control neurons (bacterial opsin
provided sustained control of action potential. Boyden 2005)
• Retinal, a requisite cofactor for opsins, would have to be added to the
cell type of interest (all vertebrate tissues contain sufficient all-trans
retinal. Douglass 2008)
Optogenetics
• Allows for high resolution temporal and spatial modulation of the
activity of a cell
• The population of neurons being targeted, the timing of the
modulation, and the nature of the modulation are all under
experimental control
Optogenetics: a diverse toolkit
• The optogenetic effect experienced by a cell will depend on
many factors
•
•
•
•
The properties of single-component opsin being used
The efficiency of the expression of that opsin
The source/wavelength/intensity of the light
The location and density of the population of neurons being
investigated
Optogenetics: a diverse toolkit
• Wide variety of opsins
Optogenetics: a diverse toolkit
Four main categories of opsins:
• Fast excitation
• Fast inhibition
• Step function
• Biochemical modulation
Optogenetics: a diverse toolkit
Biochemical modulation
• Opsin + G-protein coupled receptor
• Activated by light, but not an ion channel
• Illumination leads to intracellular signaling cascades
• Can be considered slow excitatory or slow inhibitory based on
the nature of the G protein signaling pathway
• Provide precise control of intracellular signals (cAMP, GTPase,
etc.)
Optogenetics: a diverse toolkit
Fast excitation
• Channelrhodopsins
• Cation channels
• Modified via mutagenesis with optimization in mind, but can
result in unanticipated or undesired effects
• Gain-of-function tool
Optogenetics: a diverse toolkit
Fast excitation
Optogenetics: a diverse toolkit
Fast inhibition
• Chloride & proton pumps
• Also engineered for optimal function
• Loss-of-function tool
Optogenetics: a diverse toolkit
Fast inhibition
Optogenetics: a diverse toolkit
Step function opsins (SFOs)
• Exhibit bistability
• Much slower deactivation rate
• Product of molecular engineering
• Cation channels
• Larger disparity between activating wavelength and deactivating
wavelength
Optogenetics: a diverse toolkit
Step function opsins (SFOs)
Optogenetics – a diverse toolkit
• Vast array of opsins available to be used as a result of
attempted optimization
• Point mutations
• Codon insertion/deletion/replacement
• Small changes can lead to beneficial or detrimental changes in:
• Current generation
• Opening/closing kinetics
• Sensitization/Desensitization
Optogenetics – targeting your opsin
• Viral injection
• LV , AAV
• Projection targeting
• Dendrites or axons
• Transgenic targeting
• Transgenic mouse lines that are not under recombinase-dependent
control
• Spatiotemporal targeting
• Birthdate of cells , specific layer
Optogenetics – light delivery
• Assuming you are expressing the correct opsin in the desired
cell population, you now need to somehow get light to those
cells
• There are several facets to consider and the best choice will
depend on your experiment
•
•
•
•
Excitation vs inhibition vs bistable
Wavelength
Intensity
Duration
Optogenetics – light delivery
• Brain tissue scatters and absorbs light
• Different tissues scatter and absorb light in different ways (Ex:
myelinated tissue scatters light significantly)
Optogenetics – light delivery
Different wavelengths
of light penetrate brain
tissue better than
others
Optogenetics – light delivery
• Surface targets: cell culture, brain slices, etc
• Easily accessible, spot illumination
• Deep Targets: in vivo targets that are not on the outer surface of
the brain
• Minimize damage
• Fiber optics
Optogenetics – light sources
• Variety of light sources:
• Lasers
• LEDs
• Incandescent
• Depending on where you need to deliver your
light and how, you need to select the correct light
Optogenetics – the good
• Significant experimental control
• High resolution temporal and spatial control
• Specific cell type population
• Millisecond control on time scale of cellular events like action potentials
• Wide variety of opsins available
• Can be applied to more fields than just neuroscience (cardiac
muscle, skeletal muscle, etc)
• Potential disease models
Optogenetics – the bad
• Damage to surrounding tissue (depending on location of target)
• Light absorption  heat  damage tissue or affect
physiological events
• Viral infection and/or expression of exogenous proteins can
lead to unwanted alterations in cell capacitance, physiological
activity, structural abnormalities, and toxicity
• Second and third degree currents can confound actual effects
• Causality can not always be proven
The ugly take home message
• Optogenetics is a burgeoning technique that has provided
neuroscientists with a much more finely-tuned way to
manipulate the cellular activities of a specific population of cells
they are interested in. But with any relatively new technique,
there are still kinks that need to worked out
Color-tuned Channelrhodopsins
for Multiwavelength Optogenetics
Matthias Prigge, Franziska Schneider, Satoshi P. Tsunoda, Carrie
Shilyansky, Jonas Wietek, Karl Deisseroth, and Peter Hegemann
A presentation by Daniel Blackman
Modifying ChRs to Overcome Limitations
• Limitations of ChR2s
1.
