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Saad Abbasi, Lindsey Biggs

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OPTOGENETICS and Caged compounds Saad Abbasi, Lindsey Biggs and Brennan Paedae * * * * * * * * * A-D= slice. A,B= whole cell current clamp demonstrates normal current ... – PowerPoint PPT presentation

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Title: Saad Abbasi, Lindsey Biggs


1
OPTOGENETICS and Caged compounds
  • Saad Abbasi, Lindsey Biggs
  • and Brennan Paedae

2
History of the development of optogenetics
Paper published by Chow et al, (2010) using
archaerhodopsin which completely shuts down the
cell.
Collaboration between Nagel, and Deisseroth (and
Boyden)
Three-gene phototransduction cascade used to
activate cells
Halorhodopsin used for neural silencing
Discovery and study of opsins
Paper on channelrhodopsin published by Georg
Nagel et al. (2003)
Use of nanoparticles and magnetic pulses to
activate specific cell types without such
invasive measures (Palle Lab?)
Papers published using OptoXR, light activated
GPCRs which modulate intracellular signalling
(Aiden,2009) and use in live primates (Han et al.
2009)
First published paper using optogenetics (Boyden
et al., 2005) on cultured hippocampal neurons,
followed by papers from several other labs
Halorhodopsin and neural chloride levels
Photo Halobacterium salinarum http//www.biochem.
mpg.de/en/eg/oesterhelt/web_page_list/Org_Hasal/in
dex.html
3
1970-1980s 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 1970s (Boyden, 2011)
  • Halorhodopsin chloride pump that maintains salt
    concentration discovered in late 1970s (Boyden,
    2011)
  • Sensory rhodopsin 1phototactic response
  • to orange light discovered in 1980s
    (Boyden, 2011)
  • Sensory rhodopsin 2 phototactic response
  • to blue light.
  • 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.mpi-magdeburg.mpg.de/research/groups/mn
a/smallnet.html?pp1
http//www.biochem.mpg.de/en/eg/oesterhelt/web_pag
e_list/Org_Hasal/index.html
4
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.

5
History of the development of optogenetics
Paper published by Chow et al, (2010) using
archaerhodopsin which completely shuts down the
cell.
Collaboration between Nagel, and Deisseroth (and
Boyden)
Halorhodopsin used for neural silencing
Discovery and study of opsins
Paper on channelrhodopsin published by Georg
Nagel et al. (2003)
Use of nanoparticles and magnetic pulses to
activate specific cell types without such
invasive measures (Palle Lab?)
Papers published using OptoXR, light activated
GPCRs which modulate intracellular signalling
(Aiden,2009) and use in live primates (Han et al.
2009)
First published paper using optogenetics (Boyden
et al., 2005) on cultured hippocampal neurons,
followed by papers from several other labs
Halorhodopsin and neural chloride levels
Photo chARGe in cultured hippocampal neuron (GFP
tagged) (Zemelman, 2002)
6
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.

7
History of the development of optogenetics
Paper published by Chow et al, (2010) using
archaerhodopsin which completely shuts down the
cell.
Collaboration between Nagel, and Deisseroth (and
Boyden)
Halorhodopsin used for neural silencing
Discovery and study of opsins
Let there be light! Channelrhopsin 2 is light
sensitive.
Use of nanoparticles and magnetic pulses to
activate specific cell types without such
invasive measures (Palle Lab?)
Papers published using OptoXR, light activated
GPCRs which modulate intracellular signalling
(Aiden,2009) and use in live primates (Han et al.
2009)
First published paper using optogenetics (Boyden
et al., 2005) on cultured hippocampal neurons,
followed by papers from several other labs
Halorhodopsin and neural chloride levels
Photo ChR2 conjugated to RFP http//en.wikipedia.
org/wiki/Channelrhodopsin
8
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
9
History of the development of optogenetics
Paper published by Chow et al, (2010) using
archaerhodopsin which completely shuts down the
cell.
Collaboration between Nagel, and Deisseroth (and
Boyden)
Halorhodopsin used for neural silencing
Discovery and study of opsins
Let there be light! Channelrhopsin 2 is light
sensitive.
First published paper using optogenetics (Boyden
et al., 2005).
Use of nanoparticles and magnetic pulses to
activate specific cell types without such
invasive measures (Palle Lab?)
Papers published using OptoXR, light activated
GPCRs which modulate intracellular signalling
(Aiden,2009) and use in live primates (Han et al.
2009)
Halorhodopsin and neural chloride levels
Photo ChR2 response to light (cultured
hippocampal neuron) (Boyden, 2011 from Boyden et
al. 2005 paper)
10
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

