OPTOGENETICS and
Caged compounds
Saad Abbasi, Lindsey Biggs
and Brennan Paedae
History of the development of optogenetics
Three-gene
phototransduc
tion cascade
used to
activate cells
Discovery and
study of opsins
1970’s
Collaboration
between
Nagel, and
Deisseroth
(and Boyden)
Halorhodopsin
used for neural
silencing
Paper
published by
Chow et al,
(2010) using
archaerhodops
in which
completely
shuts down
the cell.
1999
Halorhodopsin
and neural
chloride levels
Photo: Halobacterium salinarum
http://www.biochem.mpg.de/en/eg/oester
helt/web_page_list/Org_Hasal/index.html
Paper on
channelrhodo
psin published
by Georg
Nagel et al.
(2003)
First published
paper using
optogenetics
(Boyden et al.,
2005) on
cultured
hippocampal
neurons,
followed by
papers from
several other
labs
Papers
published
using OptoXR,
light activated
GPCR’s which
modulate
intracellular
signalling
(Aiden,2009)
and use in live
primates (Han
et al. 2009)
Use of
nanoparticles
and magnetic
pulses to
activate
specific cell
types without
such invasive
measures
(Palle Lab?)
1970-1980’s
Discovery and study of opsins
 Halobacterium salinarum:
 Motile organisms
 Can live with light as the only energy source (Bacteriorhodopsin)
 4 retinal proteins:
 Bacteriorhodopsin: light driven proton pump that converts light to energy source [discovered
in early 1970’s (Boyden, 2011)]
 Halorhodopsin: chloride pump that maintains salt concentration [discovered in late 1970’s
(Boyden, 2011)]
 Sensory rhodopsin 1:phototactic response
to orange light [discovered in 1980’s (Boyden, 2011)]
http://www.mpi Sensory rhodopsin 2: phototactic response
magdeburg.mp
g.de/research/g
to blue light.
roups/mna/sma
llnet.html?pp=1
 BUT….. The organisms expressing
these rhodopsins function in environments
with high salt concentrations, so there was
little optimism for function in neural tissue.
http://www.biochem.mpg.de
/en/eg/oesterhelt/web_page
_list/Org_Hasal/index.html
1999
Halorhopsin and neural chloride levels
 Okuno et al. re-opened the possibility of using rhodopsins in
neural tissue with his 1999 paper.
 Compared to H. salinarum, rhodopsins from Natronomona
pharaonis functioned best at chloride concentrations that are
similar to concentrations seen in neural tissue.
 BUT….. It was still unknown whether these rhodopsins could
be expressed and functional in neural tissue.
History of the development of optogenetics
Three-gene
phototransduc
tion cascade
used to
activate cells
Discovery and
study of opsins
1970’s
1999
Collaboration
between
Nagel, and
Deisseroth
(and Boyden)
Halorhodopsin
used for neural
silencing
Paper
published by
Chow et al,
(2010) using
archaerhodops
in which
completely
shuts down
the cell.
2002
Halorhodopsin
and neural
chloride levels
Photo: chARGe in cultured hippocampal
neuron (GFP tagged)
(Zemelman, 2002)
Paper on
channelrhodo
psin published
by Georg
Nagel et al.
(2003)
First published
paper using
optogenetics
(Boyden et al.,
2005) on
cultured
hippocampal
neurons,
followed by
papers from
several other
labs
Papers
published
using OptoXR,
light activated
GPCR’s which
modulate
intracellular
signalling
(Aiden,2009)
and use in live
primates (Han
et al. 2009)
Use of
nanoparticles
and magnetic
pulses to
activate
specific cell
types without
such invasive
measures
(Palle Lab?)
2002
chARGe may be the answer!
 Gero Miesenbrook and colleagues created a three-gene
Drosophilia phototransduction cascade that could be expressed in
cultured hippocampal neurons. When exposed to light, cells
expressing chARGe were more active. (Zemelman, 2002).
 chARGe= Arrestin-2 rhodopsin coupled to alpha subunit of g-protein.
 However, the activation of neurons was not instantaneous, but took
several seconds.