2.
3.
4.
5.
Low expression
Small conductance
Inappropriate kinetics
Partial inactivation
Ion selectivity
• Retinal pocket changes affect absorption, kinetics, and membrane
targeting
1.
2.
3.
4.
Glu123Thr/Ala (ChETA variants)
Cys128Ser or Asp156Ala
Glu90, Glu123, Leu132, or His134
C1 and C2 helix swapping
What Previous Studies Have Taught Us
• Volvox C1 (V1) absorption max
• Pyramidal neurons
• Dual-color activation
• Spectrally separated absorption
• Large photocurrents
• Different operational sensitivities
Spanning the Spectrum
• Operational Light Sensitivity
• Calcium indicators or voltage sensors
• Molecular engineering
• Helix Swapping
• Global rearrangements
Global Structural Rearrangements
Prigge et al (2012)
Imaging and Analysis
Prigge et al (2012)
Fast and Slow-Cycling
Prigge et al (2012)
AP Firing in Hippocampal Neurons
Prigge et al (2012)
Dual Light Excitation and Ion Selectivity
Prigge et al (2012)
Current-voltage relationships
Prigge et al (2012)
Conclusion
• Identified helices H6 & H7 responsible for absorption
differences, and helices H1 & H7 for membrane integration
• Achieved independent activation of distinct neural populations
through AA mutations:
•
•
•
•
ChR2 with absorption maxima 461-492 nm (blue)
C1V1 chimeric variant with absorption maxima 526-545 nm (green)
C1V1-B & C2-LC-TC provide better expression & lower inactivation
Further color tuning possible through exploring further AA mutations
Fast-conducting
mechanoreceptors
contribute to withdrawal
behavior in normal and
nerve injured rats
Ririe DG, et al. 2014. Pain.
Membrane Biophysics – Fall 2014
Background - Pain
• Transduction of noxious (or potentially noxious) stimuli from
periphery to central nervous system
• Complex integration with emotional and cognitive signals 
perception of pain
Background - Pain
A-fibers – “first pain”
C-fibers – “second pain”
Julius & Basbaum 2001
Background - Pain
Ablation of C-fibers does not eliminate pain behavior
Other afferents might be responsible for at least some portion of ongoing pain
Background - Experiment
• Neurons of interest: fast-conducting, myelinated, nociceptive,
high-threshold mechanoreceptors = AHTMRs
• Investigate: what is the role of these neurons in pain signal
transduction under normal conditions and under neuropathic
conditions?
Background - Experiment
• Selectively inhibit these specific neurons (AMHTRs) using
expression of light-activated proton pump
• Male Sprague-Dawley rats: intrathecal injection of AAV8 with
CAG/ArchT/GFP tag
Light activates proton pump
Pumps protons intra  extra
Hyperpolarizes cell / Reduce
excitability / Inhibit activity
First things first…
Expression of GFP-ArchT in soma, dendrites and axons
Expression maximized around 4 weeks post-injection
NF200: marker for myelinated cells
IB4: marker for unmyelinated cells
GFP co-expressed
almost exclusively with
NF200
NF200: myelinated
IB4: unmyelinated
In vitro recordings
• Excised DRG four weeks after injection
• Single-electrode, continuous-current clamp recording of GFP
positive cells
• 0.1 nA – 4.0 nA in 0.1 nA increments until an AP was evoked to
determine threshold
In vitro recordings
Light exposure increased threshold
Light exposure hyperpolarized cells
In vivo recordings
• 3 – 8 weeks after injection
• L4 DRG ganglia exposed
• Receptive field, conduction velocity, and electrophysiological
profile determined
In vivo recordings
In vivo recordings
Only AHTMRs affected by light
LTMR: myelinated low threshold mechanoreceptors
AHTMR: myelinated high-threshold mechanoreceptors
CHTMR: unmyelinated high-threshold mechanoreceptors
In vivo recordings
AMHTR recordings: inhibition of AP when soma exposed to light
In vivo recordings
Stimulus at receptive field in paw results in AP
AP eliminated upon illumination of DRG
In vivo recordings (transcutaneous)
Same idea: still recording from DRG, but light is now targeted at receptive field in paw
In vivo recordings (transcutaneous)
AMHTR recording: transcutaneous light still inhibits AP firing
In vivo recordings (transcutaneous)
In vivo recordings (transcutaneous)
Dose-response curve
So far, so good
• Intrathecal injection of viral vector with GFP-ArchTCAG only expresses in AMHTRs
• Expression of GFP-ArchT allows for optical inhibition
• In vitro: illumination decreases membrane potential and
increases threshold
• In vivo:
• stimulate soma + illuminate soma = inhibition
• stimulate receptive field + illuminate soma = inhibition
• stimulate RF + illuminate RF = inhibition
• Under “normal” conditions; what happens following a
nerve injury?
