Saad Abbasi, Lindsey Biggs and Brennan Paedae

OPTOGENETICS andCaged compounds

1970’s 1999

Three-gene phototransduction cascade used to activate cells

History of the development of optogenetics

Collaboration between Nagel, and Deisseroth (and Boyden)

Halorhodopsin used for neural silencing

Paper published by Chow et al, (2010) using archaerhodopsin which completely shuts down the cell.

Use of nanoparticles and magnetic pulses to activate specific cell types without such invasive measures (Palle Lab?)

Papers published using OptoXR, light activated GPCR’s which modulate intracellular signalling (Aiden,2009) and use in live primates (Han et al. 2009)

Halorhodopsin and neural chloride levels

Discovery and study of opsins

Paper on channelrhodopsin 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

Photo: Halobacterium salinarumhttp://www.biochem.mpg.de/en/eg/oesterhelt/web_page_list/Org_Hasal/index.html

1970-1980’sDiscovery and study of opsins

Halobacterium salinarum:Motile organismsCan 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)] Sensory rhodopsin 2: phototactic response to blue light.

BUT….. The organisms expressing these rhodopsins function in environmentswith 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

http://www.mpi-magdeburg.mpg.de/research/groups/mna/smallnet.html?pp=1

1999Halorhopsin and neural chloride levelsOkuno 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.

1970’s 1999 2002

Three-gene phototransduction cascade used to activate cells

History of the development of optogenetics

Collaboration between Nagel, and Deisseroth (and Boyden)

Halorhodopsin used for neural silencing

Paper published by Chow et al, (2010) using archaerhodopsin which completely shuts down the cell.

Use of nanoparticles and magnetic pulses to activate specific cell types without such invasive measures (Palle Lab?)

Papers published using OptoXR, light activated GPCR’s which modulate intracellular signalling (Aiden,2009) and use in live primates (Han et al. 2009)

Halorhodopsin and neural chloride levels

Discovery and study of opsins

Paper on channelrhodopsin 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

Photo: chARGe in cultured hippocampal neuron (GFP tagged)(Zemelman, 2002)

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.

2003 20041970’s 1999 2002

Three-gene phototransduction cascade used to activate cells

History of the development of optogenetics

Collaboration between Nagel, and Deisseroth (and Boyden)

Halorhodopsin used for neural silencing

Paper published by Chow et al, (2010) using archaerhodopsin which completely shuts down the cell.

Use of nanoparticles and magnetic pulses to activate specific cell types without such invasive measures (Palle Lab?)

Papers published using OptoXR, light activated GPCR’s which modulate intracellular signalling (Aiden,2009) and use in live primates (Han et al. 2009)

Halorhodopsin and neural chloride levels

Discovery and study of opsins

Let there be light! Channelrhopsin 2 is light sensitive.

First published paper using optogenetics (Boyden et al., 2005) on cultured hippocampal neurons, followed by papers from several other labs

Photo: ChR2 conjugated to RFPhttp://en.wikipedia.org/wiki/Channelrhodopsin

2003 and 2004Channelrhodopsin-2Chlamydomona 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

2003 2004 20051970’s 1999 2002

Three-gene phototransduction cascade used to activate cells

History of the development of optogenetics

Collaboration between Nagel, and Deisseroth (and Boyden)

Halorhodopsin used for neural silencing

Paper published by Chow et al, (2010) using archaerhodopsin which completely shuts down the cell.

Use of nanoparticles and magnetic pulses to activate specific cell types without such invasive measures (Palle Lab?)

Papers published using OptoXR, light activated GPCR’s which modulate intracellular signalling (Aiden,2009) and use in live primates (Han et al. 2009)

Halorhodopsin and neural chloride levels

Discovery and study of opsins

Let there be light! Channelrhopsin 2 is light sensitive.

First published paper using optogenetics (Boyden et al., 2005).

Photo: ChR2 response to light (cultured hippocampal neuron)(Boyden, 2011 from Boyden et al. 2005 paper)

2005First 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 circuitsHerlitze and Landmesser labs-11/05 chick

spinal cordNagel and Gottschalk labs-12/05; behaving

wormPan Lab- 4/06; retina

(Boyden, et al. 2005)

2003 2004 2005 20071970’s 1999 2002

Three-gene phototransduction cascade used to activate cells

History of the development of optogenetics

Collaboration between Nagel, and Deisseroth (and Boyden)

Paper published by Chow et al, (2010) using archaerhodopsin which completely shuts down the cell.

