Science Fiction Made Real?

Tarjani Agrawal Ruchi Ghildiyal Kamini Sehrawat Avantika Lal

Mind 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.

Neurons
Cells in the nervous system Brain and spinal cord Process and transmit information by electrical and chemical signals

www.holisticeducator.com

Action Potential
Electrical signal generated by neurons Polarized excess positive ions outside Membrane has ion channels Ion channels open Influx of positive ions Depolarization Spreads through neuron
www.chm.bris.ac.uk

Life 7 Purves

Synapse
Junction between neurons Action potential Neurotransmitter release Binds to receptors on the postsynaptic cell. Ion channels open in the postsynaptic cell. Excitatory or inhibitory
www.georgiapainphysicians.com

Historical Approaches
Surgery and stimulating electrodes Limited resolution Cumbersome Cannot target specific neuron type

Voltage and Ca2+ sensitive dyes Nonspecific

www.thedevicesite.com

Early Optogenetics
Optics + genetics Light mediated uncaging
Light unblocks neurotransmitter

Chemical modification of ion channels

Light changes protein conformation
www.public.asu.edu

Channelrhodopsin-2
Light-gated cation channel. Contains all-trans-retinal Absorbs blue light (max =480 nm). All-transto 13-cis-retinal. Conformational change Pore opening Cation influx Action potential
Modified from commons.wikimedia.org

Cell Specific Promoters

A promoter is a DNA sequence that facilitates expression of a gene. If the promoter sequence is present before the gene, the gene can give rise to a protein. Cell-specific promoters - active only in a certain set of cells. Cell specific promoter+ ChR2+fluorescent protein

Lentiviral Delivery
Spherical enveloped retroviruses Lentiviral delivery - High production, low immunogenicity, fast Cell specific promoter+ ChR2+fluorescent protein in lentivirus Injected into brain Virus inserts genes into cellular genome
len.epfl.ch

Mind Control?
Promoter expressed in few cells These cells have ChR2 and fluoresce. Optical fiber - Blue light Action potential in specific neurons These neurons control specific behaviors Control the neurons Control the mind.

Light - Controlled Length

ChR2 in neurons that produce GABA(inhibitory) in C. elegans
Blue light Body wall muscles relax Elongation

Neurons that produce acetylcholine (excitatory)

Blue light Contraction
GABA ACh www.zmescience.com

Sleep-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

P2X2: 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

Remote Controlled Flies

Escape reflex rapid leg extension and flying Controlled by 2 command neurons Light mediated excitation of command neurons Lift-off!
devcell.bio.uci.edu

Movement in Flies
Loss of dopamine-producing neurons in flies = impaired movement Light-induced activity of dopamine-producing neurons Locomotor activity increased Explored different routes

Songs of the Fly

Males produce a song which attracts females. Male flies produce a protein Fru which may regulate excitability or synaptic properties of neurons involved in song production. Optical stimulation of these neurons in females. Male courtship song. Females have circuitry for the song but normally turned off. Male and female brains wired similarly differences arise from master switches

www.istockphoto.com

NpHR
Light-driven chloride pump Contains all trans retinal Absorbs yellow light (max = 580 nm) Cl- entry Hyperpolarization Inhibits action potential
www.biochem.mpg.de

The Two-Channel System

Excitation spectrum of NpHR is shifted 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

Tandem Control in C.elegans

NpHR and ChR2 in acetylcholineproducing neurons of C.elegans Blue light Body wall muscle contraction Shortening Yellow light Muscle relaxation Elongation
www.zmescience.com

Applications
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

Therapeutic Applications
Diabetes Photoreceptor degeneration

Parkinsons Disease

Epilepsy Sleep Disorders

Mood and behavior

Application in Type II Diabetes

Defective insulin release from pancreatic cells

ChR2 in insulin producing cells Blue light Depolarization Ca2+ entry Insulin release

Application in Parkinsons Disease

Loss of dopamine producing neurons Dopamine regulates movement by stimulating parts of the basal ganglia. Treated by electrical stimulation of the basal ganglia. Optogenetic treatment is less cumbersome and would probably have less side effects.