Nerve injury investigation
• Following nerve injury, afferents become hyperexcitable
• May contribute to chronic pain
• Inhibition of AMHTRs may be beneficial for reducing pain after a
nerve injury
• However, there is no basis to assume that inhibition after a
nerve injury will be the same as inhibition in the “normal” state
Nerve injury investigation
• Induce hyperexcitability via partial ligation of L5 nerve
• Investigate the effects of light on hyperexcitability
Nerve injury investigation
Evidence of hyperexcitability
Increased receptive field
Nerve injury investigation
AMHTR: sham
AMHTR: pSNL
Nerve injury investigation
pSNL: reduction in mechanical threshold and reduction in mechanical withdraw threshold
Nerve injury investigation
Baseline vs pSNL = hyperalgesia
Laser ON vs Laser OFF = reduction of hyperalgesia
Nerve injury investigation
LTMR
AHTMR
CHTMR
Only AHTMRs inhibited by transcutaneous light on receptive field
Nerve injury investigation - summary
• pSNL resulted in an increased receptive field, increased
sensitivity, reduced mechanical threshold, and reduced
withdraw threshold
• Optogentic inhibition reduced hyperalgesia
• Transcutaneous light only inhibited AMHTRs
Conclusions
• AMHTRs, which have been established and accepted as “first”
pain fibers, seem to play a role in chronic pain as well
• Evidenced by correlation between withdraw threshold and
AMHTR sensitivity
• Also demonstrated behavioral changes under normal conditions
and under conditions that simulate pathological nerve damage
Stimulating Cardiac Muscle by Light
Cardiac Optogenetics by Cell Delivery
Zhiheng Jia, MS; Virginijus Valiunas, PhD; Zongju Lu, PhD; Harold Bien,
MD, PhD; Huilin Liu, MS; Hong-Zhang Wang, PhD; Barbara Rosati, PhD;
Peter R. Brink, PhD; Ira S. Cohen, MD, PhD; Emilia Entcheva, PhD
A presentation by Daniel Blackman
Tandem Cell Unit (TCU)
Zhiheng et al (2011)
Proof-of-Principle Cell Delivery System
• Cell line using ChR2 plasmid
• Whole-cell and dual-patch techniques
• Gap-junction uncoupling
• Electrical vs optical stimulation
Developing and Characterizing the
System
Zhiheng et al (2011)
Validation of the TCU Concept
Zhiheng et al (2011)
Dual-Patch and Carbenoxolone (CBX)
Zhiheng et al (2011)
Optical Mapping System
Zhiheng et al (2011)
Optical Mapping
Zhiheng et al (2011)
TCU Benefits and In Vivo Consideration
• TCU vs Direct:
1. Donor cells (DCs) = nonexcitable and no major repolarization
currents
2. DCs have high membrane impedance (small IR currents)
3. Reduced DC-CM vs CM-CM coupling
• Optical excitation improvements:
1. Higher membrane impedance DCs
2. Lower membrane impedance CM neighbors
3. Better source-neighbor coupling
Conclusion
• High spatiotemperal resolution of excitation without affecting
essential properties
• Potential investigations into optical pacemakers
• Five main features of this study:
1.
2.
3.
4.
5.
Desired pace rate achieved
Finer manipulation of excitation, repolarization, and AP shape
In vivo applications (vs viral transfection)
Biocompatibility of optic fibers (vs metal electrodes)
Improved energy consumption (vs electrical pacemakers)