Download 24 Optogenetics - how to use light to manipulate neuronal networks

Optogenetics
how to use light to manipulate neuronal networks
Alexandra Götz
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http://www.stanford.edu/group/dlab/optogenetics/
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Outline
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Background
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Neuron and ion channels
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Action potential
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Light sensitive channels
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Channel induction
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ReaChr
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Experiment
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Results
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ReaChR in Drosophila
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CNS neurons
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Wing extension reflex
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Changes due to social factors
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Summary
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Future challenges
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Neuron and ion channels
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Action potential
(9)
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Light sensitive channels
(3)
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Light sensitive channels
(3)
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How are the light sensitive channels induced in
the brain?
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(8)
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ReaChR
Red-activable Channelrhodopsin
Improved signaling characteristics e.g. greater
photocurrent and faster kinetics
λ~590-630nm (can penetrate deeper into tissue)
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Experiments without tissue damage possible
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Drosophila as testsubject?
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Experiment
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Study freely moving test
subjects
Led light with different:
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wavelength
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cw vs pulsed
ReaChR channels in
different neuron types
(1)
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ReaChR in Drosophila
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Gr5a receptor neurons
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studying PER (proboscis extension reflex)
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comparison of different channel types
ReaChR channel:
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Only light sensitive channel
leading to robust response to
green (530nm) and red
(627nm) light
dTrpA1 channel (thermal
activated):
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No response at high
surrounding T
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ReaChR in Drosophila
continuous illumination:
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PER decays
exponentially
➔ depolarization
block in
Gr5a neurons (cf.
thermal channels)
pulsed illumination (1Hz):
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repeated spiking when light
is activated
(2)
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CNS neuron activation:
Testing different neuron
types in CNS (central
nervous system) with red
and blue light-sensitive
channels
Various behavioral
responses expected in
channels e.g: wing
extension reflex (male
courtship song)
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response for ReaChR
channel (green and red)
no response for blue
channel (CNS too deep in
tissue)
LED tuned behavior;
experiments tunable with:
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light intensity
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pulse frequency
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Wing extension reflex
2 types of neurons known to trigger wing extension reflex,
P1 and pIP10, but their role is not nown yet:
(1)
(2)
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Wing extension reflex
2 types of neurons known to trigger wing extension reflex,
P1 and pIP10, but their role is not nown yet:
(1)
(2)
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Wing extension reflex
neuron type
control type
behavior
response
P1 neuron
probabilistic
during illumination
period
17,5 ± 27,5sec
pIP10
deterministic
triggered with LED
start
0,08 ± 0,04sec
But both neuron types with ReaChR led to intensity
independant behavior response.
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Changes due to social factors:
Probabilistic behavior of neurons leads to assumption that
external factors could lead to changes in response.
Test: single-housing males for 7 days
Results:
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lower LED intensity leads to wing extension
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higher responses to stimulation
Induction of ReaChR in both neurons separately indicates:
P1 neurons are responsible for encanced sensitivity
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Summary
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ReaChR is fitting opsin for Drosophila studies
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high-throughput screening of behavior possible
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reduced visual artifacts due to red illumination
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intensity and frequency dependent behavior
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probabilistic (P1) vs. deterministic (pIP10)
response
social isolation can affect optogenetic behavior
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Future challenges:
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finding other social impact factors to P1 or other
neuron types
ReaChR and other opsin engineering for faster
kinetis and smaller activation light ranges
create red-shifted inhibitory opsins
study of interacting neurons activated with
different wavelengths
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Thanks for listening.
Questions?
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References:
(1)Inagaki et al., Nat Methods. 2014 March; 'Optogenetic control of freely behaving adult
Drosophila using a red-shifted channelrhodopsin'
(2) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151318/ ; Supplementary info; (12.12.2014)
(3)Fenno et al., Annual Rev. Neuroscience 2011; 'The Developement and Application of
Optogenetics'
(4)Lin et al.; Nat Neurosci. 2013 October; 'ReaChR: A red-shifted variant of channelrhodopsin
enables deep transcriptional optogentic excitation'
(5)Yizhar et al.; Neuron 2011 July; 'Optogenetics in Neural Systems'
(6)http://en.wikipedia.org/wiki/Membrane_potential
(7)http://www.stanford.edu/group/dlab/optogenetics/
(8)http://optogenetics.weebly.com/why--how.html
(9)http://hyperphysics.phy-astr.gsu.edu/hbase/biology/actpot.html
(10)https://www.youtube.com/watch?v=I64X7vHSHOE
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