 A temporally precise method of activation was still
necessary.
History of the development of optogenetics
Three-gene
phototransduc
tion cascade
used to
activate cells
Discovery and
study of opsins
1970’s
Collaboration
between
Nagel, and
Deisseroth
(and Boyden)
1999
Halorhodopsin
and neural
chloride levels
Photo: ChR2
conjugated to RFP
http://en.wikipedia.org/
wiki/Channelrhodopsin
2002
2003
Let there be
light!
Channelrhopsin
2 is light
sensitive.
Halorhodopsin
used for neural
silencing
Paper
published by
Chow et al,
(2010) using
archaerhodops
in which
completely
shuts down
the cell.
2004
First published
paper using
optogenetics
(Boyden et al.,
2005) on
cultured
hippocampal
neurons,
followed by
papers from
several other
labs
Papers
published
using OptoXR,
light activated
GPCR’s which
modulate
intracellular
signalling
(Aiden,2009)
and use in live
primates (Han
et al. 2009)
Use of
nanoparticles
and magnetic
pulses to
activate
specific cell
types without
such invasive
measures
(Palle Lab?)
2003 and 2004
Channelrhodopsin-2
 Chlamydomona reinhardtii use
channelrhodopsin-2 (ChR2) to drive
phototaxis (Sineshchekov et al. 2002).
 ChR2 is a light gated cation channel which
produces movement in C. reinhardtii.
 Nagel and colleaques used ChR2 in
oocytes and HEK cells to show that it
could be used to depolarize cells via
illumination (Nagel et al. 2003).
 In 2004, a collaboration between Georg
Nagel, Karl Deisseroth (and Edward
Boyden) began.
 Nagel had since discovered that not much
all-trans retinal needed to be added to the
cultures for ChR2 function.
Boyden, 2011
History of the development of optogenetics
Three-gene
phototransduc
tion cascade
used to
activate cells
Discovery and
study of opsins
1970’s
Collaboration
between
Nagel, and
Deisseroth
(and Boyden)
1999
Halorhodopsin
and neural
chloride levels
Photo: ChR2 response
to light (cultured
hippocampal neuron)
(Boyden, 2011 from
Boyden et al. 2005
paper)
2002
2003
Let there be
light!
Channelrhopsin
2 is light
sensitive.
2004
Halorhodopsin
used for neural
silencing
Paper
published by
Chow et al,
(2010) using
archaerhodops
in which
completely
shuts down
the cell.
2005
First published
paper using
optogenetics
(Boyden et al.,
2005).
Papers
published
using OptoXR,
light activated
GPCR’s which
modulate
intracellular
signalling
(Aiden,2009)
and use in live
primates (Han
et al. 2009)
Use of
nanoparticles
and magnetic
pulses to
activate
specific cell
types without
such invasive
measures
(Palle Lab?)
2005
First published paper using optogenetics
 In 2005, Edward Boyden, Karl Deisseroth and
colleagues published the first paper using
optogentics in cultured mammalian hippocampal
neurons.
 ChR2 was expressed, and functional in neurons.
 Current produced by ChR2 activation was enough to
produce action potentials.
 ChR2 had a low rate of inactivation and quick recovery
time.
 Several other labs published papers using similar
techniques soon after. These methods had been on
the minds of many research groups!




Yawo Lab – 11/05- intact mammal brain circuits
Herlitze and Landmesser labs-11/05 chick spinal cord
Nagel and Gottschalk labs-12/05; behaving worm
Pan Lab- 4/06; retina
(Boyden, et al. 2005)
History of the development of optogenetics
Three-gene
phototransduc
tion cascade
used to
activate cells
Discovery and
study of opsins
1970’s
Collaboration
between
Nagel, and
Deisseroth
(and Boyden)
1999
Halorhodopsin
and neural
chloride levels
Photo: Halorhodpsin
Lief et al., 2011
2002
2003
Let there be
light!
Channelrhopsin
2 is light
sensitive.
2004
Halorhodopsin
used for neural
silencing
2005
First published
paper using
optogenetics
(Boyden et al.,
2005).
