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7/31/2019 8623072 Mind Control Science Fiction Made Real
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Science Fiction Made Real?Tarjani Agrawal
Ruchi Ghildiyal
Kamini Sehrawat
Avantika Lal
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Mind ControlMind Control
A dream of philosophers, science-fiction
writers, dictators and parents.
Also neuroscientists, because observation
is insufficient to find out how the brain
works.
It is necessary to manipulate specific
neurons, stimulating or silencing them.
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NeuronsNeurons
Cells in the nervous
system
Brain and spinal cord
Process and transmit
information by electricaland chemical signals
www.holisticeducator.com
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Action PotentialAction Potential
Electrical signal generated byneurons
Polarized excess positive ionsoutside
Membrane has ion channels
Ion channels open
Influx of positive ions
Depolarization
Spreads through neuron www.chm.bris.ac.uk
Life 7 Purves
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SynapseSynapse
Junction between neurons
Action potential
Neurotransmitter release
Binds to receptors on thepostsynaptic cell.
Ion channels open in thepostsynaptic cell.
Excitatory or inhibitory
www.georgiapainphysicians.com
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Historical ApproachesHistorical Approaches
Surgery and stimulating electrodes
Limited resolution
Cumbersome
Cannot target specific neuron
type
Voltage and Ca2+ sensitive dyes
Nonspecific
www.thedevicesite.com
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Early OptogeneticsEarly Optogenetics
Optics + genetics
Light mediated uncaging
Light unblocksneurotransmitter
Chemical modification of
ion channels
Light changes protein
conformationwww.public.asu.edu
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Channelrhodopsin-2Channelrhodopsin-2
Light-gated cation channel.
Contains all-trans-retinal
Absorbs blue light (max =480 nm).
All-trans to 13-cis-retinal.
Conformational change
Pore opening
Cation influx
Action potential
Modified from commons.wikimedia.org
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Cell Specific PromotersCell Specific Promoters
A promoter is a DNA sequence that facilitatesexpression of a gene.
If the promoter sequence is
present before the gene, thegene can give rise to a protein.
Cell-specific promoters - active only in a certain set of
cells.
Cell specific promoter+ ChR2+fluorescent protein
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Lentiviral DeliveryLentiviral Delivery
Spherical envelopedretroviruses
Lentiviral delivery - Highproduction, lowimmunogenicity, fast
Cell specific promoter+ChR2+fluorescent protein inlentivirus
Injected into brain
Virus inserts genes intocellular genome
len.epfl.ch
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Mind Control?Mind Control?
Promoter expressed in few cells
These cells have ChR2 andfluoresce.
Optical fiber - Blue light
Action potential in specific neurons
These neurons control specific
behaviors
Control the neurons Control themind.
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Light - Controlled LengthLight - Controlled Length
ChR2 in neurons thatproduce GABA(inhibitory) inC. elegans
Blue light
Body wall muscles relax
Elongation
Neurons that produce
acetylcholine (excitatory)
Blue light
Contraction
www.zmescience.com
GABA ACh
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Sleep-Wake TransitionSleep-Wake Transition
Hypocretin protein thought to be
involved in awakening
ChR2 in hypocretin producing
neurons of mice
Blue light
Action potential
Hypocretin release
Increased wakefulness
softexpressions.com, www.mkbt.org.uk
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P2X2: An ATP-Gated ChannelP2X2: An ATP-Gated Channel Cation channel, opens on binding ATP
Targeted using cell-specific promoter
ATP made inert by attaching a photoremovable
blocking group.
Introduced into brain
UV light
ATP released
Channel opens
Cation entry
Action Potential
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Remote Controlled FliesRemote Controlled Flies
Escape reflex rapid legextension and flying
Controlled by 2 commandneurons
Light mediated excitation
of command neurons
Lift-off!
devcell.bio.uci.edu
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Movement in FliesMovement in Flies
Loss of dopamine-producingneurons in flies = impairedmovement
Light-induced activity ofdopamine-producing neurons
Locomotor activity increased
Explored different routes
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Songs of the FlySongs of the Fly Males produce a song which
attracts females.
Male flies produce a protein Fruwhich may regulate excitability orsynaptic properties of neuronsinvolved in song production.
Optical stimulation of these neuronsin females.
Male courtship song.
Females have circuitry for the songbut normally turned off.
