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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
Early Optogenetics
Optics + genetics Light mediated uncaging
Light unblocks neurotransmitter
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
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.
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
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
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
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
ChR2 in insulin producing cells Blue light Depolarization Ca2+ entry Insulin release
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.
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 .
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.
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
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.