Paper
published by
Chow et al,
(2010) using
archaerhodops
in which
completely
shuts down
the cell.
2007
Papers
published
using OptoXR,
light activated
GPCR’s which
modulate
intracellular
signalling
(Aiden,2009)
and use in live
primates (Han
et al. 2009)
Use of
nanoparticles
and magnetic
pulses to
activate
specific cell
types without
such invasive
measures
(Palle Lab?)
2007
N. pharaonis Halorhodpsin
 March 2007-Xue Han and Boyden
published data showing that N. Pharaonis
halorhodopsin could be used for neural
silencing. (Cl- channel)
 Few weeks later, Karl et al published a
paper showing the same conclusion and
that it could be used to modify behavior
in C. elegans.
 BUT…..halorhodopsin had low
magnitude currents, would get stuck in
inactivation phase after long stimulation,
and had a slow recovery period.
(Boyden, 2011)
History of the development of optogenetics
Three-gene
phototransduc
tion cascade
used to
activate cells
Discovery and
study of opsins
1970’s
Collaboration
between
Nagel, and
Deisseroth
(and Boyden)
1999
Halorhodopsin
and neural
chloride levels
2002
2003
Let there be
light!
Channelrhopsin
2 is light
sensitive.
2004
Paper
published by
Chow et al,
(2010) using
archaerhodops
in which
completely
shuts down
the cell.
Halorhodopsin
used for neural
silencing
2005
First published
paper using
optogenetics
(Boyden et al.,
2005).
2007
2009
Papers
published on
optogenetics
in primates.
Use of
nanoparticles
and magnetic
pulses to
activate
specific cell
types without
such invasive
measures
(Palle Lab?)
2009
Optogenetics in primates
 Boyden and Desimone published research on primate brains,
suggesting these methods could someday be used for clinical
purposes.
 Conclusions:
 ChR2 can be expressed in maquaqe monkeys to modulate
activity in specific subsets of neurons, without inducing neuron
death and immune responses.
History of the development of optogenetics
Three-gene
phototransduc
tion cascade
used to
activate cells
Discovery and
study of opsins
1970’s
Collaboration
between
Nagel, and
Deisseroth
(and Boyden)
1999
Halorhodopsin
and neural
chloride levels
2002
2003
Let there be
light!
Channelrhopsin
2 is light
sensitive.
2004
Archaerhodop
sin completely
silences
neurons.
Halorhodopsin
used for neural
silencing
2005
First published
paper using
optogenetics
(Boyden et al.,
2005).
2007
2009
Papers
published on
optogenetics
in primates.
2010
Use of
nanoparticles
and magnetic
pulses to
activate
specific cell
types without
such invasive
measures
(Palle Lab?)
2010
Arechearhodopsin
 The solution to the limitations of
halorhodopsin:
Archearhodopsin
 The paper published in Jan 2010 by
Chow and Boyden showed that
archearhodopsin:
 Could completely shut down the cell.
 Had rapid recovery after long stimulation
 hyperpolarize the cell by pumping protons
out of the cell (Chow et al. 2010).
History of the development of optogenetics
Three-gene
phototransduc
tion cascade
used to
activate cells
Discovery and
study of opsins
1970’s
Collaboration
between
Nagel, and
Deisseroth
(and Boyden)
1999
Halorhodopsin
and neural
chloride levels
Photo:Arnd Pralle,
physics prof. at Univ of
Buffalo.
Research on magnetic
nanoparticles
2002
2003
Let there be
light!
Channelrhopsin
2 is light
sensitive.
2004
Archaerhodop
sin completely
silences
neurons.
Halorhodopsin
used for neural
silencing
2005
First published
paper using
optogenetics
(Boyden et al.,
2005).
2007
2009
Papers
published on
optogenetics
in primates.
2010
2011
Use of
nanoparticles and
magnetic pulses to
activate specific cell
types without such
invasive measures
Parkinson’s Disease
 Degenerative disorder of the CNS.
 Most cases occur after the age of 50
 Causes:
 Death of cells in the Substantia nigra which produce dopamine.
