8623072 mind control science fiction made real

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.

8623072 mind control science fiction made real

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