11
History of the development of optogenetics
Paper published by Chow et al, (2010) using
archaerhodopsin which completely shuts down the
cell.
Collaboration between Nagel, and Deisseroth (and
Boyden)
Halorhodopsin used for neural silencing
Discovery and study of opsins
Let there be light! Channelrhopsin 2 is light
sensitive.
First published paper using optogenetics (Boyden
et al., 2005).
Use of nanoparticles and magnetic pulses to
activate specific cell types without such
invasive measures (Palle Lab?)
Papers published using OptoXR, light activated
GPCRs which modulate intracellular signalling
(Aiden,2009) and use in live primates (Han et al.
2009)
Halorhodopsin and neural chloride levels
Photo Halorhodpsin Lief et al., 2011
12
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)
13
History of the development of optogenetics
Paper published by Chow et al, (2010) using
archaerhodopsin which completely shuts down the
cell.
Collaboration between Nagel, and Deisseroth (and
Boyden)
Halorhodopsin used for neural silencing
Discovery and study of opsins
Papers published on optogenetics in primates.
Let there be light! Channelrhopsin 2 is light
sensitive.
First published paper using optogenetics (Boyden
et al., 2005).
Use of nanoparticles and magnetic pulses to
activate specific cell types without such
invasive measures (Palle Lab?)
Halorhodopsin and neural chloride levels
14
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.

15
History of the development of optogenetics
Collaboration between Nagel, and Deisseroth (and
Boyden)
Archaerhodopsin completely silences neurons.
Halorhodopsin used for neural silencing
Discovery and study of opsins
Papers published on optogenetics in primates.
Let there be light! Channelrhopsin 2 is light
sensitive.
First published paper using optogenetics (Boyden
et al., 2005).
Use of nanoparticles and magnetic pulses to
activate specific cell types without such
invasive measures (Palle Lab?)
Halorhodopsin and neural chloride levels
16
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).

17
History of the development of optogenetics
Collaboration between Nagel, and Deisseroth (and
Boyden)
Archaerhodopsin completely silences neurons.
Halorhodopsin used for neural silencing
Discovery and study of opsins
Papers published on optogenetics in primates.
Let there be light! Channelrhopsin 2 is light
sensitive.
First published paper using optogenetics (Boyden
et al., 2005).
Use of nanoparticles and magnetic pulses to
activate specific cell types without such
invasive measures
Halorhodopsin and neural chloride levels
PhotoArnd Pralle, physics prof. at Univ of
Buffalo. Research on magnetic nanoparticles
18
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19
Parkinsons 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
20
Basal ganglia circuitry
Substantia nigra pars reticulata
Substantia nigra pars reticulata
http//www.ncbi.nlm.nih.gov/books/NBK10847/
21
  • D1-Cre mice expressed ChR2-YFP in striatum and
    fibers projecting through globus pallidus to SNr.
  • D2-Cre mice, ChR2-YFP cells bodies were seen in
    striatum, projecting to globus pallidus.
  • ChR2 was expressed in medium spiny neurons
    (DARPP-32 MSN marker)

Supp. Fig. 1
22
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.

23
In vivo laser stimulation and recording
Silicon probe with integrated laser-couple
optical fiber.
24
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.
25
Bilateral stimulation of indirect pathway
26
  • Bilateral 6-OHDA injections caused loss of
    dopaminergic innervation in dorsomedial striatum
    and Parkinsonian-like motor deficits.
  • Activation of the direct pathway completely
    restored pre-lesion motor behaviors.
  • Decreased freezing
  • Increased locomotor activity.

27
Restoration of motor behaviors by direct pathway
activation
28
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.)

29
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30
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31
  • 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)

32
OPTOGENETICS
http//www.fmls-institute.de/index.php?idneurobio
chemistry
http//blogs.physicstoday.org/industry09/
https//encrypted-tbn0.google.com/images?qtbnANd
9GcQL5mvVzT07u1TnAgnMC7QStw5V2BJ3-5ytYFM5lVolKD34Q
koEKQ
http//www.aan.com/elibrary/neurologytoday/?event
home.showArticleidovid.com/bib/ovftdb/00132985-
201107070-00006
http//czechfood.blogspot.com/2011/07/optogenetics
-optogenetica.html
  • Lindsey Biggs and Brennan Paedae

http//www.stanford.edu/shenoy/GroupResearchPubli
cations.htm
https//encrypted-tbn1.google.com/images?qtbnANd
9GcQVAQDbKMATn9SkXr45jkleAr9O9HqOum4wjihhLB4161Okh
NO68w
http//www.nytimes.com/2011/05/17/science/17optics
.html?_r1
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