 Cause of cell loss is unknown, but is genetic is some cases.
 Symptoms:
 Movement-related: shaking (tremor), rigidity, slow movements, difficulty
walking and with gait postural instability.
 Cognitive and behavioral problems in more advanced stages: dementia,
sensory, sleep and emotional problems.
 Diagnosis is usually based on symptoms, with neuroimaging used for
confirmation.
 Treatments:
 L-Dopa (which can cross the BBB) and other dopamine agonists.
 With the loss of DA producing neurons, these treatments become ineffective and can
cause diskinesia (involuntary writhing movements)
 Deep-brain stimulation and lesion surgery are used as a last resort.
www.wikipedia.org
Basal ganglia circuitry
Substantia nigra
pars reticulata
Substantia nigra
pars reticulata
Substantia
nigra pars
reticulata
http://www.ncbi.nlm.nih.gov/books/NBK10847/
 D1-Cre mice expressed
ChR2-YFP in striatum
and fibers projecting
through globus pallidus
to SNr.
 D2-Cre mice, ChR2YFP cells bodies were
seen in striatum,
projecting to globus
pallidus.
 ChR2 was expressed in
medium spiny neurons
(DARPP-32 MSN
marker)
Supp. Fig. 1
Whole cell slice electrophysiology
 Whole cell slice eletrophysiology was used to verify ChR2
expression in D1 and D2 specific neurons.
 Current-firing relationship for direct and indirect pathways
were consistent with previous data (a,b)
 470 nm illumination of the ChR2 expressing neurosn
produced light-evoked inward current and increased spiking.
Silicon probe with integrated laser-couple optical fiber.
In vivo laser stimulation and recording
Behavioral data
 Activation of direct
pathway (D1) increased
percent of time in
ambulation, and
decreased freezing and
fine motor movements.
 Activation of indirect
pathway (D2) decreased
ambulation and fine
movements and
increased the time spent
freezing.
Causal relationship between direct
pathway in increasing motor behavior
and between indirect pathway and
increased freezing responses.
Bilateral stimulation of indirect
pathway
 Bilateral 6-OHDA injections
caused loss of dopaminergic
innervation in dorsomedial
striatum and Parkinsonianlike motor deficits.
 Activation of the direct
pathway completely restored
pre-lesion motor behaviors.
 Decreased freezing
 Increased locomotor activity.
Restoration of motor behaviors by
direct pathway activation
Conclusions:
 This study provides evidence in a behaving animal that
activation of the direct and indirect pathways are modulating
motor activity as previously suggested.
 This technique offers temporally precise activation of the
circuitry (compared to pharmacological blockade, lesions, or
transgenic mice).
 This technique also allows for a quick return to baseline firing
rates and activity.
 Activation of the direct pathway in basal ganglia can ameliorate
motor deficits caused by loss of striatal neurons (which are
modulated by DA release from substantia nigra.)
 Halorhopsin from H. salinarum functions best at high
chloride levels. (Left)
 Halorhopsin from N. pharaonis functions best at lower
chloride concentrations, which are similar to neural tissue.
 (Okuno et al., 1999)
http://www.fmlsinstitute.de/index.php?id=neurobioche
mistry
http://www.aan.com/elibrary/n
eurologytoday/?event=home.s
howArticle&id=ovid.com:/bib/o
vftdb/00132985-20110707000006
http://blogs.phys
icstoday.org/ind
ustry09/
https://encryptedtbn0.google.com/images?q=tbn:AN
d9GcQL5mvVzT07u1TnAgnMC7Q
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OPTOGENETICS
Lindsey Biggs and Brennan Paedae
http://czechfood.
blogspot.com/20
11/07/optogeneti
csoptogenetica.htm
l
http://www.stanford.edu/~s
henoy/GroupResearchPubl
ications.htm
http://www.nytimes.com/2011/05/17/science/17optics.html?_r=1
https://encryptedtbn1.google.com/images?q=tbn:ANd9Gc
QVAQDbKMATn9SkXr45jkleAr9O9HqOu
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