Use of nanoparticles and magnetic pulses to activate specific cell types without such invasive measures (Palle Lab?)

Papers published using OptoXR, light activated GPCR’s which modulate intracellular signalling (Aiden,2009) and use in live primates (Han et al. 2009)

Halorhodopsin and neural chloride levels

Discovery and study of opsins

Let there be light! Channelrhopsin 2 is light sensitive.

First published paper using optogenetics (Boyden et al., 2005).

Photo: Halorhodpsin Lief et al., 2011

Halorhodopsin used for neural silencing

2007N. 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)

2003 2004 2005 2007 20091970’s 1999 2002

Three-gene phototransduction cascade used to activate cells

History of the development of optogenetics

Collaboration between Nagel, and Deisseroth (and Boyden)

Paper published by Chow et al, (2010) using archaerhodopsin which completely shuts down the cell.

Use of nanoparticles and magnetic pulses to activate specific cell types without such invasive measures (Palle Lab?)

Papers published on optogenetics in primates.

Halorhodopsin and neural chloride levels

Discovery and study of opsins

Let there be light! Channelrhopsin 2 is light sensitive.

First published paper using optogenetics (Boyden et al., 2005).

Halorhodopsin used for neural silencing

2009Optogenetics in primatesBoyden 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.

2003 2004 2005 2007 2009 20101970’s 1999 2002

Three-gene phototransduction cascade used to activate cells

History of the development of optogenetics

Collaboration between Nagel, and Deisseroth (and Boyden)

Archaerhodopsin completely silences neurons.

Use of nanoparticles and magnetic pulses to activate specific cell types without such invasive measures (Palle Lab?)

Papers published on optogenetics in primates.

Halorhodopsin and neural chloride levels

Discovery and study of opsins

Let there be light! Channelrhopsin 2 is light sensitive.

First published paper using optogenetics (Boyden et al., 2005).

Halorhodopsin used for neural silencing

2010Arechearhodopsin

The solution to the limitations of halorhodopsin:

ArchearhodopsinThe paper published in Jan 2010

by Chow and Boyden showed that archearhodopsin:Could completely shut down the

cell. Had rapid recovery after long

stimulationhyperpolarize the cell by

pumping protons out of the cell (Chow et al. 2010).

20112003 2004 2005 2007 2009 20101970’s 1999 2002

Three-gene phototransduction cascade used to activate cells

History of the development of optogenetics

Collaboration between Nagel, and Deisseroth (and Boyden)

Archaerhodopsin completely silences neurons.

Use of nanoparticles and magnetic pulses to activate specific cell types without such invasive measures

Papers published on optogenetics in primates.

Halorhodopsin and neural chloride levels

Discovery and study of opsins

Let there be light! Channelrhopsin 2 is light sensitive.

First published paper using optogenetics (Boyden et al., 2005).

Photo:Arnd Pralle, physics prof. at Univ of Buffalo.Research on magnetic nanoparticles

Halorhodopsin used for neural silencing

Parkinson’s DiseaseDegenerative disorder of the CNS.Most cases occur after the age of 50Causes:

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

http://www.ncbi.nlm.nih.gov/books/NBK10847/

Substantia nigra pars reticulata

Substantia nigra pars reticulata

Substantia nigra pars reticulata

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

Whole cell slice electrophysiologyWhole 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.

In vivo laser stimulation and recording

Silicon probe with integrated laser-couple optical fiber.

Behavioral dataActivation 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 Parkinsonian-like motor deficits.

Activation of the direct pathway completely restored pre-lesion motor behaviors. Decreased freezingIncreased 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)

Lindsey Biggs and Brennan Paedae

OPTOGENETICS

http://www.nytimes.com/2011/05/17/science/17optics.html?_r=1

https://encrypted-tbn0.google.com/images?q=tbn:ANd9GcQL5mvVzT07u1TnAgnMC7QStw5V2BJ3-5ytYFM5lVolKD34QkoEKQ

https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcQVAQDbKMATn9SkXr45jkleAr9O9HqOum4wjihhLB4161OkhNO68w

http://www.fmls-institute.de/index.php?id=neurobiochemistry

http://www.aan.com/elibrary/neurologytoday/?event=home.showArticle&id=ovid.com:/bib/ovftdb/00132985-201107070-00006

http://blogs.physicstoday.org/industry09/

http://czechfood.blogspot.com/2011/07/optogenetics-optogenetica.html

http://www.stanford.edu/~shenoy/GroupResearchPublications.htm