Male and female brains wired
similarly differences arise frommaster switches www.istockphoto.com
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NpHRNpHR
Light-driven chloride pump
Contains all trans retinal
Absorbs yellow light (max = 580 nm)
Cl- entry
Hyperpolarization
Inhibits action potential
www.biochem.mpg.de
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The Two-Channel SystemThe Two-Channel System
Excitation spectrum of NpHR isshifted with respect to that of ChR2
Coexpression of both proteins in a
neuron
Different wavelengths of light
selectively activate or inactivate the
cell.
Two-knob remote control to
increase/decrease activity of
specific neurons
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Tandem Control inTandem Control in C.elegansC.elegans
NpHR and ChR2 in acetylcholine-producing neurons of C.elegans
Blue light
Body wall musclecontraction
Shortening
Yellow lightYellow light
Muscle relaxation
Elongation
www.zmescience.com
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ApplicationsApplications
So, when are we going to
invade another country with
an army of remote-controlled
flies?
Switch specific neurons on or
off at will and see their
function.
Most promising method to
understand how the brain
works. aycu37.webshots.com
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Therapeutic ApplicationsTherapeutic Applications
Mood andMood andbehaviorbehavior
ParkinsonsParkinsons
DiseaseDisease
PhotoreceptorPhotoreceptor
degenerationdegenerationDiabetesDiabetes
SleepSleep
DisordersDisorders
EpilepsyEpilepsy
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Application in Type II DiabetesApplication in Type II Diabetes
Defective insulin releasefrom pancreatic cells
ChR2 in insulin producing cells
Blue light
Depolarization
Ca2+ entry
Insulin release
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ReferencesReferences Adamantidis A.R., Zhang F., Aravanis A. M., Deisseroth K., de Lecea L. (2007), Neural substrates of awakening
probed with optogenetic control of hypocretin neurons,Nature 450(7168):420-4
Boyden E., Zhang F., Bamberg E., Nagel G., Deisseroth K. (2007), Millisecond-timescale, genetically targetedoptical control of neural activity,Nature Neuroscience 8, 1263 - 1268
Husser M., Smith S. L. (2007), Controlling neural circuits with light, Nature 446, 617-619
Knpfel T. (2008), Expanding the toolbox for remote control of neuronal circuits, Nature Methods - 5, 293-295
Liewald J., Brauner M., Stephens G., Bouhours M., Schultheis C., Zhen M., Gottschalk A (2008) , Optogenetic
analysis of synaptic function, Nature Methods 5, 895 - 902
Lima S.Q., Miesenbck G., (2005), Remote Control of Behavior through Genetically Targeted Photostimulationof Neurons, Cell, Vol. 121, 141152.
Lin B., Koizumi A., Tanaka N., Panda S., Masland R. (2008), Restoration of visual function in retinal
degeneration mice by ectopic expression of melanopsin,PNAS105 (41), 16009-16014
Miesenbock G. (2008), Lighting up the Brain, Scientific American India, 3(10), 34-41
Zhang F., Wang L., Boyden E., Deisseroth K. ( 2006), Channelrhodopsin-2 and optical control of excitable cells,Nature Methods 3(10), 785-792
Zhang F., Wang L., Brauner M., Liewald J., Kay K., Watzke N., Wood P., Bamberg E., Nagel G., Gottschalk A.,Deisseroth K. (2007), Multimodal fast optical interrogation of neural circuitry,Nature 446, 633-639
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Fura-2
Fura-2 is a ratiometric fluorescentdye which binds to free intracellular
calcium. It was the first highly used
dye for calcium imaging and
remains very popular. Fura-2 is
excited at 340 nm (Ca complex)
and 380 nm (free anion) of light,
and the ratio of the emissions at
those wavelengths is directly
correlated to the amount of
intracellular calcium. The use of the
ratio automatically cancels outconfounding variables, such as
variable dye concentration and cell
thickness, making Fura-2 one of
the most preferred tools to quantify
calcium levels.
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Light-mediated neurotransmitter uncaging A compound such as glutamate or GABA is inactivated by a blocking
moiety (eg. 2-nitrobenzyl phosphate, 1-(2-nitro) phenylethylphosphate.
light releases the blocking moiety.
Populations of neurons could be stimulated with a temporal precisionon the order of tens of milliseconds and spatial resolution down to fiftymicrometers.
Uncaging releases a ligand that activates endogenous receptorpathways in target neurons.