References
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 targeted optical 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 Photostimulation of 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, PNAS 105 (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, Nature446, 633-639

Fura-2
Fura-2 is a ratiometric fluorescent dye 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 340nm (Ca complex) and 380nm (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 out confounding variables, such as variable dye concentration and cell thickness, making Fura-2 one of the most preferred tools to quantify calcium levels.

Light-mediated neurotransmitter uncaging

A compound such as glutamate or GABA is inactivated by a blocking moiety (eg. 2-nitrobenzyl phosphate, 1-(2-nitro) phenylethyl phosphate. light releases the blocking moiety. Populations of neurons could be stimulated with a temporal precision on the order of tens of milliseconds and spatial resolution down to fifty micrometers. Uncaging releases a ligand that activates endogenous receptor pathways in target neurons.

Chemically modified ion channels and receptors

light-activated forms of the Shaker potassium channel and the iGluR6 receptor 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) wavelength illumination can alternate the azobenzene arm between cis and trans isomers, toggling the ligand in and out of the proteins target site (the pore or 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 for in vivo work (as with uncaging methods), the azobenzene compound must be supplemented.

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 .

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.

Light-controlled length
Introduced ChR2 + YFP using two cell typespecific promoters: (i) Punc-47, which normally drives expression of the vesicular GABA transporter in GABAergic neurons (from the integrated transgene zxIs3); (ii) Punc-17, which normally drives expression of the vesicular 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 by 450490 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 from paralysis after 3 s. Remained partially elongated even after 60 s, suggesting sustained GABA release. Elongation abolished in mutants that lacked the vGAT. photostimulated cholinergic neurons in worms expressing zxIs6 - 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 wildtype worms showed no effect when illuminated.

Neural substrates of awakening probed with optogenetic control of hypocretin neurons

hypocretin neurons excite brain nuclei with important roles in wakefulness Hcrt::ChR2-mCherry (red) lentiviruses stereotactically delivered to lateral hypothalamus. Hcrt = mouse prepro-hypocretin gene promoter. Cells in hypothalamus slices co-expressing ChR2mCherry exhibited photocurrents on illumination with blue light. mice implanted with EEG and EMG electrodes. Wakefulness = desynchronized low-amplitude EEG/EMG activity. Without blue light, the sleepwake cycle of ChR2 animals was not different from control (Hcrt-mCherry) animals. optical fibre + 20-mW 488-nm diode laser to deliver light into the lateral hypothalamus. Photostimulation (5 and 30 Hz) during sleep reduced the latency to wakefulness of Hcrt::ChR2-mCherry animals compared to control animals. Ten-second continuous light stimulation elicited a similar effect. duration of the waking events after light delivery were not significantly different. Hcrt receptor inhibitor SB334867 blocked the effect. photostimulations less effective in Hcrt knockout mice.

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-2nitrophenyl)ethyl]-ATP P2X2 expressed in GF neurons by cell-specific promoter UV light releases ATP

Escape movements in 82% trials

Dopamine induced motion

Promoter of the tyrosine hydroxylase gene for dopamine biosynthesis 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) and also reward assessment. Altered reward expectation might be responsible for more adventurous exploration.

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 communicates species-specific information, allowing females to recognize conspecific males. examination of the mesothoracic ganglion (Msg), revealed that expression of the sexdetermination 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 behaviour and formation of the muscle of Lawrence (MOL). Fru protein is not detected in the female CNS.

Application in photoreceptor degeneration Loss of photoreceptor cells, as happens in a number of human diseases, leads to irreversible blindness. Lin et al. (2008) used a viral vector to express 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

Application in Epilepsy
Mutations in genes coding for ion channels Altered inhibition/excitation balance in neurons Might be ameliorated by activating NpHR and/or ChR2 targeted to specific cell types.