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Chemically modified ion channels and
receptors
light-activated forms of the Shaker potassium channel and the iGluR6receptor by attaching a photoswitch close to the active site of each receptor
the photoswitch consists of a photoisomerizable azobenzene group,covalently attached to an agonist.
Switching between long (580 nm) and short (380 nm) wavelengthillumination can alternate the azobenzene arm between cis and transisomers, toggling the ligand in and out of the proteins target site (the poreor ligand-binding domain).
the same azobenzene linker can work with multiple receptors and channels.
takes seconds for the cell to respond to light stimulation.
But posing challenges forin vivo work (as with uncaging methods), theazobenzene compound must be supplemented.
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Lentiviral Delivery
Spherical Enveloped Retroviruses
Viral integrase inserts viral genome into cellular genome at a
random position.
Cancer concerns
For safety reasons lentiviral vectors never carry the genes required
for their replication.
To produce a lentivirus, several plasmids are transfected into a
packaging cell line. One or more plasmids encode the virion
proteins, such as the capsid and the reverse transcriptase. Another
plasmid contains the genetic material to be delivered by the vector.
It is transcribed to produce the viral genome .
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Effect of sustained illumination
upon absorption of a photon of light, the
retinal molecule flips toward the
cytoplasmic side, losing a proton to the
nearby Asp side chain.
If NpHR is illuminated for 10 min., the
efficacy very slightly decreases due to the
accumulation of the deprotonated form.
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Light-controlled length Introduced ChR2 + YFP using two cell typespecific
promoters:(i) Punc-47, which normally drives expression of thevesicular GABA transporter in GABAergic neurons (from theintegrated transgene zxIs3);(ii) Punc-17, which normally drives expression of thevesicular acetylcholine transporter in cholinergic neurons(from the integrated transgene zxIs6).
Grew worms on medium containing all-trans retinal.
activated ChR2-YFP in zxIs3 (GABA) worms swimming by450490 nm light - swimming behavior blocked. On a solid substrate, illuminated zxIs3 worms exhibited
paralysis, because of relaxation of body-wall muscle cells.The body elongated up to 104% within 350 ms. Reversible.
During 10 s of illumination, worms partially recovered fromparalysis after 3 s. Remained partially elongated even after60 s, suggesting sustained GABA release.
Elongation abolished in mutants that lacked the vGAT.
photostimulated cholinergic neurons in worms expressingzxIs6 - rapid contraction to 92.0% of the initial length.Contractions reached a maximal level after 200300 ms.Contractions were sustained during 60 s of illumination.
Transgenic worms grown without all-trans retinal and wild-type worms showed no effect when illuminated.
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Drosophila experiments Giant fiber system controls motor neurons innervating flight musculature
P2X2 ATP activated cation channel
ATP rendered inert by blocking group - P3-[1-(4,5-dimethoxy-2-nitrophenyl)ethyl]-ATP
P2X2 expressed in GF neurons by cell-specific promoter
UV light releases ATP
Escape movements in 82% trials
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Dopamine induced motion
Promoter of the tyrosine hydroxylase gene for dopaminebiosynthesis
17 mw/mm2, 4 150 ms pulses spaced 1.5 s apart
Reduced frequency of pausing, shorter pauses
Average speed unchanged
Flies ventured into the center of the field more often
Dopaminergic neurons facilitate movement (increased activity) andalso reward assessment. Altered reward expectation might beresponsible for more adventurous exploration.
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Songs of the fly
Courtship song is male-specific. It consists of a humming sound and a rhythmically
patterned pulse song, which stimulate the female to mate. Pulse song communicatesspecies-specific information, allowing females to recognize conspecific males.
examination of the mesothoracic ganglion (Msg), revealed that expression of the sex-determination gene fruitless (fru) is necessary for song production
Fru has been postulated to be a neural sex-determination factor that directs the
development of at least two male-specific characteristics, namely courtship behaviourand formation of the muscle of Lawrence (MOL).
Fru protein is not detected in the female CNS.
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Application in photoreceptor degeneration
Loss of photoreceptor cells, as happens ina number of human diseases, leads to
irreversible blindness.
Lin et al. (2008) used a viral vector toexpress in a large number of retinal
ganglion cells the light sensitive protein
melanopsin photoresponses in these cells even after
degeneration of the photoreceptors
enhancement of visual function
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Application in Epilepsy
Mutations in genes coding for ion channels
Altered inhibition/excitation balance inneurons
Might be ameliorated by activating NpHRand/or ChR2 targeted to specific cell
types.